ALLOGENEIC CELL COMPOSITIONS AND METHODS OF USE

FIELD OF THE DISCLOSURE

The disclosure is directed to molecular biology, and more, specifically, to chimeric receptors, allogeneic cell compositions, methods of making and methods of using the same.

INCORPORATION-BY-REFERENCE OF SEQUENCE LISTING

The contents of the file named “POTH-046_001WO_SequenceListing.txt”, which was created on Sep. 5, 2019, and is 55.7 MB in size are hereby incorporated by reference in their entirety.

BACKGROUND OF THE INVENTION

There has been a long-felt but unmet need in the art for an allogeneic cell composition that overcomes the challenges presented by eliminating genes involved in a graft versus host response and host versus graft response. The disclosure provides allogeneic cell compositions, methods of making and methods of using these compositions which comprise non-naturally occurring structural improvements to restore responsiveness of allogeneic cells to environmental stimuli as well as reduce or prevent rejection by natural killer cell-mediated cytotoxicity.

SUMMARY OF THE INVENTION

The present disclosure provides a non-naturally occurring chimeric stimulatory receptor (CSR) comprising: (a) an ectodomain comprising a activation component, wherein the activation component is isolated or derived from a first protein; (b) a transmembrane domain; and (c) an endodomain comprising at least one signal transduction domain, wherein the at least one signal transduction domain is isolated or derived from a second protein; wherein the first protein and the second protein are not identical.

The activation component can comprise a portion of one or more of a component of a T-cell Receptor (TCR), a component of a TCR complex, a component of a TCR co-receptor, a component of a TCR co-stimulatory protein, a component of a TCR inhibitory protein, a cytokine receptor, and a chemokine receptor to which an agonist of the activation component binds. The activation component can comprise a CD2 extracellular domain or a portion thereof to which an agonist binds.

The signal transduction domain can comprise one or more of a component of a human signal transduction domain, T-cell Receptor (TCR), a component of a TCR complex, a component of a TCR co-receptor, a component of a TCR co-stimulatory protein, a component of a TCR inhibitory protein, a cytokine receptor, and a chemokine receptor. The signal transduction domain can comprise a CD3 protein or a portion thereof. The CD3 protein can comprise a CD3ζ protein or a portion thereof.

The endodomain can further comprise a cytoplasmic domain. The cytoplasmic domain can be isolated or derived from a third protein. The first protein and the third protein can be identical. The ectodomain can further comprise a signal peptide. The signal peptide can be derived from a fourth protein. The first protein and the fourth protein can be identical. The transmembrane domain can be isolated or derived from a fifth protein. The first protein and the fifth protein can be identical.

In some aspects, the activation component does not bind a naturally-occurring molecule. In some aspects, the activation component binds a naturally-occurring molecule but the CSR does not transduce a signal upon binding of the activation component to a naturally-occurring molecule. In some aspects, the activation component binds to a non-naturally occurring molecule. In some aspects, the activation component does not bind a naturally-occurring molecule but binds a non-naturally occurring molecule. The CSR can selectively transduces a signal upon binding of the activation component to a non-naturally occurring molecule. In a preferred aspect, the present disclosure provides a non-naturally occurring chimeric stimulatory receptor (CSR) comprising: (a) an ectodomain comprising a signal peptide and an activation component, wherein the signal peptide comprises a CD2 signal peptide or a portion thereof and wherein the activation component comprises a CD2 extracellular domain or a portion thereof to which an agonist binds; (b) a transmembrane domain, wherein the transmembrane domain comprises a CD2 transmembrane domain or a portion thereof; and (c) an endodomain comprising a cytoplasmic domain and at least one signal transduction domain, wherein the cytoplasmic domain comprises a CD2 cytoplasmic domain or a portion thereof and wherein the at least one signal transduction domain comprises a CD3ζ protein or a portion thereof. In some aspects, the non-naturally CSR comprises an amino acid sequence at least 80%, at least 90%, at least 95% or at least 99% identical to SEQ ID NO:17062. In a preferred aspect, the non-naturally occurring CSR comprises an amino acid sequence of SEQ ID NO:17062.

The present disclosure also provides a non-naturally occurring chimeric stimulatory receptor (CSR) wherein the ectodomain comprises a modification. The modification can comprise a mutation or a truncation of the amino acid sequence of the activation component or the first protein when compared to a wild type sequence of the activation component or the first protein. The mutation or a truncation of the amino acid sequence of the activation component can comprise a mutation or truncation of a CD2 extracellular domain or a portion thereof to which an agonist binds. The mutation or truncation of the CD2 extracellular domain can reduce or eliminate binding with naturally occurring CD58. In some aspects, the CD2 extracellular domain comprising the mutation or truncation comprises an amino acid sequence at least 80%, at least 90%, at least 95% or at least 99% identical to SEQ ID NO:17119. In a preferred aspect, the CD2 extracellular domain comprising the mutation or truncation comprises an amino acid sequence of SEQ ID NO:17119.

In a preferred aspect, the present disclosure provides non-naturally occurring chimeric stimulatory receptor (CSR) comprising: (a) an ectodomain comprising a signal peptide and an activation component, wherein the signal peptide comprises a CD2 signal peptide or a portion thereof and wherein the activation component comprises a CD2 extracellular domain or a portion thereof to which an agonist binds and wherein the CD2 extracellular domain or a portion thereof to which an agonist binds comprises a mutation or truncation; (b) a transmembrane domain, wherein the transmembrane domain comprises a CD2 transmembrane domain or a portion thereof; and (c) an endodomain comprising a cytoplasmic domain and at least one signal transduction domain, wherein the cytoplasmic domain comprises a CD2 cytoplasmic domain or a portion thereof and wherein the at least one signal transduction domain comprises a CD3ζ protein or a portion thereof. In some aspects, the non-naturally CSR comprises an amino acid sequence at least 80%, at least 90%, at least 95% or at least 99% identical to SEQ ID NO:17118. In a preferred aspect, the non-naturally occurring CSR comprises an amino acid sequence of SEQ ID NO:17118.

The present disclosure provides a nucleic acid sequence encoding any CSR disclosed herein. The present disclosure provides a vector comprising a nucleic acid sequence encoding any CSR disclosed herein. The present disclosure provides a transposon comprising a nucleic acid sequence encoding any CSR disclosed herein.

The present disclosure provides a cell comprising any CSR disclosed herein. The present disclosure provides a cell comprising a nucleic acid sequence encoding any CSR disclosed herein. The present disclosure provides a cell comprising a vector comprising a nucleic acid sequence encoding any CSR disclosed herein. The present disclosure provides a cell comprising a transposon comprising a nucleic acid sequence encoding any CSR disclosed herein.

A modified cell disclosed herein can be an allogeneic cell or an autologous cell. In some preferred aspects, the modified cell is an allogeneic cell. In some preferred aspects, the modified cell is an allogeneic T-cell or a modified allogeneic CAR T-cell.

The present disclosure provides a composition comprising any CSR disclosed herein. The present disclosure provides a composition comprising a nucleic acid sequence encoding any CSR disclosed herein. The present disclosure provides a composition comprising a vector comprising a nucleic acid sequence encoding any CSR disclosed herein. The present disclosure provides a composition comprising a transposon comprising a nucleic acid sequence encoding any CSR disclosed herein. The present disclosure provides a composition comprising a modified cell disclosed herein or a composition comprising a plurality of modified cells disclosed herein.

The present disclosure provides a modified T lymphocyte (T-cell), comprising: (a) a modification of an endogenous sequence encoding a T-cell Receptor (TCR), wherein the modification reduces or eliminates a level of expression or activity of the TCR; and (b) a chimeric stimulatory receptor (CSR) comprising: (i) an ectodomain comprising an activation component, wherein the activation component is isolated or derived from a first protein; (ii) a transmembrane domain; and (iii) an endodomain comprising at least one signal transduction domain, wherein the at least one signal transduction domain is isolated or derived from a second protein; wherein the first protein and the second protein are not identical.

The modified T-cell can further comprise an inducible proapoptotic polypeptide. The modified T-cell can further comprise a modification of an endogenous sequence encoding Beta-2-Microglobulin (B2M), wherein the modification reduces or eliminates a level of expression or activity of a major histocompatibility complex (MHC) class I (MHC-I).

The modified T-cell can further comprise a non-naturally occurring polypeptide comprising an HLA class I histocompatibility antigen, alpha chain E (HLA-E) polypeptide. The non-naturally occurring polypeptide comprising a HLA-E polypeptide can further comprise a B2M signal peptide. The non-naturally occurring polypeptide comprising a HLA-E polypeptide can further comprise a B2M polypeptide. The non-naturally occurring polypeptide comprising an HLA-E polypeptide can further comprise a linker, wherein the linker is positioned between the B2M polypeptide and the HLA-E polypeptide. The non-naturally occurring polypeptide comprising an HLA-E polypeptide can further comprise a peptide and a B2M polypeptide. The non-naturally occurring polypeptide comprising an HLA-E can further comprise a first linker positioned between the B2M signal peptide and the peptide, and a second linker positioned between the B2M polypeptide and the peptide encoding the HLA-E.

The modified T-cell can further comprise a non-naturally occurring antigen receptor, a sequence encoding a therapeutic polypeptide, or a combination thereof. The non-naturally occurring antigen receptor can comprise a chimeric antigen receptor (CAR).

The CSR can be transiently expressed in the modified T-cell. The CSR can be stably expressed in the modified T-cell. The polypeptide comprising the HLA-E polypeptide can be transiently expressed in the modified T-cell. The polypeptide comprising the HLA-E polypeptide can be stably expressed in the modified T-cell. The inducible proapoptotic polypeptide can be transiently expressed in the modified T-cell. The inducible proapoptotic polypeptide can be stably expressed in the modified T-cell. The non-naturally occurring antigen receptor or a sequence encoding a therapeutic protein can be transiently expressed in the modified T-cell. The non-naturally occurring antigen receptor or a sequence encoding a therapeutic protein can be stably expressed in the modified T-cell.

The modified T-cell can be an autologous cell. The modified T-cell can be an allogeneic cell. The modified T-cell can be an early memory T cell, a stem cell-like T cell, a stem memory T cell (T_SCM), a central memory T cell (T_CM) or a stem cell-like T cell.

The present disclosure provides a composition comprising any modified T-cell disclosed herein. The present disclosure also provides a composition comprising a population of modified T lymphocytes (T-cells), wherein a plurality of the modified T-cells of the population comprise the CSR disclosed herein. The present disclosure also provides a composition comprising a population of T lymphocytes (T-cells), wherein a plurality of the T-cells of the population comprise the modified T-cell disclosed herein.

The present disclosure provides methods of treating a disease or disorder comprising administering to a subject in need thereof a therapeutically-effective amount of any composition disclosed herein; or a composition for use in the treatment of a disease or disorder. In one aspect, the composition is a modified T-cell or population of modified T-cells as disclosed herein. The present disclosure also a method of treating a disease or disorder comprising administering to a subject in need thereof a therapeutically-effective amount of a composition disclosed herein and at least one non-naturally occurring molecule that binds the CSR.

The present disclosure provides a method of producing a population of modified T-cells comprising, consisting essential of, or consisting of introducing into a plurality of primary human T-cells a composition comprising the CSR of the present disclosure or a sequence encoding the same to produce a plurality of modified T-cells under conditions that stably express the CSR within the plurality of modified T-cells and preserve desirable stem-like properties of the plurality of modified T-cells. The present disclosure provides a composition comprising a population of modified T-cells produced by the method. In some aspects, at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% of the population comprising the CSR expresses one or more cell-surface marker(s) of a stem memory T cell (T_SCM) or a T_SCM-like cell; and wherein the one or more cell-surface marker(s) comprise CD45RA and CD62L. some aspects, at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% of the population expresses one or more cell-surface marker(s) of a central memory T cell (T_CM) or a T_CM-like cell; and wherein the one or more cell-surface marker(s) comprise CD45RO and CD62L. The composition can be for use in the treatment of a disease or disorder. The present disclosure also provides for use of a composition produced by the method for the treatment of a disease or disorder. The present disclosure further provides a method of treating a disease or disorder comprising administering to a subject in need thereof a therapeutically-effective amount of the composition produced by the method. The method of treating can further comprising administering an activator composition to the subject to activate the population of modified T-cells in vivo, to induce cell division of the population of modified T-cells in vivo, or a combination thereof.

The present disclosure provides a method of producing a population of modified T-cells comprising, consisting essential of, or consisting of introducing into a plurality of primary human T-cells a composition comprising the CSR of the present disclosure or a sequence encoding the same to produce a plurality of modified T-cells under conditions that transiently express the CSR within the plurality of modified T-cells and preserve desirable stem-like properties of the plurality of modified T-cells. The present disclosure provides a composition comprising a population of modified T-cells produced by the method. In some aspects, at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% of the population comprising the CSR expresses one or more cell-surface marker(s) of a stem memory T cell (T_SCM) or a T_SCM-like cell; and wherein the one or more cell-surface marker(s) comprise CD45RA and CD62L. some aspects, at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% of the population expresses one or more cell-surface marker(s) of a central memory T cell (T_CM) or a T_CM-like cell; and wherein the one or more cell-surface marker(s) comprise CD45RO and CD62L. The composition can be for use in the treatment of a disease or disorder. The present disclosure also provides for use of a composition produced by the method for the treatment of a disease or disorder. The present disclosure further provides a method of treating a disease or disorder comprising administering to a subject in need thereof a therapeutically-effective amount of the composition produced by the method. In some aspects, the modified T-cells within the population of modified T-cells administered to the subject no longer express the CSR.

The present disclosure provides a method of expanding a population of modified T-cells comprising introducing into a plurality of primary human T-cells a composition comprising the CSR of the present disclosure or a sequence encoding the same to produce a plurality of modified T-cells under conditions that stably express the CSR within the plurality of modified T-cells and preserve desirable stem-like properties of the plurality of modified T-cells and contacting the cells with an activator composition to produce a plurality of activated modified T-cells, wherein expansion of the plurality of modified T-cells is at least two fold higher than the expansion of a plurality of wild-type T-cells not stably expressing the CSR under the same conditions. In some aspects, at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% of the population comprising the CSR expresses one or more cell-surface marker(s) of a stem memory T cell (T_SCM) or a T_SCM-like cell; and wherein the one or more cell-surface marker(s) comprise CD45RA and CD62L. some aspects, at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% of the population expresses one or more cell-surface marker(s) of a central memory T cell (T_CM) or a T_CM-like cell; and wherein the one or more cell-surface marker(s) comprise CD45RO and CD62L. The present disclosure provides a composition comprising a population of modified T-cells expanded by the method. The composition can be for use in the treatment of a disease or disorder. The present disclosure also provides for use of a composition expanded by the method for the treatment of a disease or disorder. The present disclosure further provides a method of treating a disease or disorder comprising administering to a subject in need thereof a therapeutically-effective amount of the composition expanded by the method. The method of treating can further comprising administering an activator composition to the subject to activate the population of modified T-cells in vivo, to induce cell division of the population of modified T-cells in vivo, or a combination thereof.

The present disclosure provides a method of expanding a population of modified T-cells comprising introducing into a plurality of primary human T-cells a composition comprising the CSR of the present disclosure or a sequence encoding the same to produce a plurality of modified T-cells under conditions that transiently express the CSR within the plurality of modified T-cells and preserve desirable stem-like properties of the plurality of modified T-cells and contacting the cells with an activator composition to produce a plurality of activated modified T-cells, wherein expansion of the plurality of modified T-cells is at least two fold higher than the expansion of a plurality of wild-type T-cells not transiently expressing the CSR under the same conditions. The present disclosure provides a composition comprising a population of modified T-cells expanded by the method. In some aspects, at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% of the population comprising the CSR expresses one or more cell-surface marker(s) of a stem memory T cell (T_SCM) or a T_SCM-like cell; and wherein the one or more cell-surface marker(s) comprise CD45RA and CD62L. some aspects, at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% of the population expresses one or more cell-surface marker(s) of a central memory T cell (T_CM) or a T_CM-like cell; and wherein the one or more cell-surface marker(s) comprise CD45RO and CD62L. The composition can be for use in the treatment of a disease or disorder. The present disclosure also provides for use of a composition expanded by the method for the treatment of a disease or disorder. The present disclosure further provides a method of treating a disease or disorder comprising administering to a subject in need thereof a therapeutically-effective amount of the composition expanded by the method. In some aspects, the modified T-cells within the population of modified T-cells administered to the subject no longer express the CSR.

Any of the above aspects can be combined with any other aspect.

Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs. In the Specification, the singular forms also include the plural unless the context clearly dictates otherwise; as examples, the terms “a,” “an,” and “the” are understood to be singular or plural and the term “or” is understood to be inclusive. By way of example, “an element” means one or more element. Throughout the specification the word “comprising,” or variations such as “comprises” or “comprising,” will be understood to imply the inclusion of a stated element, integer or step, or group of elements, integers or steps, but not the exclusion of any other element, integer or step, or group of elements, integers or steps. About can be understood as within 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, 0.5%, 0.1%, 0.05%, or 0.01% of the stated value. Unless otherwise clear from the context, all numerical values provided herein are modified by the term “about.”

Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present disclosure, suitable methods and materials are described below. All publications, patent applications, patents, and other references mentioned herein are incorporated by reference in their entirety. The references cited herein are not admitted to be prior art to the claimed invention. In the case of conflict, the present Specification, including definitions, will control. In addition, the materials, methods, and examples are illustrative only and are not intended to be limiting. Other features and advantages of the disclosure will be apparent from the following detailed description and claims.

BRIEF DESCRIPTION OF THE DRAWINGS

The patent or application file contains at least one drawing executed in color. Copies of this patent or patent application publication with color drawing(s) will be provided by the Office upon request and payment of the necessary fee.

FIG. 1 is a schematic diagram depicting a T-cell receptor (TCR) and co-receptors CD28 and CD2.

FIG. 2 is a schematic diagram depicting primary and secondary co-stimulation is delivered to T-cell via binding of agonist mAbs (anti-CD3, anti-CD28, and anti-CD2). Full T-cell activation critically depends on TCR engagement in conjunction with a second signal by co-stimulatory receptors that boost the immune response. Primary and secondary co-stimulation can be delivered to T-cell via treatment with and engagement of surface receptors with agonist mAbs (E.g. anti-CD3, anti-CD28, and anti-CD2).

FIG. 3 is a schematic diagram showing that, in absence of TCR, only secondary co-stimulation is delivered to T-cell via binding of agonist mAbs. Since full T-cell activation is critically dependent on primary stimulation via CD3ζ in conjunction with a second signal by co-stimulatory receptors, T cell activation and expansion is suboptimal and thus reduced.

FIG. 4 is a schematic diagram showing that, in absence of TCR, stimulation is enhanced with expression of Chimeric Stimulatory Receptors (CSRs). In the absence of TCR, but in the presence of surface-expressed CSR/s, primary and secondary co-stimulatory signals are delivered when T cell is treated with standard agonist mAbs. Since a fuller T-cell activation is achieved via CSR-mediated stimulatory signals, T cell activation and expansion is enhanced.

FIG. 5 is a schematic diagram depicting an exemplary CSR CD28z of the disclosure.

FIG. 6 is a schematic diagram depicting an exemplary CSR CD2z of the disclosure.

FIG. 7 is a schematic of a strategy for mutation of CSR CD2z to eliminate natural ligand (CD58) binding. A panel of CSR CD2z mutants was designed within the extracellular domain of CD2. The goal of this panel was to identify mutants that no longer bind CD58 but retain their receptivity to being bound by the anti-CD2 activator reagent. This may be desirable for two main reasons: 1) CD58 expression by activated T cells may interact with the wild type (WT) CD2z CSR and possibly interfere with the optimal performance of the CSR, and 2) since the WT CD2z CSR might function as a natural ligand CAR, it is possible that T cells expressing the CSR may mediate cytotoxic activity against CD58-expressing cells, including activated T cells. Thus, a mutant CD2z CSR that cannot interact with CD58 but retains its ability to bind activating anti-CD2 reagent for optimal cell expansion is desired.

FIG. 8 is a schematic diagram depicting an exemplary CSR CD2z-D111H of the disclosure. A D111H mutation is within the CD2 extracellular domain of the CSR CD2z-D111H construct.

FIGS. 9A-9B are a series of plots showing that piggyBac® delivery of CSR enhances the expansion of TCRb/b2M double-knockout CAR-T cells. Pan T cells isolated from normal donor blood were genetically modified using the piggyBac® DNA modification system in combination with the Cas-CLOVER™ gene-editing system. Cells were electroporated in a single reaction with a transposon encoding a CAR, selection gene and a CSR (either CD28z or CD2z), an mRNA encoding the super piggyBac™ transposase enzyme, an mRNA encoding Cas-CLOVER™, and multiple guide RNA (gRNA) targeting TCRb and b2M in order to knockout the TCR and MHCI (double-knockout; DKO). The cells were subsequently stimulated with agonist mAbs anti-CD2, anti-CD3 and anti-CD28, and were later selected for genetic modification over the course of a 16 day culture period. At the end of the initial culture period all T cells expressed the CAR, indicating successful selection for genetically-modified cells (data not shown). In the samples expressing either CD2z or CD28z CSR, a greater degree of expansion of the DKO cells was observed as a greater frequency of the CAR alone DKO cells (FIGS. 9A and 9B). In DKO CAR-T cell samples expressing either CD2z or CD28z CSR, at least a two fold expansion of the cells was observed in comparison to DKO CAR-T cells alone.

FIGS. 10A-10B are a series of plots showing that CSR CD2z or CD28z in purified DKO CAR-T cells results in enhanced expansion upon re-stimulation. After initial genetic modification and a first round of stimulation and expansion, cells from each group (Mock (WT CAR-T cells), DKO CAR-T cells, DKO CAR-T cells+CD2z CSR, and DKO CAR-T cells+CD28z CSR) were purified for TCR⁻MHCI⁻ cells using magnetic beads. The purified cells were then re-stimulated using anti-CD2, anti-CD3, and anti-CD28 agonist mAbs. At the end of the 14 day culture period, TCR and MHCI expression (A) as well as magnitude of cell population expansion (B) was determined. After this secondary expansion, all purified DKO cells, including those expressing either CD2z or CD28z CSR, were still extremely pure for DKO cells (>98.8% DKO). DKO CAR-T cells expressing either CD2z or CD28z CSR resulted in enhanced expansion when compared to those not expressing either CSR.

FIG. 11 is a graph showing that cytokine supplementation can further expand purified DKO CAR-T cells expressing CSR upon re-stimulation. After initial genetic modification and a first round of stimulation and expansion, cells expressing CSRs were purified for DKO cells using magnetic beads. The purified cells were then re-stimulated using anti-CD2, anti-CD3, and anti-CD28 agonist mAbs in the presence exogenous purified recombinant IL7 and IL15. At the end of the 14 day culture period, magnitude of cell population expansion was determined. After a secondary expansion, all purified DKO cells, including those expressing either CD2z or CD28z CSR, were still extremely pure for TCR⁻MHCI⁻ cells (>98.8% double knockout (data not shown)). In addition, cells grew robustly in the presence of IL7 and IL15, which was greater than that without supplementation. These data demonstrate that exogenous cytokines may be added to further expand WT CAR-T cells expressing CSR.

FIG. 12 is a graph showing that surface expression of CAR is not significantly affected by co-expression of CSR in DKO cells. After secondary expansion, cells (Mock (WT T cells), WT CAR-T cells, DKO CAR-T cells, DKO CAR-T cells+CD2z CSR, and DKO CAR-T cells+CD28z CSR) were stained for the surface-expression of CAR and compared to control WT CAR-T cells and Mock T cells. Expression of CD2z or CD28z CSR does not have a significant impact on expression of CAR molecule on the surface of T cells.

FIG. 13 is a graph showing that expression of CSRs does not significantly affect DKO CAR-T cell cytotoxicity in vitro. After secondary expansion, cells (Mock (WT T cells), WT CAR-T cells, DKO CAR-T cells, DKO CAR-T cells+CD2z CSR, and DKO CAR-T cells+CD28z CSR) were co-cultured with engineered K562-BCMA-Luciferase (eK562-Luc.BCMA) or negative control line K562-PSMA-Luciferase (eK562-Luc.PSMA) for 48 hours at 10:1, 3:1, or 1:1 E:T ratios. Luciferase signal was measured to determine cytotoxicity. Killing of eK562-Luc.PSMA is shown in dotted lines, while killing of eK562-Luc.BCMA is shown in solid lines. All CAR⁺ T cells expressed an anti-BCMA specific CAR. DKO CAR-T cells exhibit similar in vitro cytotoxicity as WT CAR-TCR cells. This activity is not significantly affected by CD2z or CD28z CSR co-expression.

FIG. 14 is a graph showing that expression of CSRs does not significantly affect DKO CAR-T cell secretion of IFNg in vitro. Supernatants from the 48 hour killing assay were assayed for secreted IFNg as a measure of antigen-specific functionality of the BCMA CAR T cells. All CAR-T cells, either with or without CD2z or CD28z CSR expression secrete IFNg in response to co-culture with target cells expressing BCMA (eK562-Luc.BCMA), but not those expressing an irrelevant target (eK562-Luc.PSMA).

FIG. 15 is a series of plots showing that expression of CSRs does not significantly affect DKO CAR-T cell proliferation in vitro. Mock (WT T-cells), WT CAR-T cells, DKO CAR-T cells, DKO CAR-T cells+CD2z CSR, and DKO CAR-T cells+CD28z CSR cells were labelled with Cell Trace Violet (CTV), which is diluted as cells proliferate. The cells were co-cultured for 5 days with eK562-Luc.PSMA or eK562-Luc.BCMA cells at a 1:2 E:T ratio. All CAR-T cells, either with or without CD2z or CD28z proliferate in response to target cells expressing BCMA (eK562-Luc.BCMA) but not those expressing an irrelevant antigen (eK562-Luc.PSMA).

FIG. 16 is a pair of graphs showing that the memory phenotype of DKO CAR-T is not significantly affected with CD2z CSR co-expression. WT CAR-T cells, DKO CAR-T cells, DKO CAR-T cells+CD2z, and DKO CAR-T cells+CD28z were stained for expression of surface CD45RA, CD45RO, and CD62L to define Tscm, Tcm, Tem, and Teff cells; Tscm (CD45RA⁺CD45RO⁻CD62L⁺), Tcm (CD45RA⁻CD45RO⁺CD62L⁺), Tem (CD45RA⁻CD45RO⁺CD62L⁻), Teff (CD45RA⁺CD45RO⁻CD62L⁻). WT and DKO CAR-T cells with or without CD2z are comprised predominantly of exceptionally high levels of favorable Tscm and Tcm cells. However, when CD28z is expressed in DKO CAR-T cells, the phenotype is significantly more differentiated, favoring Tcm and Tem cells. This phenotype may have a negative impact on the in vivo functionality of these CAR T cells since they appear to be more differentiated.

FIG. 17 is a series of graphs showing that the expression of activation/exhaustion markers in DKO CAR-T is not significantly affected with CD2z CSR co-expression. Mock (WT T cells), WT CAR-T cells, DKO CAR-T cells, DKO CAR-T cells+CD2z, and DKO CAR-T cells+CD28z were examined by flow cytometry for the expression of important exhaustion molecules Lag3, PD1, and Tim3. WT and DKO CAR-T cells with or without CD2z have little to no expression of exhaustion molecules when compared to mock T cells. However, expression of CD28z CSR in DKO CAR-T during the expansion process leads to significant upregulation of exhaustion markers Lag3, PD1, and Tim3. This phenotype may have a negative impact on the in vivo functionality of these CAR T cells since they appear to be more exhausted. By contrast, CD2z expression has little to no effect on the exhaustion phenotype of DKO CAR-T cells while significantly enhancing the expansion capability of the cells.

FIG. 18 is a graph showing that delivery of CSR enhances the expansion of CAR-T cells. CSRs were delivered to CAR-T cells either transiently by mRNA or stably by piggyBac®. Pan T cells isolated from the blood of a normal donor were genetically modified using the piggyBac® DNA modification system and the standard Poseida process. Cells were co-electroporated in a single reaction with mRNA encoding the Super piggyBac™ transposase enzyme (SPB), a transposon encoding a BCMA CAR and selection gene, along with an additional mRNA encoding a CSR (either CD28z or CD2z; resulting in transient expression) or a CD19 mRNA control, or, with a transposon encoding a BCMA CAR, selection gene and a CSR (either CD28z or CD2z; resulting in stable expression). The cells were subsequently stimulated with agonist mAbs anti-CD2, anti-CD3 and anti-CD28, and were later selected for genetic modification over the course of a 19 day culture period. At the end of the initial culture period all T cells expressed the CAR, indicating successful selection for genetically-modified cells (data not shown). Bars represent total live CAR-T cells in well and numbers indicate fold-enhancement of expansion above CAR-T cells produced in the absence of a CSR or a CD19 mRNA control. In the samples expressing either CD2z or CD28z CSR, either transiently or stably, a greater degree of expansion of the CAR-T cells.

FIG. 19 is a series of bar graphs showing that expression of CSRs does not significantly affect CAR-T cell cytotoxicity. CSRs were delivered to CAR-T cells either transiently by mRNA or stably by piggyBac®. Pan T cells isolated from the blood of a normal donor were genetically modified using the piggyBac® DNA modification system and the standard Poseida process. Cells were co-electroporated in a single reaction with mRNA encoding the Super piggyBac™ transposase enzyme (SPB), a transposon encoding a BCMA CAR and selection gene, along with an additional mRNA encoding a CSR (either CD28z or CD2z; resulting in transient expression), or, with a transposon encoding a BCMA CAR, selection gene and a CSR (either CD28z or CD2z; resulting in stable expression). The cells were subsequently stimulated with agonist mAbs anti-CD2, anti-CD3 and anti-CD28, and were later selected for genetic modification over the course of a 19 day culture period. At the end of the initial culture period all T cells expressed the CAR, indicating successful selection for genetically-modified cells (data not shown). To assess CAR-T cell ability to kill, cells were co-cultured with engineered K562-BCMA-Luciferase (eK562-Luc.BCMA) or negative control line K562-Luciferase (eK562-Luc) for 48 hours at 10:1, 3:1, or 1:1 E:T ratios. Luciferase signal was measured to determine cytotoxicity. Killing of eK562-Luc is shown in bar graph on left, while killing of eK562-Luc.BCMA is shown in bar graph on right. All CAR⁺ T cells expressed an anti-BCMA specific CAR and exhibited similar in vitro cytotoxicity against BCMA+ target cells. In summary, this activity was not significantly affected by transient or stable CSR co-expression.

FIG. 20 is a schematic diagram showing that, in presence of TCR, stimulation is enhanced with expression of Chimeric Stimulatory Receptors (CSRs). In the presence of surface-expressed CSR/s, either transiently or stably expressed, enhanced primary and secondary co-stimulatory signals are delivered when T cell is treated with reagents displaying agonist mAbs. In one aspect, this schematic diagram represents an autologous cell. Since a fuller T-cell activation is achieved via CSR-mediated stimulatory signals, T cell activation and expansion is enhanced.

FIG. 21 is a series of graphs showing that CSRs are expressed on the surface of T cells and do not lead to cellular activation in the absence of exogenous stimulation. Pan T cells from normal blood donors were stimulated with anti-CD3/anti-CD28 beads in standard T cell culture media, then rested. These cells were then electroporated (BTX ECM 830 electroporator @ 500V for 700 μs) with 10 μg of mRNA encoding either CD28 CSR, CD2 CSR, or wild-type CD19 control. Two days later the electroporated cells were examined by flow cytometry for surface-expression of each molecule and data are shown as stacked histograms. In addition, cell size (FSC-A) and CD69 expression was evaluated as a possible indication of cellular activation above the Mock electroporated control cells. Increased surface expression of CD28, CD2, and CD19 were detected in T cells electroporated either with CD28z CSR, CD2z CSR or CD19, respectively. Expression of these molecules on the surface of T cells did not intrinsically activate the cells in the absence of exogenous stimulation.

FIG. 22 is a series of line graphs showing that CSR molecules can be delivered transiently during manufacturing for the enhanced expansion of CAR-T cells. Pan T cells isolated from healthy donor blood were genetically modified using the piggyBac® DNA modification system in combination with the Cas-CLOVER™ gene-editing system (CC) for the production of allogeneic (Allo) CAR-T cells, or without CC gene-editing for the production of autologous (Auto) CAR-T cells; auto CAR-T cells were produced by nucleofection of an mRNA encoding the super piggyBac® transposase enzyme (SPB) and a transposon encoding a CAR, selection gene and a safety switch. For production of Allo CAR-T, cells were electroporated (EP) in a single reaction with an mRNA encoding the SPB enzyme, an mRNA encoding CC, multiple guide RNAs (gRNA) targeting TCRb and b2M for the knockout of TCR and MHCI, and a transposon encoding either a CAR, selection gene and the CSR CD2z, or a transposon encoding a CAR, selection gene and a safety switch that did not encode a CSR. For CAR-T cells that did not receive a CSR encoded in the transposon for stable integration, the CD2z CSR was provided to the cells transiently as an mRNA only once in the initial EP reaction, at varying amounts of 5 μg, 10 μg, and 20 μg of mRNA in a 100 μl EP reaction. Following EP, all cells were subsequently stimulated with a cocktail of agonist mAbs anti-CD2, anti-CD3 and anti-CD28, and were later selected for genetic modification over the course of a 19-day culture period using the selection gene. At the end of the initial culture period, all T cells expressed the CAR, indicating successful selection for genetically-modified cells (data not shown). Data for each is shown in line graph at various days of production. In the samples where the CD2z CSR was provided stably (as encoded in the transposon (Stable)) or transiently (as encoded in mRNA (mRNA)), a greater degree of expansion of the CAR-T cells was observed as compared to the CAR-T cells produced without a CSR. These data show that the CSR can be delivered transiently as mRNA during manufacturing for enhanced expansion of both autologous and allogeneic CAR-T products.

FIG. 23A is a bar graph showing CSR CD2z mutant staining data. A panel of CSR CD2z mutants was designed, constructed, and tested for surface expression and binding to several anti-CD2 antibody reagents. To do so, each mutant was synthesized, subcloned into an in-house mRNA production vector, and then high-quality mRNA was produced for each. K562 cells were electroporated with 9 μg of mRNA, and surface-expression of each molecule was analyzed by flow cytometry the next day and data are shown as bar graphs. Each molecule was stained with anti-CD2 activator reagent, anti-CD2 monoclonal antibody (clone TS1/8), or anti-CD2 polyclonal antibody reagent (goat anti-human CD2). Variable binding was observed for each construct and data are summarized in FIG. 23C.

FIG. 23B is a series of bar graphs showing CSR CD2z mutant degranulation data. The panel of CSR CD2z mutants was tested for the capability of mediating degranulation against CD58-positive cell targets. T cell degranulation is a surrogate of T cell killing that can be measured by FACS staining for intracellular CD107a expression following coculture with target cell lines expressing target antigen. Specifically, pan T cells from normal blood donors were stimulated with anti-CD3/anti-CD28 beads in standard T cell culture media, then rested. These cells were then electroporated (BTX ECM 830 electroporator @ 500V for 700 μs) with 9 μg of mRNA expressing CSR CD2z mutants and cultured overnight. The next day, the cells were cocultured for 4-6 hours in the presence of various target cell lines. Positive target cell lines included K562 cells or Rat2 cells that were electroporated or lipofected, respectively, with mRNA encoding human CD58, while negative controls were either Rat2 cells that were not electroporated or CSR CD2z mutant expressing T cells alone. Only T cells expressing CSR CD2z mutants that recognized surface-expressed human CD58 were capable of degranulating at levels above background. Little reactivity was observed for the D111H, K67R/Y110D, K67R/Q70K/Y110D/D111H, Delta K106-120, CD3z deletion and mock control, and data are summarized in FIG. 23C.

FIG. 23C is a summary of staining and degranulation data. Data from surface-expression and binding studies, as well as those from degranulation experiments for each CSR CD2z mutant is summarized in the table. Two candidates that are expressed on the surface and/or retain binding to the anti-CD2 activator reagent that do not mediate anti-CD58 degranulation activity are the D111H and K67R/Y1101D CSR CD2z mutants. Only the D111H mutant is strongly bound by all staining reagents on the cell surface while completely abrogating anti-CD58 degranulation activity.

FIG. 23D is a series of flow cytometry plots showing the expression of CD48, CD58 or CD59 on K562 and Rat2 cells. To confirm possible ligands for the CSR WT CD2z molecule, a panel of known and suspected ligands including human CD48, CD58, and CD59 were tested. Degranulation of engineered T cells was evaluated against the cell lines K562 and Rat2 that were made to overexpress the target ligands and confirmed for expression by FACS staining. Red histograms are unstained cells and blue histograms are cells that were electroporated/lipofected with mRNA and then stained for expression of the respective marker by FACS.

FIG. 23E is a bar graph showing that CSR CD2z recognizes human CD58, but not CD48 or CD59. To confirm possible ligands for the CSR WT CD2z molecule, a panel of known and suspected ligands including human CD48, CD58, and CD59 were tested. Degranulation of engineered T cells was evaluated against the cell lines K562 and Rat2 that were made to overexpress the target ligands and confirmed for expression by FACS staining. Cells were electroporated/lipofected with mRNA and then stained for expression of the respective marker by FACS. As a control, a BCMA CAR was included as well as a K562 cell line overexpressing BCMA. In addition, T cells transfected with GFP were also included as a control. T cell degranulation is a surrogate of T cell killing that can be measured by FACS staining for intracellular CD107a expression following coculture with target cell lines expressing target antigen. Pan T cells from normal blood donors were stimulated with anti-CD3/anti-CD28 beads in standard T cell culture media, then rested. These T cells were then electroporated with mRNA expressing CSR WT CD2z, BCMA CAR, or GFP and cultured overnight. The next day, the cells were cocultured for 4-6 hours in the presence of the various target cell lines that were electroporate/lipofected with mRNA encoding human CD48, CD58 or CD59, while negative controls were either K562 or Rat2 cells that were not electroporated/lipofected, or each of the electroporated T cells alone. T cells expressing either the CSR WT CD2z or BCMA CAR were capable of degranulating at levels above background when cocultured with cell lines overexpressing human CD58 or BCMA, respectively, and not against human CD48 or CD59. Little reactivity was observed for the T cells expressing GFP.

FIG. 24A is a bar graph showing that the delivery of CSR CD2z-D111H mutant enhances the expansion of Allo CAR-T cells. Pan T cells isolated from healthy donor blood were genetically modified using the piggyBac® DNA modification system in combination with the Cas-CLOVER™ gene-editing system (CC) for the production of allogeneic (Allo) CAR-T cells, or without CC gene-editing, as a control, for the production of autologous (Auto) CAR-T without a CSR (No CSR); auto CAR-T cells were produced by nucleofection of an mRNA encoding the super piggyBac™ transposase enzyme (SPB) and a transposon encoding a CAR, selection gene and a safety switch. For production of Allo CAR-T, cells were electroporated (EP) in a single reaction with an mRNA encoding the SPB enzyme, an mRNA encoding CC, multiple guide RNAs (gRNA) targeting TCRb and b2M for the knockout of TCR and MHCI, and a transposon encoding either a CAR, selection gene and either the WT or mutant (D111H) CSR CD2z, or a transposon encoding a CAR, selection gene and a safety switch that did not encode a CSR. For the latter, Allo CAR-T cells that did not receive a CSR encoded in the transposon for stable integration, the WT or mutant (D111H) CSR CD2z was provided to the cells transiently as an mRNA only once in the initial EP reaction. Following EP, all cells were subsequently stimulated with a cocktail of agonist mAbs anti-CD2, anti-CD3 and anti-CD28, and were later selected for genetic modification over the course of up to a 15-day culture period using the selection gene. At the end of the initial culture period, all T cells expressed the CAR, indicating successful selection for genetically-modified cells (data not shown), and then all non-edited TCR-positive cells were depleted via negative selection to yield a population of Allo CAR-T cells that were >99% TCR-negative (data not shown). All samples were performed in duplicate, except the Auto (No CSR) control, and data for peak expansion for each (day of peak expansion is displayed) is shown in bar graph where error bars represent standard deviation. In the samples where either the WT or mutant (D111H) CD2z was provided stably (as encoded in the transposon (Stable)) or transiently (as encoded in mRNA (mRNA)), a greater degree of expansion of the Allo CAR-T cells was observed as compared to the Allo CAR-T cells produced without a CSR.

FIG. 24B is a series of bar graphs showing that the delivery of CSR CD2z-D111H mutant does not inhibit gene editing. Pan T cells isolated from healthy donor blood were genetically modified using the piggyBac® DNA modification system in combination with the Cas-CLOVER™ gene-editing system (CC) to produce allogeneic (Allo) CAR-T cells. Cells were electroporated (EP) in a single reaction with an mRNA encoding the SPB enzyme, an mRNA encoding CC, multiple guide RNA (gRNA) targeting TCRb and b2M for the knockout of TCR and MHCI, and a transposon encoding either a CAR, selection gene and either the WT or mutant (D111H) CSR CD2z, or a transposon encoding a CAR, selection gene and a safety switch that did not encode a CSR. For the latter, cells that did not receive a CSR encoded in the transposon for stable integration, the WT or mutant (D111H) CSR CD2z was provided transiently as an mRNA only once in the initial EP reaction. Following EP, all cells were subsequently stimulated with a cocktail of agonist mAbs anti-CD2, anti-CD3 and anti-CD28, and were later selected for genetic modification over the course of up to a 14-day culture period using the selection gene. At the end of the initial culture period, all T cells expressed the CAR, indicating successful selection for genetically-modified cells (data not shown). All samples were performed in duplicate, and data is shown in bar graph where error bars represent standard deviation. In the samples where either the WT or mutant (D111H) CD2z was provided stably (as encoded in the transposon (Stable)) or transiently (as encoded in mRNA (mRNA)), a similar or greater degree of gene editing of the Allo CAR-T cells was observed as compared to the Allo CAR-T cells produced without a CSR.

FIG. 24C is a bar graph showing that the memory phenotype of Allo CAR-T is not significantly affected by delivery of CD2z CSRs. Allo CAR-T cells with no CSR and Allo CAR-Ts with CSR that was delivered either stably or transiently were stained for expression of surface CD45RA, CD45RO, and CD62L to define Tscm, Tcm, Tem, and Teff cells; Tscm (CD45RA⁺CD45RO⁻CD62L), Tcm (CD45RA⁻CD45RO⁺CD62L⁺), Tem (CD45RA⁻CD45RO⁺CD62L⁻), Teff (CD45RA⁻CD45RO⁻CD62L⁻). All samples were performed in duplicate, and data is shown in bar graph where error bars represent standard deviation. Delivery of CSRs did not dramatically affect the levels of favorable Tscm and Tcm cells in the products.

FIG. 25 is a schematic diagram depicting an exemplary HLA-bGBE composition of the disclosure.

FIG. 26 is a schematic diagram depicting an exemplary HLA-gBE composition of the disclosure.

FIG. 27 is a pair of graphs showing that expression of single-chain HLA-E diminishes NK cell-mediated cytotoxicity against HLA-deficient T cells. B2M and TCRαβ was knocked-out of T cells (Jurkat) using CRISPR. B2M/TCRαβ double-knockout (DKO) T cells were electroporated with mRNA encoding an HLA-E molecule (HLA-bGBE), expressed on a single chain with B2M and the peptide VMAPRETLIL (SEQ ID NO: 17127) (B2M/peptide/HLA-E). DKO T cells electroporated with varying amounts of mRNA encoding single chain HLA-E were used as targets for artificial antigen presenting cell (aAPC)-expanded NK cells in a 3 hour co-culture. % cytotoxicity was calculated based on the number of target cells remaining after 3 hours compared to target cells alone. These data demonstrate that surface expression of HLA-E in DKO T cells reduces the total level of cell killing by NK cells in a dose-dependent manner.

FIG. 28 is a listing of gRNA sequences (from top to bottom) and primer sequences (from top to bottom)

FIG. 29 is a series of flow cytometry plots showing that targeted knockout of endogenous HLA-ABC, but not HLA-E. Since we showed that surface expression of HLA-E in MHCI KO T cells can increase their resistance to NK cell-mediated cytotoxicity, we explored additional strategies beyond introduction of a single-chain HLA-E gene. To do so, multiple guide RNA (gRNA) were designed to disrupt the expression of the main targets of host versus graft (HvG), HLA-A, HLA-B and HLA-C, while minimizing disruption of endogenous HLA-E. Specifically, guides were designed to target a conserved region occurring in all the three MHCI protein targets, but not in HLA-E. Pan human T cells were electroporated with mRNA encoding CRISPR Cas9 in combination with various gRNAs and efficiency of MHCI knockout was measured by surface HLA-A and HLA-E expression. FACS analysis of HLA-A and HLA-E expression was performed after a single round of T cell expansion and data are displayed below. These data demonstrate that gene-editing technology can be used to target disruption of MHCI while retaining levels of endogenous HLA-E on the surface of gene-edited T cells.

FIG. 30 is a schematic diagram of the missing-self hypothesis of natural killer mediated toxicity towards MHCI-KO cells.

FIG. 31 is a schematic depiction of the Csy4-T2A-Clo051-G4Slinker-dCas9 construct map (Embodiment 2).

FIG. 32 is a schematic depiction of the pRT1-Clo051-dCas9 Double NLS construct map (Embodiment 1).

FIG. 33 is a schematic diagram showing an exemplary method for the production of allogeneic CAR-Ts of the disclosure.

FIG. 34A is a graph showing high efficiency gene editing of endogenous TCRa in proliferating Jurkat cells and in resting primary human pan T cells as an exemplary method for the production of allogeneic and universal CAR-Ts using Cas-CLOVER™ (an RNA-guided fusion protein comprising a dCas9-Clo051). Cas-CLOVER system disrupted TCRa expression in rapidly proliferating Jurkat T cells and non-dividing resting T cells at comparably high levels.

FIG. 34B is a series of flow cytometry graphs showing efficient gene editing of endogenous TCRa, TCRb, and B2M in resting primary human pan T cells using Cas-CLOVER™. Critical targets TCRa, TCRB, and B2M that mediate alloreactivity were efficiently edited by Cas-CLOVER in resting human T cells.

FIG. 35 is a series of flow cytometry plots showing that Cas-CLOVER can be multiplexed by co-delivering reagents for TCRβ and β2M into primary human T cells. TCRβ/β2M double knock-out (DKO) cells were further enriched using antibody-beads based purification, and purified cells were analyzed by FACS for downregulation of surface expressed CD3 and β2M.

FIG. 36 is a series of graphs demonstrating reduced alloreactivity after KO of TCR and MHCI. Alloreactivities of WT or DKO (TCR and MHCI) CAR-T cells was analyzed by mixed lymphocyte reaction (MLR) and IFNγ by ELISpot assay. On the left, WT or gene-edited DKO CAR-T cells were labeled with celltrace violet (CTV) and mixed at 1:1 ratio with irradiated peripheral blood mononuclear cells (PBMC)s and incubated for 12 days or 20 hr before analysis of proliferation or activation-induced secretion of IFNγ by ELISpot assay, respectively. WT or DKO CAR-T cells were incubated with PBMCs from either allogenic (Donor #1 PBMC and Donor #2 PBMC) or autologous (Autologous PBMC) donors at 1:1 ratio. After 12 days, CTV dye dilution was assessed by FACS and results showed significant proliferation of WT CAR-T cells when incubated with allogeneic PBMCs; proliferative rates of 40% and 39% by WT CAR-T cells was observed when cultured with allogeneic PBMCs from two different donors in comparison to only 2% when WT CAR-T cells were incubated with autologous PBMCs. On the other hand, DKO CAR-T cells did not proliferate when incubated with allogeneic PBMCs, demonstrating that KO of TCR and MHCI resulted in the elimination of graft-versus-host alloreactivity. This was also true in the short-term IFNγ by ELISpot assay (lower left) which showed that only WT CAR-T cells became activated and secreted IFNγ when incubated with allogeneic PBMCs, but not the DKO CAR-T cells. On the right, irradiated WT or DKO CAR-T cells were mixed at 1:1 ratio with PBMCs labeled with CFSE and incubated for 12 days or 20 hr before analysis of proliferation or activation-induced secretion of IFNγ by ELISpot assay, respectively. After 12 days, CFSE dye dilution was assessed by FACS and showed significant proliferation of PBMCs (most likely T cells) when incubated with allogeneic CAR-T cells; 37% and 9% of PBMCs proliferated in comparison to only 2% when incubated with autologous CAR-T cells. On the other hand, PBMCs did not proliferate above background when incubated with allogeneic CAR-T cells, demonstrating that KO of TCR and MHCI resulted in the elimination of host-versus-graft alloreactivity. This was also true in the short-term IFNγ by ELISpot assay (lower left) which showed that only WT CAR-T cells caused activation and secretion of IFNγ by PBMCs when incubated with allogeneic CAR-Ts, not the DKO CAR-T cells.

FIG. 37 is a series of graphs showing that DKO and WT CAR-Ts have similar CAR-expression and stem-like phenotypes. Gene editing does not affect CAR-T cell phenotype. BCMA CAR-expressing TCRβ/β2M DKO and WT T cells were analyzed for phenotype. CAR expression was comparable in WT and DKO. WT and DKO CAR-T cells were analyzed by FACS for expression of CD45RA and CD62L, markers for T stem cell memory (TSCM). These data demonstrate that gene editing of allo CAR-Ts does not significantly reduce the composition of memory CAR-T cells, retaining the exceptionally high and predominantly TSCM phenotype.

FIG. 38 is a series of graphs showing that DKO CAR-Ts are highly functional. Gene editing does not affect CAR-T cell functionality. BCMA CAR-expressing TCRβ/β2M DKO and WT T cells were analyzed for function. Proliferation against H929 (BCMA+) tumor lines was assessed by mixing CAR-T cells with H929 cells, incubated for 7 days, and analyzed for tumor-specific proliferation by FACS. Cytotoxicity and IFNg secretion against H929 (BCMA+) tumor lines was assessed by mixing CAR-T cells with H929 cells at various ratios, incubated for 24 hrs and analyzed for tumor-specific killing by FACS. Cytotoxicity data are normalized to the tumor cell only sample. These data show that gene editing to produce DKO CAR-T cells does not significantly affect their functional capacity.

FIG. 39A is a schematic diagram showing preclinical evaluation of the P-PSMA-101 transposon when delivered by a full-length plasmid (FLP) versus a nanotransposon (NT) at ‘stress’ doses using the Murine Xenograft Model. The murine xenograft model using a luciferase-expressing LNCaP cell line (LNCaP.luc) injected subcutaneously (SC) into NSG mice was utilized to assess in vivo anti-tumor efficacy of the P-PSMA-101 transposon as delivered by a full-length plasmid (FLP) or a nanotransposon (NT) at two different ‘stress’ doses (2.5×10{circumflex over ( )}6 or 4×10{circumflex over ( )}6) of total CAR-T cells from two different normal donors. All CAR-T cells were produced using piggyBac® (PB) delivery of P-PSMA-101 transposon using either FLP or NT delivery. Mice were injected in the axilla with LNCaP and treated when tumors were established (100-200 mm³by caliper measurement). Mice were treated with two different ‘stress’ doses (2.5×10{circumflex over ( )}6 or 4×10{circumflex over ( )}6) of P-PSMA-101 CAR-Ts by IV injection for greater resolution in detecting possible functional differences in efficacy between transposon delivery by the FLP and the NT.

FIG. 39B are a series of graphs showing the tumor volume assessment of mice treated as described in FIG. 34A. Tumor volume assessment by caliper measurement for control mice (black), Donor #1 FLP mice (red), Donor #1 NT mice (blue), Donor #2 FLP mice (orange), and Donor #2 NT mice (green) as displayed as group averages with error bars (top) and individual mice (bottom). The y-axis shows the tumor volume (mm³) assessed by caliper measurement. The x-axis shows the number of days post T cell treatment. Delivered by NT, P-PSMA-101 transposon at a ‘stress’ dose demonstrated enhanced anti-tumor efficacy as measured by caliper in comparison to the FLP and control mice against established SC LNCaP.luc solid tumors.

DETAILED DESCRIPTION OF THE INVENTION

The present disclosure provides a non-naturally occurring chimeric stimulatory receptor (CSR) comprising, consisting essential of, or consisting of: (a) an ectodomain comprising a activation component, wherein the activation component is isolated or derived from a first protein; (b) a transmembrane domain; and (c) an endodomain comprising at least one signal transduction domain, wherein the at least one signal transduction domain is isolated or derived from a second protein, wherein the first protein and the second protein are not identical.

The activation component can comprise, consist essential of, or consist of: one or more of a component of a human transmembrane receptor, a human cell-surface receptor, a T-cell Receptor (TCR), a component of a TCR complex, a component of a TCR co-receptor, a component of a TCR co-stimulatory protein, a component of a TCR inhibitory protein, a cytokine receptor, or a chemokine receptor. The activation component can comprise, consist essential of, or consist of: a portion of one or more of a component of a T-cell Receptor (TCR), a component of a TCR complex, a component of a TCR co-receptor, a component of a TCR co-stimulatory protein, a component of a TCR inhibitory protein, a cytokine receptor, or a chemokine receptor to which an agonist of the activation component binds.

The ectodomain can comprise, consist essential of, or consist of: a CD2 extracellular domain or a portion thereof to which an agonist binds or the ectodomain can comprise, consist essential of, or consist of: a CD28 extracellular domain or a portion thereof to which an agonist binds. The activation component can comprise, consist essential of, or consist of: a CD2 extracellular domain or a portion thereof to which an agonist binds or the activation component can comprise, consist essential of, or consist of: a CD28 extracellular domain or a portion thereof to which an agonist binds. The CD2 extracellular domain to which an agonist binds comprises, consists essential of, or consists of the amino acid sequence at least 95% identical to the amino acid sequence of SEQ ID NO: 17111. The CD2 extracellular domain to which an agonist binds comprises, consists essential of, or consists of the amino acid sequence at least 99% identical to the amino acid sequence of SEQ ID NO: 17111. The CD2 extracellular domain to which an agonist binds comprises, consists essential of, or consists of the amino acid sequence of SEQ ID NO: 17111. The CD28 extracellular domain to which an agonist binds comprises, consists essential of, or consists of the amino acid sequence at least 95% identical to the amino acid sequence of SEQ ID NO: 17099. The CD28 extracellular domain to which an agonist binds comprises, consists essential of, or consists of the amino acid sequence at least 99% identical to the amino acid sequence of SEQ ID NO: 17099. The CD28 extracellular domain to which an agonist binds comprises, consists essential of, or consists of the amino acid sequence of SEQ ID NO: 17099.

The signal transduction domain can comprise, consist essential of, or consist of: one or more of a component of a human signal transduction domain, T-cell Receptor (TCR), a component of a TCR complex, a component of a TCR co-receptor, a component of a TCR co-stimulatory protein, a component of a TCR inhibitory protein, a cytokine receptor, or a chemokine receptor. The second protein can comprise, consist essential of, or consist of: a CD3 protein or a portion thereof. The signal transduction domain can comprise, consist essential of, or consist of a CD3 protein or a portion thereof. The CD3 protein can comprise, consist essential of, or consist of a CD3ζ protein or a portion thereof. The CD3ζ protein comprises, consists essential of, or consists of the amino acid sequence at least 95% identical to the amino acid sequence of SEQ ID NO: 17102. The CD3ζ protein comprises, consists essential of, or consists of the amino acid sequence at least 99% identical to the amino acid sequence of SEQ ID NO: 17102. The CD3ζ protein comprises, consists essential of, or consists of the amino acid sequence of SEQ ID NO: 17102.

The endodomain of a CSR of the present disclosure can further comprise, consist essential of, or consist of a cytoplasmic domain. The cytoplasmic domain can be isolated or derived from a third protein. In some aspects, the first protein and the third protein of a CSR of the present disclosure are identical. The cytoplasmic domain can comprise, consist essential of, or consist of: a CD2 cytoplasmic domain or a portion thereof or the cytoplasmic domain can comprise, consist essential of, or consist of: a CD28 cytoplasmic domain or a portion thereof.

The CD2 cytoplasmic domain comprises, consists essential of, or consists of the amino acid sequence at least 95% identical to the amino acid sequence of SEQ ID NO: 17113. The CD2 cytoplasmic domain comprises, consists essential of, or consists of the amino acid sequence at least 99% identical to the amino acid sequence of SEQ ID NO: 17113. The CD2 cytoplasmic domain comprises, consists essential of, or consists of the amino acid sequence of SEQ ID NO: 17113. The CD28 cytoplasmic domain comprises, consists essential of, or consists of the amino acid sequence at least 95% identical to the amino acid sequence of SEQ ID NO: 17101. The CD28 cytoplasmic domain comprises, consists essential of, or consists of the amino acid sequence at least 99% identical to the amino acid sequence of SEQ ID NO: 17101. The CD28 cytoplasmic domain comprises, consists essential of, or consists of the amino acid sequence of SEQ ID NO: 17101.

The endodomain of a CSR of the present disclosure can further comprise, consist essential of, or consist of a signal peptide. The signal peptide can be isolated or derived from a fourth protein. In some aspects, the first protein and the fourth protein of a CSR of the present disclosure are identical. The signal peptide can comprise, consist essential of, or consist of: a CD2 signal peptide or a portion thereof; the signal peptide can comprise, consist essential of, or consist of: a CD28 signal peptide or a portion thereof or the signal peptide can comprise, consist essential of, or consist of: a CD8a signal peptide or a portion thereof. The CD2 signal peptide comprises, consists essential of, or consists of the amino acid sequence at least 95% identical to the amino acid sequence of SEQ ID NO: 17110. The CD2 signal peptide comprises, consists essential of, or consists of the amino acid sequence at least 99% identical to the amino acid sequence of SEQ ID NO: 17110. The CD2 signal peptide comprises, consists essential of, or consists of the amino acid sequence of SEQ ID NO: 17110. The CD28 signal peptide comprises, consists essential of, or consists of the amino acid sequence at least 95% identical to the amino acid sequence of SEQ ID NO: 17098. The CD28 signal peptide comprises, consists essential of, or consists of the amino acid sequence at least 99% identical to the amino acid sequence of SEQ ID NO: 17098. The CD28 signal peptide comprises, consists essential of, or consists of the amino acid sequence of SEQ ID NO: 17098. The CD8a signal peptide comprises, consists essential of, or consists of the amino acid sequence at least 95% identical to the amino acid sequence of SEQ ID NO: 17037. The CD8a signal peptide comprises, consists essential of, or consists of the amino acid sequence at least 99% identical to the amino acid sequence of SEQ ID NO: 17037. The CD8a signal peptide comprises, consists essential of, or consists of the amino acid sequence of SEQ ID NO: 17037.

The transmembrane domain of a CSR of the present disclosure can be isolated or derived from a fifth protein. In some aspects, the first protein and the fifth protein of a CSR of the present disclosure are identical. The transmembrane domain can comprise, consist essential of, or consist of: a CD2 transmembrane domain or a portion thereof or the transmembrane domain can comprise, consist essential of, or consist of: a CD28 transmembrane domain or a portion thereof. The CD2 transmembrane domain comprises, consists essential of, or consists of the amino acid sequence at least 95% identical to the amino acid sequence of SEQ ID NO: 17112. The CD2 transmembrane domain comprises, consists essential of, or consists of the amino acid sequence at least 99% identical to the amino acid sequence of SEQ ID NO: 17112. The CD2 transmembrane domain comprises, consists essential of, or consists of the amino acid sequence of SEQ ID NO: 17112. The CD28 transmembrane domain comprises, consists essential of, or consists of the amino acid sequence at least 95% identical to the amino acid sequence of SEQ ID NO: 17100. The CD28 transmembrane domain comprises, consists essential of, or consists of the amino acid sequence at least 99% identical to the amino acid sequence of SEQ ID NO: 17100. The CD28 transmembrane domain comprises, consists essential of, or consists of the amino acid sequence of SEQ ID NO: 17100.

In some aspects, the activation component of the CSR of the present disclosure does not bind or is incapable of binding a naturally-occurring molecule. In some aspects, the activation component of the CSR of the present disclosure binds or is capable of binding a naturally-occurring molecule and the CSR transduces a signal upon binding of the activation component to the naturally-occurring molecule. In other aspects, the activation component of the CSR of the present disclosure can bind a naturally-occurring molecule but the CSR does not transduce a signal upon binding of the activation component to a naturally-occurring molecule. In preferred aspects, the activation component of the CSR of the present disclosure binds or is capable of binding to a non-naturally occurring molecule. The activation component of the CSR of the present disclosure selectively transduces a signal upon binding of a non-naturally occurring molecule to the activation component. In one aspect, the naturally occurring molecule is an naturally occurring agonist/activating agent for the activation component of the CSR. The naturally occurring agonist/activating agent that can bind a CSR activation component can be any naturally occurring antibody or antibody fragment. The naturally occurring antibody or antibody fragment can be a naturally occurring anti-CD3 antibody or fragment thereof, an anti-CD2 antibody or fragment thereof, an anti-CD28 antibody or fragment thereof, or any combination thereof. In some aspects, the naturally occurring agonist/activating agent that can bind a CSR activation component can be one or more of an anti-human CD3 monospecific tetrameric antibody complex, an anti-human CD2 monospecific tetrameric antibody complex, an anti-human CD28 monospecific tetrameric antibody complex, or a combination thereof. In one aspect, the non-naturally occurring molecule is an non-naturally occurring agonist/activating agent for the activation component of the CSR. The non-naturally occurring agonist/activating agent that can bind a CSR activation component can be any non-naturally occurring antibody or antibody fragment. The non-naturally occurring antibody or antibody fragment can be a non-naturally occurring anti-CD3 antibody or fragment thereof, an anti-CD2 antibody or fragment thereof, an anti-CD28 antibody or fragment thereof, or any combination thereof. In some aspects, the non-naturally occurring agonist/activating agent that can bind a CSR activation component can be one or more of an anti-human CD3 monospecific tetrameric antibody complex, an anti-human CD2 monospecific tetrameric antibody complex, an anti-human CD28 monospecific tetrameric antibody complex, or a combination thereof. In some aspects, the non-naturally occurring agonist/activating agent that can bind a CSR activation component can be selected from the group consisting of anti-CD2 monoclonal antibody, BTI-322 (Przepiorka et al., Blood 92(11):4066-4071, 1998) and humanized anti-CD2 monoclonal antibody clone AFC-TAB-104 (Siplizumab)(Bissonnette et al. Arch. Dermatol. Res. 301(6):429-442, 2009).

In some aspects, the ectodomain of the CSR of the present disclosure can comprise a modification. The modification can comprise a mutation or a truncation in the amino acid sequence of the activation component or the first protein when compared to a wild type amino acid sequence of the activation component or the first protein. The mutation or a truncation in the amino acid sequence of the activation component or the first protein can comprise a mutation or truncation of a CD2 extracellular domain or a portion thereof to which an agonist binds. The mutation or truncation of the CD2 extracellular domain reduces or eliminates binding with naturally occurring CD58.

A reduction in binding is when at least 50%, at least 75%, at least 900%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% of the binding ability of the mutated or truncated CD2 extracellular domain is reduced when compared to the naturally occurring wild-type counterpart. An elimination in binding is when 100% of the binding ability of the mutated or truncated CD2 extracellular domain is reduced when compared to the naturally occurring wild-type CD2 extracellular domain.

The mutated or truncated CD2 extracellular domain binds anti-CD2 activating agonists and anti-CD2 activating molecules but does not bind naturally occurring CD58. The mutated or truncated CD2 extracellular domain comprises, consists essential of, or consists of the amino acid sequence at least 80% identical to the amino acid sequence of SEQ ID NO: 17119. The mutated or truncated CD2 extracellular domain comprises, consists essential of, or consists of the amino acid sequence at least 85% identical to the amino acid sequence of SEQ ID NO: 17119. The mutated or truncated CD2 extracellular domain comprises, consists essential of, or consists of the amino acid sequence at least 90% identical to the amino acid sequence of SEQ ID NO: 17119. The mutated or truncated CD2 extracellular domain comprises, consists essential of, or consists of the amino acid sequence at least 95% identical to the amino acid sequence of SEQ ID NO: 17119. The mutated or truncated CD2 extracellular domain comprises, consists essential of, or consists of the amino acid sequence at least 99% identical to the amino acid sequence of SEQ ID NO: 17119. The mutated or truncated CD2 extracellular domain comprises, consists essential of, or consists of the amino acid sequence of SEQ ID NO: 17119. The CSR comprising the mutated or truncated CD2 extracellular domain comprises, consists essential of, or consists of the amino acid sequence at least 90% identical to the amino acid sequence of SEQ ID NO: 17118. The CSR comprising the mutated or truncated CD2 extracellular domain comprises, consists essential of, or consists of the amino acid sequence at least 95% identical to the amino acid sequence of SEQ ID NO: 17118. The CSR comprising the mutated or truncated CD2 extracellular domain comprises, consists essential of, or consists of the amino acid sequence at least 99% identical to the amino acid sequence of SEQ ID NO: 17118. The CSR comprising the mutated or truncated CD2 extracellular domain comprises, consists essential of, or consists of the amino acid sequence of SEQ ID NO: 17118.

The present disclosure also provides a non-naturally occurring chimeric stimulatory receptor (CSR) comprising, consisting essential of, or consisting of: (a) an ectodomain comprising a activation component, wherein the activation component is isolated or derived from a first protein and wherein the activation component binds to a non-naturally occurring molecule but does not bind a naturally-occurring molecule; (b) a transmembrane domain; and (c) an endodomain comprising at least one signal transduction domain, wherein the at least one signal transduction domain is isolated or derived from a second protein; wherein the first protein and the second protein are not identical.

The present disclosure also provides a non-naturally occurring chimeric stimulatory receptor (CSR) comprising, consisting essential of, or consisting of: (a) an ectodomain comprising a activation component, wherein the activation component is isolated or derived from a first protein; (b) a transmembrane domain; and (c) an endodomain comprising at least one signal transduction domain, wherein the at least one signal transduction domain is isolated or derived from a second protein; wherein the first protein and the second protein are not identical and wherein the CSR does not transduce a signal upon binding of a naturally-occurring molecule to the activation component.

The present disclosure also provides a non-naturally occurring chimeric stimulatory receptor (CSR) comprising, consisting essential of, or consisting of: (a) an ectodomain comprising a activation component, wherein the activation component is isolated or derived from a first protein; (b) a transmembrane domain; and (c) an endodomain comprising at least one signal transduction domain, wherein the at least one signal transduction domain is isolated or derived from a second protein; wherein the first protein and the second protein are not identical and wherein the CSR transduces a signal upon binding of a non-naturally-occurring molecule to the activation component.

The present disclosure also provides a non-naturally occurring chimeric stimulatory receptor (CSR) comprising, consisting essential of, or consisting of: (a) an ectodomain comprising a signal peptide and an activation component, wherein the signal peptide comprises a CD2 signal peptide or a portion thereof and wherein the activation component comprises a CD2 extracellular domain or a portion thereof to which an agonist binds; (b) a transmembrane domain, wherein the transmembrane domain comprises a CD2 transmembrane domain or a portion thereof; and (c) an endodomain comprising a cytoplasmic domain and at least one signal transduction domain, wherein the cytoplasmic domain comprises a CD2 cytoplasmic domain or a portion thereof and wherein the at least one signal transduction domain comprises a CD3ζ protein or a portion thereof.

The present disclosure also provides a non-naturally occurring chimeric stimulatory receptor (CSR) comprising, consisting essential of, or consisting of: (a) an ectodomain comprising a signal peptide comprising the amino acid sequence of SEQ ID NO: 17110 and an activation component comprising the amino acid sequence of SEQ ID NO: 17111; (b) a transmembrane domain of SEQ ID NO: 17112; and (c) an endodomain comprising a cytoplasmic domain comprising the amino acid sequence of SEQ ID NO: 17113 and at least one signal transduction domain comprising the amino acid sequence of SEQ ID NO: 17102. The non-naturally occurring chimeric stimulatory receptor (CSR) can comprise, consist essential of, or consist of an amino acid sequence at least 80% identical to SEQ ID NO:17062. The non-naturally occurring chimeric stimulatory receptor (CSR) can comprise, consist essential of, or consist of an amino acid sequence at least 85% identical to SEQ ID NO:17062. The non-naturally occurring chimeric stimulatory receptor (CSR) can comprise, consist essential of, or consist of an amino acid sequence at least 90% identical to SEQ ID NO:17062. The non-naturally occurring chimeric stimulatory receptor (CSR) can comprise, consist essential of, or consist of an amino acid sequence at least 95% identical to SEQ ID NO:17062. The non-naturally occurring chimeric stimulatory receptor (CSR) can comprise, consist essential of, or consist of an amino acid sequence at least 99% identical to SEQ ID NO:17062. The non-naturally occurring chimeric stimulatory receptor (CSR) can comprise, consist essential of, or consist of an amino acid sequence of SEQ ID NO:17062.

The present disclosure further provides a non-naturally occurring chimeric stimulatory receptor (CSR) comprising, consisting essential of, or consisting of: (a) an ectodomain comprising a signal peptide and an activation component, wherein the signal peptide comprises a CD2 signal peptide or a portion thereof and wherein the activation component comprises a mutation or truncation of a wild-type CD2 extracellular domain or a portion thereof to which an agonist binds; (b) a transmembrane domain, wherein the transmembrane domain comprises a CD2 transmembrane domain or a portion thereof; and (c) an endodomain comprising a cytoplasmic domain and at least one signal transduction domain, wherein the cytoplasmic domain comprises a CD2 cytoplasmic domain or a portion thereof and wherein the at least one signal transduction domain comprises a CD3ζ protein or a portion thereof. In one aspect, the mutation or truncation of the CD2 extracellular domain reduces or eliminates binding with naturally occurring CD58. In another aspect, the mutated or truncated CD2 extracellular domain binds anti-CD2 activating agonists and anti-CD2 activating molecules but does not bind naturally occurring CD58.

The present disclosure further provides a non-naturally occurring chimeric stimulatory receptor (CSR) comprising, consisting essential of, or consisting of: (a) an ectodomain comprising a signal peptide comprising the amino acid sequence of SEQ ID NO: 17110 and a activation component comprising the amino acid sequence of SEQ ID NO: 17119; (b) a transmembrane domain of SEQ ID NO: 17112; and (c) an endodomain comprising a cytoplasmic domain comprising the amino acid sequence of SEQ ID NO: 17113 and at least one signal transduction domain comprising the amino acid sequence of SEQ ID NO: 17102. The non-naturally occurring chimeric stimulatory receptor (CSR) can comprise, consist essential of, or consist of an amino acid sequence at least 80% identical to SEQ ID NO: 17118. The non-naturally occurring chimeric stimulatory receptor (CSR) can comprise, consist essential of, or consist of an amino acid sequence at least 85% identical to SEQ ID NO: 17118. The non-naturally occurring chimeric stimulatory receptor (CSR) can comprise, consist essential of, or consist of an amino acid sequence at least 90% identical to SEQ ID NO: 17118. The non-naturally occurring chimeric stimulatory receptor (CSR) can comprise, consist essential of, or consist of an acid sequence at least 95% identical to SEQ ID NO: 17118. The non-naturally occurring chimeric stimulatory receptor (CSR) can comprise, consist essential of, or consist of an acid sequence at least 99% identical to SEQ ID NO: 17118. The non-naturally occurring chimeric stimulatory receptor (CSR) can comprise, consist essential of, or consist of an acid sequence of SEQ ID NO: 17118.

The present disclosure also provides a nucleic acid sequence encoding an amino acid sequence of any chimeric stimulatory receptor (CSR) disclosed herein. The present disclosure also provides transposon, a vector, a donor sequence or a donor plasmid comprising, consisting essential of or consisting of a nucleic acid sequence encoding the amino acid sequence of any chimeric stimulatory receptor (CSR) disclosed herein. In one aspect, the vector can be a viral vector. In one aspect, a viral vector can be an an adenoviral vector, adeno-associated viral (AAV) vector, retroviral vector, lentiviral vector or a chimeric viral vector.

The present disclosure also provides a cell comprising, consisting essential of or consisting of any chimeric stimulatory receptor (CSR) disclosed herein. The present disclosure also provides a cell comprising, consisting essential of or consisting of a nucleic acid sequence encoding an amino acid sequence of any chimeric stimulatory receptor (CSR) disclosed herein. The present disclosure also provides a cell comprising, consisting essential of or consisting of a transposon, a vector, a donor sequence or a donor plasmid comprising, consisting essential of or consisting of a nucleic acid sequence encoding the amino acid sequence of any chimeric stimulatory receptor (CSR) disclosed herein. In one aspect, the vector can be a viral vector. In one aspect, a viral vector can be an an adenoviral vector, adeno-associated viral (AAV) vector, retroviral vector, lentiviral vector or a chimeric viral vector. A cell of the present disclosure comprising, consisting essential of or consisting of any chimeric stimulatory receptor (CSR) disclosed herein can be an allogeneic cell or an autologous cell. In some preferred embodiments, the cell is an allogeneic cell.

The present disclosure also provides a composition comprising, consisting essential of or consisting of any chimeric stimulatory receptor (CSR) disclosed herein. The present disclosure also provides a composition comprising, consisting essential of or consisting of a nucleic acid sequence encoding an amino acid sequence of any chimeric stimulatory receptor (CSR) disclosed herein. The present disclosure also provides a composition comprising, consisting essential of or consisting of a transposon, a vector, a donor sequence or a donor plasmid comprising, consisting essential of or consisting of a nucleic acid sequence encoding the amino acid sequence of any chimeric stimulatory receptor (CSR) disclosed herein. In one aspect, the vector can be a viral vector. In one aspect, a viral vector can be an an adenoviral vector, adeno-associated viral (AAV) vector, retroviral vector, lentiviral vector or a chimeric viral vector. The present disclosure also provides a composition comprising, consisting essential of or consisting of a cell or a plurality of cells comprising, consisting essential of or consisting of any chimeric stimulatory receptor (CSR) disclosed herein.

The present disclosure provides a modified cell comprising, consisting essential of, or consisting of a chimeric stimulatory receptor (CSR) comprising, consisting essential of, or consisting of: (i) an ectodomain comprising an activation component, wherein the activation component is isolated or derived from a first protein; (ii) a transmembrane domain; and (iii) an endodomain comprising at least one signal transduction domain, wherein the at least one signal transduction domain is isolated or derived from a second protein; wherein the first protein and the second protein are not identical.

The present disclosure also provides a modified cell comprising, consisting essential of, or consisting of (a) a chimeric stimulatory receptor (CSR) comprising: (i) an ectodomain comprising an activation component, wherein the activation component is isolated or derived from a first protein; (ii) a transmembrane domain; and (iii) an endodomain comprising at least one signal transduction domain, wherein the at least one signal transduction domain is isolated or derived from a second protein; wherein the first protein and the second protein are not identical; and (b) an inducible proapoptotic polypeptide.

The present disclosure also provides a modified cell comprising, consisting essential of, or consisting of: (a) a chimeric stimulatory receptor (CSR) comprising: (i) an ectodomain comprising an activation component, wherein the activation component is isolated or derived from a first protein; (ii) a transmembrane domain; and (iii) an endodomain comprising at least one signal transduction domain, wherein the at least one signal transduction domain is isolated or derived from a second protein; wherein the first protein and the second protein are not identical; (b) a sequence encoding an inducible proapoptotic polypeptide; and wherein the cell is a T-cell, (c) a modification of an endogenous sequence encoding a T-cell Receptor (TCR), wherein the modification reduces or eliminates a level of expression or activity of the TCR.

The present disclosure provides a modified cell comprising, consisting essential of, or consisting of: (a) a modification of an endogenous sequence encoding Beta-2-Microglobulin (B2M), wherein the modification reduces or eliminates a level of expression or activity of a major histocompatibility complex (MHC) class I (MHC-I); and (b) a non-naturally occurring sequence comprising an HLA class I histocompatibility antigen, alpha chain E (HLA-E) polypeptide.

The present disclosure provides a modified T lymphocyte (T-cell) comprising, consisting essential of, or consisting of: (a) a modification of an endogenous sequence encoding a T-cell Receptor (TCR), wherein the modification reduces or eliminates a level of expression or activity of the TCR; and (b) chimeric stimulatory receptor (CSR) comprising: (i) an ectodomain comprising an activation component, wherein the activation component is isolated or derived from a first protein; (ii) a transmembrane domain; and (iii) an endodomain comprising at least one signal transduction domain, wherein the at least one signal transduction domain is isolated or derived from a second protein; wherein the first protein and the second protein are not identical.

The present disclosure provides a modified T lymphocyte (T-cell) comprising, consisting essential of, or consisting of: (a) a modification of an endogenous sequence encoding a T-cell Receptor (TCR), wherein the modification reduces or eliminates a level of expression or activity of the TCR; (b) chimeric stimulatory receptor (CSR) comprising: (i) an ectodomain comprising an activation component, wherein the activation component is isolated or derived from a first protein; (ii) a transmembrane domain; and (iii) an endodomain comprising at least one signal transduction domain, wherein the at least one signal transduction domain is isolated or derived from a second protein; wherein the first protein and the second protein are not identical; and (c) a non-naturally occurring chimeric antigen receptor.

The present disclosure provides a modified T lymphocyte (T-cell) comprising, consisting essential of, or consisting of: (a) a modification of an endogenous sequence encoding a T-cell Receptor (TCR), wherein the modification reduces or eliminates a level of expression or activity of the TCR; (b) a modification of an endogenous sequence encoding Beta-2-Microglobulin (B2M), wherein the modification reduces or eliminates a level of expression or activity of a major histocompatibility complex (MHC) class I (MHC-I); and (c) a chimeric stimulatory receptor (CSR) comprising: (i) an ectodomain comprising an activation component, wherein the activation component is isolated or derived from a first protein; (ii) a transmembrane domain; and (iii) an endodomain comprising at least one signal transduction domain, wherein the at least one signal transduction domain is isolated or derived from a second protein; wherein the first protein and the second protein are not identical.

The present disclosure provides a modified T lymphocyte (T-cell) comprising, consisting essential of, or consisting of: (a) a modification of an endogenous sequence encoding a T-cell Receptor (TCR), wherein the modification reduces or eliminates a level of expression or activity of the TCR; (b) a modification of an endogenous sequence encoding Beta-2-Microglobulin (B2M), wherein the modification reduces or eliminates a level of expression or activity of a major histocompatibility complex (MHC) class I (MHC-I); (c) a chimeric stimulatory receptor (CSR) comprising: (i) an ectodomain comprising an activation component, wherein the activation component is isolated or derived from a first protein; (ii) a transmembrane domain; and (iii) an endodomain comprising at least one signal transduction domain, wherein the at least one signal transduction domain is isolated or derived from a second protein; wherein the first protein and the second protein are not identical; and (d) a non-naturally occurring chimeric antigen receptor.

The present disclosure also provides a modified T lymphocyte (T-cell) comprising, consisting essential of, or consisting of: (a) a modification of an endogenous sequence encoding a T-cell Receptor (TCR), wherein the modification reduces or eliminates a level of expression or activity of the TCR; (b) a modification of an endogenous sequence encoding Beta-2-Microglobulin (B2M), wherein the modification reduces or eliminates a level of expression or activity of a major histocompatibility complex (MHC) class I (MHC-I); (c) a non-naturally occurring sequence comprising an HLA class I histocompatibility antigen, alpha chain E (HLA-E); and (d) a chimeric stimulatory receptor (CSR) comprising: (i) an ectodomain comprising an activation component, wherein the activation component is isolated or derived from a first protein; (ii) a transmembrane domain; and (iii) an endodomain comprising at least one signal transduction domain, wherein the at least one signal transduction domain is isolated or derived from a second protein; wherein the first protein and the second protein are not identical.

The present disclosure also provides a modified T lymphocyte (T-cell) comprising, consisting essential of, or consisting of: (a) a modification of an endogenous sequence encoding a T-cell Receptor (TCR), wherein the modification reduces or eliminates a level of expression or activity of the TCR; (b) a modification of an endogenous sequence encoding Beta-2-Microglobulin (B2M), wherein the modification reduces or eliminates a level of expression or activity of a major histocompatibility complex (MHC) class I (MHC-I); (c) a non-naturally occurring sequence comprising an HLA class I histocompatibility antigen, alpha chain E (HLA-E); (d) a chimeric stimulatory receptor (CSR) comprising: (i) an ectodomain comprising an activation component, wherein the activation component is isolated or derived from a first protein; (ii) a transmembrane domain; and (iii) an endodomain comprising at least one signal transduction domain, wherein the at least one signal transduction domain is isolated or derived from a second protein; wherein the first protein and the second protein are not identical; and (e) a non-naturally occurring chimeric antigen receptor.

The present disclosure also provides a modified T lymphocyte (T-cell), consisting essential of, or consisting of: (a) a modification of an endogenous sequence encoding a T-cell Receptor (TCR), wherein the modification reduces or eliminates a level of expression or activity of the TCR; (b) a modification that reduces or eliminates a level of expression or activity of a HLA class I histocompatibility antigen, alpha chain A (HLA-A), HLA class I histocompatibility antigen, alpha chain B (HLA-B), HLA class I histocompatibility antigen, alpha chain C (HLA-C), or a combination thereof; and (c) a chimeric stimulatory receptor (CSR) comprising: (i) an ectodomain comprising an activation component, wherein the activation component is isolated or derived from a first protein; (ii) a transmembrane domain; and (iii) an endodomain comprising at least one signal transduction domain, wherein the at least one signal transduction domain is isolated or derived from a second protein; wherein the first protein and the second protein are not identical.

The present disclosure also provides a modified T lymphocyte (T-cell), consisting essential of, or consisting of: (a) a modification of an endogenous sequence encoding a T-cell Receptor (TCR), wherein the modification reduces or eliminates a level of expression or activity of the TCR; (b) a modification that reduces or eliminates a level of expression or activity of a HLA class I histocompatibility antigen, alpha chain A (HLA-A), HLA class I histocompatibility antigen, alpha chain B (HLA-B), HLA class I histocompatibility antigen, alpha chain C (HLA-C), or a combination thereof; (c) a non-naturally occurring sequence comprising an HLA class I histocompatibility antigen, alpha chain E (HLA-E); and (d) a chimeric stimulatory receptor (CSR) comprising: (i) an ectodomain comprising an activation component, wherein the activation component is isolated or derived from a first protein; (ii) a transmembrane domain; and (iii) an endodomain comprising at least one signal transduction domain, wherein the at least one signal transduction domain is isolated or derived from a second protein; wherein the first protein and the second protein are not identical.

A modified cell of the present disclosure (preferably a modified T-cell of the present disclosure) can further comprise, consist essential of, or consist of an inducible proapoptotic polypeptide. The inducible proapoptotic polypeptide comprises, consists essential of, or consists of the amino acid sequence at least 95% identical to the amino acid sequence of SEQ ID NO: 14641. The inducible proapoptotic polypeptide comprises, consists essential of, or consists of the amino acid sequence at least 99% identical to the amino acid sequence of SEQ ID NO: 14641. The inducible proapoptotic polypeptide comprises, consists essential of, or consists of the amino acid sequence of SEQ ID NO: 14641.

A modified cell of the present disclosure (preferably a modified T-cell of the present disclosure) can further comprise, consist essential of, or consist of a modification of an endogenous sequence encoding Beta-2-Microglobulin (B2M), wherein the modification reduces or eliminates a level of expression or activity of a major histocompatibility complex (MHC) class I (MHC-I). A reduction of a level of expression or activity is when at least 50%, at least 75%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% of the expression of the MHC-I in a cell or the functional activity of the MHC-I in a cell is reduced when compared to the naturally occurring wild-type counterpart of the cell. A reduction of a level of expression or activity is when at least 50%, at least 75%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% of the expression of the MHC-I in a T-cell or the functional activity of the MHC-I in a T-cell is reduced when compared to a naturally occurring wild-type T-cell. An elimination a level of expression or activity is when 100% of the expression of the MHC-I in a cell or the functional activity of the MHC-I in a cell is reduced when compared to the naturally occurring wild-type counterpart of the cell. An elimination a level of expression or activity is when 100% of the expression of the MHC-I in a T-cell or the functional activity of the MHC-I in a T-cell is reduced when compared to the naturally occurring wild-type T-cell.

A modified cell of the present disclosure (preferably a modified T-cell of the present disclosure) can further comprise, consist essential of, or consist of a non-naturally occurring polypeptide comprising an HLA class I histocompatibility antigen, alpha chain E (HLA-E). The HLA-E polypeptide comprises, consists essential of, or consists of the amino acid sequence at least 95% identical to the amino acid sequence of SEQ ID NO: 17131. The HLA-E polypeptide comprises, consists essential of, or consists of the amino acid sequence at least 99% identical to the amino acid sequence of SEQ ID NO: 17131. The HLA-E polypeptide comprises, consists essential of, or consists of the amino acid sequence of SEQ ID NO: 17131.

The non-naturally occurring polypeptide comprising a HLA-E can further comprise, consist essential of, or consist of a B2M signal peptide. The B2M signal peptide comprises, consists essential of, or consists of the amino acid sequence at least 95% identical to the amino acid sequence of SEQ ID NO: 17126. The B2M signal peptide comprises, consists essential of, or consists of the amino acid sequence at least 99% identical to the amino acid sequence of SEQ ID NO: 17131. The B2M signal peptide comprises, consists essential of, or consists of the amino acid sequence of SEQ ID NO: 17131.

The non-naturally occurring polypeptide comprising a HLA-E can further comprise, consist essential of, or consist of a B2M polypeptide. The B2M polypeptide comprises, consists essential of, or consists of the amino acid sequence at least 95% identical to the amino acid sequence of SEQ ID NO: 17129. The B2M polypeptide comprises, consists essential of, or consists of the amino acid sequence at least 99% identical to the amino acid sequence of SEQ ID NO: 17129. The B2M polypeptide comprises, consists essential of, or consists of the amino acid sequence of SEQ ID NO: 17129.

The non-naturally occurring polypeptide comprising a HLA-E can further comprise, consist essential of, or consist of a linker molecule (referred to herein as a linker). The non-naturally occurring polypeptide comprising a HLA-E can further comprise, consist essential of, or consist of a linker, wherein the linker is positioned between the B2M polypeptide and the HLA-E polypeptide. The linker comprises, consists essential of, or consists of the amino acid sequence at least 95% identical to the amino acid sequence of SEQ ID NO: 17130. The linker comprises, consists essential of, or consists of the amino acid sequence at least 99% identical to the amino acid sequence of SEQ ID NO: 17130. The linker comprises, consists essential of, or consists of the amino acid sequence of SEQ ID NO: 17130.

The non-naturally occurring polypeptide comprising a HLA-E can further comprise, consist essential of, or consist of a peptide and a B2M polypeptide. The peptide comprises, consists essential of, or consists of the amino acid sequence at least 95% identical to the amino acid sequence of SEQ ID NO: 17127. The peptide comprises, consists essential of, or consists of the amino acid sequence at least 99% identical to the amino acid sequence of SEQ ID NO: 17127. The peptide comprises, consists essential of, or consists of the amino acid sequence of SEQ ID NO: 17127.

The non-naturally occurring polypeptide comprising a HLA-E can further comprise, consist essential of, or consist of a first linker positioned between the B2M signal peptide and the peptide, and a second linker positioned between the B2M polypeptide and the HLA-E polypeptide. The first linker comprises, consists essential of, or consists of the amino acid sequence at least 95% identical to the amino acid sequence of SEQ ID NO: 17128. The first linker comprises, consists essential of, or consists of the amino acid sequence at least 99% identical to the amino acid sequence of SEQ ID NO: 17128. The first linker comprises, consists essential of, or consists of the amino acid sequence of SEQ ID NO: 17128. The second linker comprises, consists essential of, or consists of the amino acid sequence at least 95% identical to the amino acid sequence of SEQ ID NO: 17130. The second linker comprises, consists essential of, or consists of the amino acid sequence at least 99% identical to the amino acid sequence of SEQ ID NO: 17130. The second linker comprises, consists essential of, or consists of the amino acid sequence of SEQ ID NO: 17130.

In one aspect, the non-naturally occurring polypeptide comprising an HLA-E comprises, consists essential of, or consists of a B2M signal peptide, a peptide, a first linker, a B2M polypeptide, a second linker and an HLA-E polypeptide. The peptide can be positioned between the B2M signal peptide and the first linker, the B2M polypeptide can be positioned between the first linker and the second linker and the second linker can be positioned between the B2M polypeptide and the HLA-E polypeptide. The non-naturally occurring polypeptide comprising an HLA-E comprises, consists essential of, or consists of the amino acid sequence at least 95% identical to the amino acid sequence of SEQ ID NO: 17064. The non-naturally occurring polypeptide comprising an HLA-E comprises, consists essential of, or consists of the amino acid sequence at least 99% identical to the amino acid sequence of SEQ ID NO: 17064. The non-naturally occurring polypeptide comprising an HLA-E comprises, consists essential of, or consists of the amino acid sequence of SEQ ID NO: 17064. The non-naturally occurring polypeptide comprising an HLA-E can be encoded by the nucleic acid have the sequence of SEQ ID NO: 17065.

In one aspect, the non-naturally occurring polypeptide comprising an HLA-E comprises, consists essential of, or consists of a B2M signal peptide, a B2M polypeptide, a linker and an HLA-E polypeptide. The B2M polypeptide can be positioned between the B2M signal peptide and the linker, the linker can be positioned between the B2M polypeptide and the HLA-E polypeptide. The non-naturally occurring polypeptide comprising an HLA-E comprises, consists essential of, or consists of the amino acid sequence at least 95% identical to the amino acid sequence of SEQ ID NO: 17066. The non-naturally occurring polypeptide comprising an HLA-E comprises, consists essential of, or consists of the amino acid sequence at least 99% identical to the amino acid sequence of SEQ ID NO: 17066. The non-naturally occurring polypeptide comprising an HLA-E comprises, consists essential of, or consists of the amino acid sequence of SEQ ID NO: 17066. The non-naturally occurring polypeptide comprising an HLA-E can be encoded by the nucleic acid have the sequence of SEQ ID NO: 17067.

In one aspect, the non-naturally occurring polypeptide comprising an HLA-E comprises, consists essential of, or consists of a B2M signal peptide and an HLA-E polypeptide. The B2M signal peptide can be positioned before (e.g. 5′ in the context of a nucleic acid sequence or amino terminus in the context of an amino acid sequence) HLA-E polypeptide. The non-naturally occurring polypeptide comprising an HLA-E comprises, consists essential of, or consists of the amino acid sequence at least 95% identical to the amino acid sequence of SEQ ID NO: 17068. The non-naturally occurring polypeptide comprising an HLA-E comprises, consists essential of, or consists of the amino acid sequence at least 99% identical to the amino acid sequence of SEQ ID NO: 17068. The non-naturally occurring polypeptide comprising an HLA-E comprises, consists essential of, or consists of the amino acid sequence of SEQ ID NO: 17068. The non-naturally occurring polypeptide comprising an HLA-E can be encoded by the nucleic acid have the sequence of SEQ ID NO: 17069.

A modified cell of the present disclosure (preferably a modified T-cell of the present disclosure) can further comprise, consist essential of, or consist of a non-naturally occurring antigen receptor, a sequence encoding a therapeutic polypeptide, or a combination thereof. In a preferred aspect, the non-naturally occurring antigen receptor comprises, consists essential of or consists of a chimeric antigen receptor (CAR). The CAR comprise, consist essential of, or consist of (a) an ectodomain comprising an antigen recognition region, (b) a transmembrane domain, and (c) an endodomain comprising at least one costimulatory domain. The ectodomain of the CAR can further comprise, consist essential of, or consist of a signal peptide. The ectodomain of the CAR can further comprise, consist essential of, or consist of a hinge between the antigen recognition region and the transmembrane domain. The endodomain of the CAR can further comprise, consist essential of, or consist of a human CD3ζ endodomain. The at least one costimulatory domain of the CAR can further comprise, consist essential of, or consist of a human 4-1BB, CD28, CD40, ICOS, MyD88, OX-40 intracellular segment, or any combination thereof. In a preferred aspect, at least one costimulatory domain comprises a human CD28 and/or a 4-1BB costimulatory domain.

A modified cell of the present disclosure can be an immune cell or an immune cell precursor. The immune cell can be a lymphoid progenitor cell, a natural killer (NK) cell, a cytokine induced killer (CIK) cell, a T lymphocyte (T-cell), a B lymphocyte (B-cell) or an antigen presenting cell (APC). In preferred aspects, the immune cell is a T cell, an early memory T cell, a stem cell-like T cell, a stem memory T cell (T_SCM), a central memory T cell (T_CM) or a stem cell-like T cell. The immune cell precursor can a hematopoietic stem cell (HSC). The modified cell can be a stem cell, a differentiated cell, a somatic cell or an antigen presenting cell (APC). The modified cell can be an autologous cell or an allogeneic cell. In one aspect, the cell is a modified allogeneic T-cell. In another aspect, the cell is modified allogeneic T-cell expressing a chimeric antigen receptor (CAR), a CAR T-cell.

A modified cell of the present disclosure (preferably a modified T-cell of the present disclosure) can express a CSR of the present disclosure transiently or stably. In one aspect, a CSR of the present disclosure is transiently expressed in a modified cell of the present disclosure (preferably a modified T-cell of the present disclosure). In one aspect, a CSR of the present disclosure is stably expressed in a modified cell of the present disclosure (preferably a modified T-cell of the present disclosure).

A modified cell of the present disclosure (preferably a modified T-cell of the present disclosure) can express a non-naturally occurring polypeptide comprising the HLA-E of the present disclosure transiently or stably. In one aspect, a non-naturally occurring polypeptide comprising the HLA-E of the present disclosure is transiently expressed in a modified cell of the present disclosure (preferably a modified T-cell of the present disclosure). In one aspect, a non-naturally occurring polypeptide comprising the HLA-E of the present disclosure is stably expressed in a modified cell of the present disclosure (preferably a modified T-cell of the present disclosure).

A modified cell of the present disclosure (preferably a modified T-cell of the present disclosure) can express an inducible proapoptotic polypeptide of the present disclosure transiently or stably. In one aspect, an inducible proapoptotic polypeptide of the present disclosure is transiently expressed in a modified cell of the present disclosure (preferably a modified T-cell of the present disclosure). In a preferred aspect, an inducible proapoptotic polypeptide of the present disclosure is stably expressed in a modified cell of the present disclosure (preferably a modified T-cell of the present disclosure).

A modified cell of the present disclosure (preferably a modified T-cell of the present disclosure) can express a non-naturally occurring antigen receptor or a sequence encoding a therapeutic protein of the present disclosure transiently or stably. In one aspect, a non-naturally occurring antigen receptor or a sequence encoding a therapeutic protein of the present disclosure is transiently expressed in a modified cell of the present disclosure (preferably a modified T-cell of the present disclosure). In a preferred aspect, a non-naturally occurring antigen receptor or a sequence encoding a therapeutic protein of the present disclosure is stably expressed in a modified cell of the present disclosure (preferably a modified T-cell of the present disclosure).

In one aspect, a CSR of the present disclosure is stably expressed, the inducible proapoptotic polypeptide of the present disclosure is stably expressed and a non-naturally occurring antigen receptor or a sequence encoding a therapeutic protein is stably expressed in a modified cell of the present disclosure (preferably a modified T-cell of the present disclosure).

In one aspect, a CSR of the present disclosure is stably expressed, a non-naturally occurring polypeptide comprising the HLA-E of the present disclosure is stably expressed, the inducible proapoptotic polypeptide of the present disclosure is stably expressed and a non-naturally occurring antigen receptor or a sequence encoding a therapeutic protein is stably expressed in a modified cell of the present disclosure (preferably a modified T-cell of the present disclosure).

In one aspect, a CSR of the present disclosure is stably expressed, a non-naturally occurring polypeptide comprising the HLA-E of the present disclosure is transiently expressed, the inducible proapoptotic polypeptide of the present disclosure is stably expressed and a non-naturally occurring antigen receptor or a sequence encoding a therapeutic protein is stably expressed in a modified cell of the present disclosure (preferably a modified T-cell of the present disclosure).

In one aspect, a CSR of the present disclosure is transiently expressed, the inducible proapoptotic polypeptide of the present disclosure is stably expressed and the non-naturally occurring antigen receptor or a sequence encoding a therapeutic protein is stably expressed in a modified cell of the present disclosure (preferably a modified T-cell of the present disclosure).

In one aspect, a CSR of the present disclosure is transiently expressed, a non-naturally occurring polypeptide comprising the HLA-E of the present disclosure is transiently expressed, the inducible proapoptotic polypeptide of the present disclosure is stably expressed and a non-naturally occurring antigen receptor or a sequence encoding a therapeutic protein is stably expressed in a modified cell of the present disclosure (preferably a modified T-cell of the present disclosure).

In one aspect, a CSR of the present disclosure is transiently expressed, a non-naturally occurring polypeptide comprising the HLA-E of the present disclosure is stably expressed, the inducible proapoptotic polypeptide of the present disclosure is stably expressed and a non-naturally occurring antigen receptor or a sequence encoding a therapeutic protein is stably expressed in a modified cell of the present disclosure (preferably a modified T-cell of the present disclosure).

The present disclosure provides a modified cell (preferably a modified T-cell comprising, consisting essential of, or consisting of (a) a modification of an endogenous sequence encoding a T-cell Receptor (TCR), wherein the modification reduces or eliminates a level of expression or activity of the TCR; and (b) a sequence encoding a chimeric stimulatory receptor (CSR) comprising: (i) an ectodomain comprising an activation component, wherein the activation component is isolated or derived from a first protein; (ii) a transmembrane domain; and (iii) an endodomain comprising at least one signal transduction domain, wherein the at least one signal transduction domain is isolated or derived from a second protein; wherein the first protein and the second protein are not identical.

The modified cell further can further comprise, consist essential of or consist of a sequence encoding an inducible proapoptotic polypeptide. The modified cell can further comprise, consist essential of or consist of a sequence encoding a non-naturally occurring antigen receptor, a sequence encoding a therapeutic polypeptide, or a combination thereof. The non-naturally occurring antigen receptor can comprise, consist essential of or consist of a chimeric antigen receptor (CAR).

A transposon, a vector, a donor sequence or a donor plasmid can comprise, consist essential of or consist of the sequence encoding the CSR, the sequence encoding the inducible proapoptotic polypeptide, or a combination thereof. The transposon, the vector, the donor sequence or the donor plasmid can further comprise, consist essential of or consist of a sequence encoding a non-naturally occurring antigen receptor or a sequence encoding a therapeutic protein. The transposon, the vector, the donor sequence, or the donor plasmid can further comprise, consist essential of or consist of a sequence encoding a selection marker. The transposon can be a piggyBac® transposon, a piggy-Bac® like transposon, a Sleeping Beauty transposon, a Helraiser transposon, a Tol2 transposon or a TcBuster transposon. The sequence encoding the CSR can be transiently expressed in the cell. The sequence encoding the CSR can be stably expressed in the cell. The sequence encoding the inducible proapoptotic polypeptide can be stably expressed in the cell. The sequence encoding a non-naturally occurring antigen receptor or a sequence encoding a therapeutic protein is stably expressed in the cell. In some aspects, the sequence encoding the CSR can be transiently expressed in the cell and the sequence encoding the inducible proapoptotic polypeptide can be stably expressed in the cell. In some aspects, the sequence encoding the CSR can be stably expressed in the cell and the sequence encoding the inducible proapoptotic polypeptide can be stably expressed in the cell. In some aspects, the sequence encoding the CSR can be transiently expressed in the cell, the sequence encoding the inducible proapoptotic polypeptide can be stably expressed in the cell and sequence encoding a non-naturally occurring antigen receptor or a sequence encoding a therapeutic protein is stably expressed in the cell. In some aspects, the sequence encoding the CSR can be stably expressed in the cell, the sequence encoding the inducible proapoptotic polypeptide can be stably expressed in the cell and sequence encoding a non-naturally occurring antigen receptor or a sequence encoding a therapeutic protein is stably expressed in the cell. In one aspect, the vector can be a viral vector. In one aspect, a viral vector can be an an adenoviral vector, adeno-associated viral (AAV) vector, retroviral vector, lentiviral vector or a chimeric viral vector.

A first transposon, a first vector, a first donor sequence, or a first donor plasmid can comprise, consist essential of or consist of the sequence encoding the CSR. The first transposon, the first vector, the first donor sequence, or the first donor plasmid can further comprise, consist essential of or consist of a sequence encoding a first selection marker.

A second transposon, a second vector, a second donor sequence, or a second donor plasmid can comprise, consist essential of or consist of one or more of the sequence encoding the inducible proapoptotic polypeptide, the sequence encoding a non-naturally occurring antigen receptor, and the sequence encoding a therapeutic protein. The second transposon, the second vector, the second donor sequence, or the second donor plasmid can further comprise, consist essential of or consist of a sequence encoding a second selection marker. The first selection marker and the second selection marker are identical. The first selection marker and the second selection marker are not identical. The selection marker can comprise, consist essential of or consist of a cell surface marker. The selection marker can comprise, consist essential of or consist of a protein that is active in dividing cells and not active in non-dividing cells. The selection marker can comprise, consist essential of or consist of a metabolic marker.

In one aspect, the selection marker can comprise, consist essential of or consist of a dihydrofolate reductase (DHFR) mutein enzyme. The DHFR mutein enzyme can comprise, consist essential of or consist of the amino acid sequence of SEQ ID NO: 17012.

The DHFR mutein enzyme of SEQ ID NO: 17012 can further comprise, consist essential of or consist of a mutation at one or more of positions 80, 113, or 153. The amino acid sequence of the DHFR mutein enzyme of SEQ ID NO: 17012 can further comprise, consist essential of or consist of one or more of a substitution of a Phenylalanine (F) or a Leucine (L) at position 80; a substitution of a Leucine (L) or a Valine (V) at position 113, and a substitution of a Valine (V) or an Aspartic Acid (D) at position 153.

A modified cell of the present disclosure (preferably a modified T-cell of the present disclosure) can further comprise, consist essential of or consist of a gene editing composition. The gene editing composition can comprise, consist essential of or consist of a sequence encoding a DNA binding domain and a sequence encoding a nuclease protein or a nuclease domain thereof. The gene editing composition can be expressed transiently by the modified cell. The gene editing composition can be expressed stably by the modified cell.

The gene editing composition can comprise, consist essential of or consist of a sequence encoding a nuclease protein or a sequence encoding a nuclease domain thereof. The sequence encoding a nuclease protein or the sequence encoding a nuclease domain thereof can comprise, consist essential of or consist of a DNA sequence, an RNA sequence, or a combination thereof. The nuclease or the nuclease domain thereof can comprise, consist essential of or consist of one or more of a CRISPR/Cas protein, a Transcription Activator-Like Effector Nuclease (TALEN), a Zinc Finger Nuclease (ZFN), and an endonuclease. The CRISPR/Cas protein can comprise, consist essential of or consist of a nuclease-inactivated Cas (dCas) protein. The nuclease or the nuclease domain thereof can comprise, consist essential of or consist of a nuclease-inactivated Cas (dCas) protein and an endonuclease. The endonuclease can comprise, consist essential of or consist of a Clo051 nuclease or a nuclease domain thereof. The gene editing composition can comprise, consist essential of or consist of a fusion protein. The fusion protein can comprise, consist essential of or consist of a nuclease-inactivated Cas9 (dCas9) protein and a Clo051 nuclease or a Clo051 nuclease domain. The fusion protein can comprise, consist essential of or consist of the amino acid sequence of SEQ ID NO: 17013. The fusion protein is encoded by a nucleic acid comprising, consisting essential of or consisting of the sequence of SEQ ID NO: 17014. The fusion protein can comprise, consist essential of or consist of the amino acid sequence of SEQ ID NO: 17058. The fusion protein is encoded by a nucleic acid comprising, consisting essential of or consisting of the sequence of SEQ ID NO: 17059.

The gene editing composition can further comprise, consist essential of or consist of a guide sequence. The guide sequence can comprise, consist essential of or consist of an RNA sequence. In aspects when the modified cell is a T-cell, the guide RNA can comprise, consist essential of or consist of a sequence complementary to a target sequence encoding an endogenous TCR. The guide RNA can comprise, consist essential of or consist of a sequence complementary to a target sequence encoding a B2M polypeptide. The guide RNA can comprise, consist essential of or consist of a sequence complementary to a target sequence within a safe harbor site of a genomic DNA sequence.

The transposon, the vector, the donor sequence or the donor plasmid can further comprise, consist essential of or consist of a gene editing composition comprising a guide sequence and a sequence encoding a fusion protein comprising a sequence encoding an inactivated Cas9 (dCas9) and a sequence encoding a Clo051 nuclease or a nuclease domain thereof.

The first transposon, the first vector, the first donor sequence or the first donor plasmid can further comprise, consist essential of or consist of a gene editing composition comprising a guide sequence and a sequence encoding a fusion protein comprising a sequence encoding an inactivated Cas9 (dCas9) and a sequence encoding a Clo051 nuclease or a nuclease domain thereof.

The second transposon, the second vector, the second donor sequence or the second donor plasmid can further comprise, consist essential of or consist of a gene editing composition comprising a guide sequence and a sequence encoding a fusion protein comprising a sequence encoding an inactivated Cas9 (dCas9) and a sequence encoding a Clo051 nuclease or a nuclease domain thereof.

A third transposon, a third vector, a third donor sequence or a third donor plasmid can comprise, consist essential of or consist of a gene editing composition comprising a guide sequence and a sequence encoding a fusion protein comprising a sequence encoding an inactivated Cas9 (dCas9) and a sequence encoding a Clo051 nuclease or a nuclease domain thereof.

The Clo051 nuclease or a nuclease domain thereof can induce a single or double strand break in a target sequence. The donor sequence or a donor plasmid can integrate at a position of single or double strand break or at a position of cellular repair within a target sequence, or a combination thereof.

The present disclosure provides a composition comprising, consisting essential of, or consisting of a modified cell of the present disclosure (preferably a modified T-cell of the present disclosure).

The present disclosure provides a plurality of modified cells comprising any non-naturally occurring chimeric stimulatory receptor (CSR) disclosed herein and provides a plurality of modified cells comprising any modified cell disclosed herein. The plurality of modified cells can comprise, consist essential of, or consist of immune cells or an immune cell precursors. The plurality of immune cells can comprise, consist essential of, or consist of lymphoid progenitor cells, natural killer (NK) cells, cytokine induced killer (CIK) cells, T lymphocytes (T-cells), B lymphocytes (B-cells) or antigen presenting cells (APCs).

The present disclosure provides a composition comprising a population of modified cells, wherein a plurality of the modified cells of the population comprise any non-naturally occurring chimeric stimulatory receptor (CSR) disclosed herein and provides a composition comprising a population of modified cells, wherein a plurality of the modified cells of the population comprise any modified cell disclosed herein. The population of modified cells can comprise, consist essential of, or consist of immune cells or an immune cell precursors. The population of immune cells can comprise, consist essential of, or consist of lymphoid progenitor cells, natural killer (NK) cells, cytokine induced killer (CIK) cells, T lymphocytes (T-cells), B lymphocytes (B-cells) or antigen presenting cells (APCs). The composition can comprise a pharmaceutically-acceptable carrier.

The present disclosure provides a composition comprising a population of modified T lymphocytes (T-cells), wherein a plurality of the modified T-cells of the population comprise any non-naturally occurring chimeric stimulatory receptor (CSR) disclosed herein and provides a composition comprising a population of T lymphocytes (T-cells), wherein a plurality of the T-cells of the population comprise any modified T-cell disclosed herein. The composition can comprise a pharmaceutically-acceptable carrier.

Preferably, the present disclosure provides a composition comprising a population of T lymphocytes (T-cells), wherein a plurality of the T-cells of the population comprise a non-naturally occurring chimeric stimulatory receptor (CSR) comprising, consisting essential of, or consisting of: (a) an ectodomain comprising a activation component, wherein the activation component is isolated or derived from a first protein; (b) a transmembrane domain; and (c) an endodomain comprising at least one signal transduction domain, wherein the at least one signal transduction domain is isolated or derived from a second protein, wherein the first protein and the second protein are not identical. The composition can comprise a pharmaceutically-acceptable carrier. In some aspects, at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% of the population comprise the CSR.

The plurality of the T-cells of the population can further comprise an inducible proapoptotic polypeptide. In some aspects, at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% of the population comprise the inducible proapoptotic polypeptide.

The plurality of the T-cells of the population can further comprise a modification of an endogenous sequence encoding a T-cell Receptor (TCR), wherein the modification reduces or eliminates a level of expression or activity of the TCR. In some aspects, at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% of the population comprise the modification of the endogenous sequence encoding the TCR, wherein the modification reduces or eliminates a level of expression or activity of the TCR.

The plurality of the T-cells of the population can further comprise a modification of an endogenous sequence encoding Beta-2-Microglobulin (B2M), wherein the modification reduces or eliminates a level of expression or activity of a major histocompatibility complex (MHC) class I (MHC-I). In some aspects, at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% of the population comprise the modification of the endogenous sequence encoding B2M, wherein the modification reduces or eliminates a level of expression or activity of MHC-I.

The plurality of the T-cells of the population can further comprise a modification of an endogenous sequence encoding a T-cell Receptor (TCR), wherein the modification reduces or eliminates a level of expression or activity of the TCR and a modification of an endogenous sequence encoding Beta-2-Microglobulin (B2M), wherein the modification reduces or eliminates a level of expression or activity of a major histocompatibility complex (MHC) class I (MHC-I).

In some aspects, at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 800%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% of the population comprise both modification of the endogenous sequence encoding the TCR, wherein the modification reduces or eliminates a level of expression or activity of the TCR and the modification of the endogenous sequence encoding B2M, wherein the modification reduces or eliminates a level of expression or activity of MHC-I.

The plurality of the T-cells of the population can further comprise a non-naturally occurring sequence comprising an HLA class I histocompatibility antigen, alpha chain E (HLA-E) polypeptide. In some aspects, at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% of the population comprise the non-naturally occurring sequence comprising the HLA-E polypeptide.

The plurality of the T-cells of the population can further comprise a non-naturally occurring antigen receptor, a sequence encoding a therapeutic polypeptide, or a combination thereof. In some aspects, at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 800%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% of the population comprise the non-naturally occurring antigen receptor, the sequence encoding a therapeutic polypeptide, or a combination thereof. In preferred aspects, the non-naturally occurring antigen receptor is a chimeric antigen receptor (CAR).

The plurality of the T-cells of the population can comprise an early memory T cell, a stem cell-like T cell, a stem memory T cell (T_SCM), a central memory T cell (T_CM) or a stem cell-like T cell. In some aspects, one or more of a stem cell-like T cell, a stem cell memory T cell (T_SCM) and a central memory T cell (T_CM) comprise at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% of the population of modified T-cells.

In some aspects, at least 5%, at least 10%, at least 15%, at least 200%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% of the population comprising the CSR expresses one or more cell-surface marker(s) of a stem memory T cell (T_SCM) or a T_SCM-like cell; and wherein the one or more cell-surface marker(s) comprise CD45RA and CD62L.

In some aspects, at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% of the population expresses one or more cell-surface marker(s) of a central memory T cell (T_CM) or a T_CM-like cell; and wherein the one or more cell-surface marker(s) comprise CD45RO and CD62L.

The present disclosure provides a method of producing a modified T-cell comprising, consisting essential of, or consisting of, introducing into a primary human T-cell a composition comprising a Chimeric Stimulator Receptor (CSR) of the present disclosure or a sequence encoding the same to produce a modified T-cell under conditions that transiently express the CSR within the modified T-cell and preserve desirable stem-like properties of the modified T-cell. The primary human T-cell can be a resting primary human T-cell. The present disclosure provides a modified T-cell produced by the disclosed method. The present disclosure provides a method of administering the modified T-cell comprising the transiently expressed CSR produced by the disclosed method. In one aspect, the present disclosure provides a method of administering the modified T-cell produced by the disclosed method after the modified T-cell no longer expresses the CSR. The present disclosure provides a method of administering a modified T-cell comprising the transiently expressed CSR produced by the disclosed method to treat a disease or disorder. In one aspect, the present disclosure provides a method of administering the modified T-cell produced by the disclosed method after the modified T-cell no longer expresses the CSR to treat a disease or disorder.

The present disclosure provides a method of producing a population of modified T-cells comprising, consisting essential of, or consisting of, introducing into a plurality of primary human T-cells a composition comprising a Chimeric Stimulator Receptor (CSR) of the present disclosure or a sequence encoding the same to produce a plurality of modified T-cells under conditions that transiently express the CSR within the plurality of modified T-cells and preserve desirable stem-like properties of the plurality of modified T-cells. The primary human T-cells can comprise resting primary human T-cells. The present disclosure provides a population of modified T-cell produced by the disclosed method. The present disclosure provides a method of administering the population of modified T-cells comprising the transiently expressed CSR produced by the disclosed method. In one aspect, the present disclosure provides a method of administering the population of modified T-cells produced by the disclosed method after the plurality of T-cells no longer express the CSR. The present disclosure provides a method of administering the population of modified T-cells comprising the transiently expressed CSR produced by the disclosed method to treat a disease or disorder. In one aspect, the present disclosure provides a method of administering the population of modified T-cells produced by the disclosed method after the plurality of modified T-cells no longer express the CSR to treat a disease or disorder.

The method of producing a modified T-cell or producing a population of modified T-cells can further comprise introducing a modification of an endogenous sequence encoding a T-cell Receptor (TCR), wherein the modification reduces or eliminates a level of expression or activity of the TCR. The method of producing a modified T-cell or producing a population of modified T-cells can further comprise introducing a modification of an endogenous sequence encoding Beta-2-Microglobulin (B2M), wherein the modification reduces or eliminates a level of expression or activity of a major histocompatibility complex (MHC) class I (MHC-1). In some aspects, the method of producing a modified T-cell or producing a population of modified T-cells can further comprising introducing both a modification of an endogenous sequence encoding TCR, wherein the modification reduces or eliminates a level of expression or activity of the TCR and introducing a modification of an endogenous sequence encoding B2M, wherein the modification reduces or eliminates a level of expression or activity of MHC-1.

The method of producing a modified T-cell or producing a population of modified T-cells can further comprise introducing into the primary human T-cell or plurality of primary human T cells a composition comprising an antigen receptor, a therapeutic protein or a sequence encoding the same. In one aspect, the antigen receptor is a non-naturally occurring antigen receptor. In a preferred aspect, the method of producing a modified T-cell or producing a population of modified T-cells can further comprise introducing into the primary human T-cell or plurality of primary human T cells a composition comprising a Chimeric Antigen Receptor (CAR) or a sequence encoding the same. The method can further comprise introducing into the primary human T-cell or plurality of primary human T cells a composition comprising an inducible proapoptotic polypeptide or a sequence encoding the same. The method of producing a modified T-cell or producing a population of modified T-cells can further comprise introducing into the primary human T-cell or plurality of primary human T cells a composition comprising an antigen receptor, a therapeutic protein or a sequence encoding the same and a composition comprising an inducible proapoptotic polypeptide or a sequence encoding the same.

The method of producing a modified T-cell or producing a population of modified T-cells can further comprise contacting the modified T-cell or population of modified T-cells with an activator composition. The activator composition can comprise, consist essential of, or consist of one or more agonists or activating agents that can bind a CSR activation component of the modified T-cell or plurality of modified T-cells. The agonist/activating agent can be naturally occurring or non-naturally occurring. In preferred aspects, the agonist/activating agent is an antibody or antibody fragment. The agonist/activating agent can be one or more of an anti-CD3 antibody or fragment thereof, an anti-CD2 antibody or fragment thereof, an anti-CD28 antibody or fragment thereof, or any combination thereof. In some aspects, the agonist/activating agent that can be one or more of an anti-human CD3 monospecific tetrameric antibody complex, an anti-human CD2 monospecific tetrameric antibody complex, an anti-human CD28 monospecific tetrameric antibody complex, or a combination thereof. The agonist/activating can contact the modified T-cell or population of modified T-cells in vitro, ex vivo or in vivo. In a preferred aspect, the agonist/activating activates the modified T-cell or population of modified T-cells, induces cell division in the modified T-cell or population of modified T-cells, increases cell division (e.g., cell doubling time) in the modified T-cell or population of modified T-cells, increases fold expansion in the modified T-cell or population of modified T-cells, or any combination thereof.

The present disclosure provides a method of expanding a population of modified T-cells comprising, consisting essential of, or consisting of, introducing into a plurality of primary human T-cells a composition comprising a Chimeric Stimulator Receptor (CSR) of the present disclosure or a sequence encoding the same to produce a plurality of modified T-cells under conditions that stably express the CSR within the plurality of modified T-cells and preserve desirable stem-like properties of the plurality of modified T-cells and contacting the cells with an activator composition to produce a plurality of activated modified T-cells, wherein expansion of the plurality of modified T-cells is at least two fold higher than the expansion of a plurality of wild-type T-cells not stably expressing a CSR of the present disclosure under the same conditions. The method wherein the expansion of the plurality of modified T-cells is at least three fold, at least four fold, at least five fold, at least six fold, at least seven fold, at least eight fold, at least nine fold or at least 10 fold higher than the expansion of a plurality of wild-type T-cells not stably expressing a CSR of the present disclosure under the same conditions.

The present disclosure provides a method of expanding a population of modified T-cells comprising, consisting essential of, or consisting of, introducing into a plurality of primary human T-cells a composition comprising a Chimeric Stimulator Receptor (CSR) of the present disclosure or a sequence encoding the same to produce a plurality of modified T-cells under conditions that transiently express the CSR within the plurality of modified T-cells and preserve desirable stem-like properties of the plurality of modified T-cells and contacting the cells with an activator composition to produce a plurality of activated modified T-cells, wherein expansion of the plurality of modified T-cells is at least two fold higher than the expansion of a plurality of wild-type T-cells not transiently expressing a CSR of the present disclosure under the same conditions. The method wherein the expansion of the plurality of modified T-cells is at least three fold, at least four fold, at least five fold, at least six fold, at least seven fold, at least eight fold, at least nine fold or at least 10 fold higher than the expansion of a plurality of wild-type T-cells not transiently expressing a CSR of the present disclosure under the same conditions.

The activator composition of the methods of expanding a population of can comprise, consist essential of, or consist of one or more agonists or activating agents that can bind a CSR activation component of the modified T-cell or plurality of modified T-cells. The agonist/activating agent can be naturally occurring or non-naturally occurring. In preferred aspects, the agonist/activating agent is an antibody or antibody fragment. The agonist/activating agent can be one or more of an anti-CD3 antibody or fragment thereof, an anti-CD2 antibody or fragment thereof, an anti-CD28 antibody or fragment thereof, or any combination thereof. In some aspects, the agonist/activating agent that can be one or more of an anti-human CD3 monospecific tetrameric antibody complex, an anti-human CD2 monospecific tetrameric antibody complex, an anti-human CD28 monospecific tetrameric antibody complex, or a combination thereof.

The conditions can comprise culturing the modified T-cell or plurality of modified T-cells in a media comprising a sterol; an alkane; phosphorus and one or more of an octanoic acid, a palmitic acid, a linoleic acid, and an oleic acid. The culturing can be in vivo or ex vivo. The modified T-cell can be an allogeneic T-cell or the plurality of modified T-cells can be allogeneic T-cells. The modified T-cell can be an autologous T-cell or the plurality of modified T-cells can be autologous T-cells.

In some aspects, the media can comprise one or more of octanoic acid at a concentration of between 0.9 mg/kg to 90 mg/kg, inclusive of the endpoints; palmitic acid at a concentration of between 0.2 mg/kg to 20 mg/kg, inclusive of the endpoints; linoleic acid at a concentration of between 0.2 mg/kg to 20 mg/kg, inclusive of the endpoints; oleic acid at a concentration of 0.2 mg/kg to 20 mg/kg, inclusive of the endpoints; and a sterol at a concentration of about 0.1 mg/kg to 10 mg/kg, inclusive of the endpoints.

In some aspects, the media can comprise one or more of octanoic acid at a concentration of about 9 mg/kg, palmitic acid at a concentration of about 2 mg/kg, linoleic acid at a concentration of about 2 mg/kg, oleic acid at a concentration of about 2 mg/kg and a sterol at a concentration of about 1 mg/kg.

In some aspects, the media can comprise one or more of octanoic acid at a concentration of between 6.4 μmol/kg and 640 μmol/kg, inclusive of the endpoints; palmitic acid at a concentration of between 0.7 μmol/kg and 70 μmol/kg, inclusive of the endpoints; linoleic acid at a concentration of between 0.75 μmol/kg and 75 μmol/kg, inclusive of the endpoints; oleic acid at a concentration of between 0.75 μmol/kg and 75 μmol/kg, inclusive of the endpoints; and a sterol at a concentration of between 0.25 μmol/kg and 25 μmol/kg, inclusive of the endpoints.

In some aspects, the media can comprise one or more of octanoic acid at a concentration of about 64 μmol/kg, palmitic acid at a concentration of about 7 μmol/kg, linoleic acid at a concentration of about 7.5 μmol/kg, oleic acid at a concentration of about 7.5 μmol/kg and a sterol at a concentration of about 2.5 μmol/kg.

The present disclosure provides compositions comprising any modified T-cell produced by a method dislosed herein. The present disclosure provides compositions comprising any population of modified T-cell produced by a method dislosed herein. The present disclosure provides compositions comprising any modified T-cell expanded by a method dislosed herein. The present disclosure provides compositions comprising any population of modified T-cell expanded by a method dislosed herein.

The present disclosure provides compositions for use in the treatment of a disease or disorder disclosed herein or the use of a composition for the treatment of any disease or disorder disclosed herein. The present disclosure also provides methods of treating a disease or disorder comprising, consisting essential of, or consisting of administering to a subject in need thereof a therapeutically-effective amount of a composition disclosed herein and at least one non-naturally occurring molecule which binds to the activation component of a CSR disclosed herein. The compositions can comprise, consist essential of or consist of any of the modified cells or populations of modified cells disclosed herein. Preferably, any of the modified T-cells or CAR T-cells disclosed herein. Any non-naturally occurring molecule capable of binding to the activation component of the CSR of the present disclosure and selectively transducing a signal upon binding can be administered. Preferably, the non-naturally occurring molecule is an non-naturally CSR agonist/activating agent for the activation component. The non-naturally occurring agonist/activating agent that can bind a CSR activation component can be any non-naturally occurring antibody or antibody fragment. The non-naturally occurring antibody or antibody fragment can be a non-naturally occurring anti-CD3 antibody or fragment thereof, an anti-CD2 antibody or fragment thereof, an anti-CD28 antibody or fragment thereof, or any combination thereof. In some aspects, the non-naturally occurring agonist/activating agent that can bind a CSR activation component can be one or more of an anti-human CD3 monospecific tetrameric antibody complex, an anti-human CD2 monospecific tetrameric antibody complex, an anti-human CD28 monospecific tetrameric antibody complex, or a combination thereof. In some aspects, the non-naturally occurring agonist/activating agent that can bind an activation component can be selected from the group consisting of anti-CD2 monoclonal antibody, BTI-322 (Przepiorka et al., Blood 92(11):4066-4071, 1998) and humanized anti-CD2 monoclonal antibody clone AFC-TAB-104 (Siplizumab)(Bissonnette et al. Arch. Dermatol. Res. 301(6):429-442, 2009). In some aspects, administration of non-naturally occurring molecule capable of binding to the activation component of the CSR stimulates cell division of the modified cells in vivo. Thus, the present disclosure provides a method of stimulating cell division of a modified cell of the present disclosure in vivo by administering a non-naturally CSR agonist/activating agent for the activation component to a subject harboring the modified cell of the present disclosure.

In some aspects, the disease or disorder is a cell proliferation disease or disorder. In some aspects, the cell proliferation disease or disorder is cancer. The cancer can be a solid tumor cancer or a hematologic cancer. In some aspects, the solid tumor is prostate cancer or breast cancer. In preferred aspects, the prostate cancer is castrate-resistant prostate cancer. In some aspects, the hematologic cancer is multiple myeloma.

The modified cells or population of modified cells comprised within the disclosed compositions can be cultured in vitro or ex vivo prior to administration to a subject in need thereof. The modified cells can be allogenic modified cells or autologous modified cells. In some aspects, the cells are allogeneic modified T-cells or autologous modified T-cells. In some aspects, the cells are allogeneic modified CAR T-cells or autologous modified CAR T-cells. In some aspects, the cells are allogeneic modified CAR T-cells comprising a CSR of the present disclosure or autologous modified CAR T-cells comprising a CSR of the present disclosure.

The modified cell compositions or the compositions comprising populations of modified cells can be administered to the patient by any means known in the art. In some aspects, the composition is administered by systemic administration. In some aspects, the composition is administered by intravenous administration. The intravenous administration can be in an intravenous injection or an intravenous infusion. In some aspects, the composition is administered by local administration. In some aspects, the composition is administered by an intraspinal, intracerebroventricular, intraocular or intraosseous injection or infusion.

The therapeutically effective amount can be a single dose or multiple doses of modified cell compositions or the compositions comprising populations of modified cells. In some aspects, the therapeutically effective dose is a single dose and wherein the allogeneic cells of the composition engraft and/or persist for a sufficient time to treat the disease or disorder. In some aspects, the single dose is one of at least 2, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100 or any number of doses in between that are manufactured simultaneously

In some aspects, the uses and methods for the treatment of a disease or disorder further provide that subjects do not develop graft v host (GvH) disease, host v graft (HvG) disease, or a combination thereof, following administration of modified cell compositions disclosed herein or the compositions comprising populations of modified cells disclosed herein.

Allogeneic cells of the disclosure are engineered to prevent adverse reactions to engraftment following administration to a subject. Allogeneic cells may be any type of cell.

In some embodiments of the composition and methods of the disclosure, allogeneic cells are stem cells. In some embodiments, allogeneic cells are derived from stem cells. Exemplary stem cells include, but are not limited to, embryonic stem cells, adult stem cells, induced pluripotent stem cells (iPSCs), multipotent stem cells, pluripotent stem cells, and hematopoetic stem cells (HSCs).

In some embodiments of the composition and methods of the disclosure, allogeneic cells are differentiated somatic cells.

In some embodiments of the composition and methods of the disclosure, allogeneic cells are immune cells. In some embodiments, allogeneic cells are T lymphocytes (T cells). In some embodiments, allogeneic cells are T cells that do not express one or more components of a naturally-occurring T-cell Receptor (TCR). In some embodiments, allogeneic cells are T cells that express a non-naturally occurring antigen receptor. Alternatively, or in addition, in some embodiments, allogeneic cells are T cells that express a non-naturally occurring Chimeric Stimulatory Receptor (CSR). In some embodiments, the non-naturally occurring CSR comprises or consists of a switch receptor. In some embodiments, the switch receptor comprises an extracellular domain, a transmembrane domain, and an intracellular domain. In some embodiments, the extracellular domain of the switch receptor binds to a TCR co-stimulatory molecule and transduces a signal to the intracellular space of the allogeneic cell that recapitulates TCR signaling or TCR co-stimulatory signaling.

Chimeric Stimulatory Receptors (CSRs)

Adoptive cell compositions that are “universally” safe for administration to any patient requires a significant reduction or elimination of alloreactivity.

Towards this end, allogeneic cells of the disclosure are modified to interrupt expression or function of a T-cell Receptor (TCR) and/or a class of Major Histocompatibility Complex (MHC). The TCR mediates graft vs host (GvH) reactions whereas the MHC mediates host vs graft (HvG) reactions. In preferred embodiments, any expression and/or function of the TCR is eliminated in allogeneic cells of the disclosure to prevent T-cell mediated GvH that could cause death to the subject. Thus, in particularly preferred embodiments, the disclosure provides a pure TCR-negative allogeneic T-cell composition (e.g. each cell of the composition expresses at a level so low as to either be undetectable or non-existent).

In preferred embodiments, expression and/or function of MHC class I (MHC-I, specifically, HLA-A, HLA-B, and HLA-C) is reduced or eliminated in allogeneic cells of the disclosure to prevent HvG and, consequently, to improve engraftment of allogeneic cells of the disclosure in a subject. Improved engraftment of the allogeneic cells of the disclosure results in longer persistence of the cells, and, therefore, a larger therapeutic window for the subject. Specifically, in the allogeneic cells of the disclosure, expression and/or function of a structural element of MHC-I, Beta-2-Microglobulin (B2M), is reduced or eliminated in allogeneic cells of the disclosure.

The above strategies for generating an allogeneic cell of the disclosure induce further challenges. T Cell Receptor (TCR) knockout (KO) in T cells results in loss of expression of CD3-zeta (CD3z or CD3ζ), which is part of the TCR complex. The loss of CD3ζ in TCR-KO T-cells dramatically reduces the ability of optimally activating and expanding these cells using standard stimulation/activation reagents, including, but not limited to, agonist anti-CD3 mAb. When the expression or function of any one component of the TCR complex is interrupted, all components of the complex are lost, including TCR-alpha (TCRa), TCR-beta (TCRβ), CD3-gamma (CD3γ), CD3-epsilon (CD3ε), CD3-delta (CD3δ), and CD3-zeta (CD3ζ). Both CD3ε and CD3ζ are required for T cell activation and expansion. Agonist anti-CD3 mAbs typically recognize CD3ε and possibly another protein within the complex which, in turn, signals to CD3ζ. CD3ζ provides the primary stimulus for T cell activation (along with a secondary co-stimulatory signal) for optimal activation and expansion. Under normal conditions, full T-cell activation depends on the engagement of the TCR in conjunction with a second signal mediated by one or more co-stimulatory receptors (e.g. CD28, CD2, 4-1BBL, etc. . . . ) that boost the immune response. However, when the TCR is not present, T cell expansion is severely reduced when stimulated using standard activation/stimulation reagents, including agonist anti-CD3 mAb. In fact, T cell expansion is reduced to only 20-40% of the normal level of expansion when stimulated using standard activation/stimulation reagents, including agonist anti-CD3 mAb.

The disclosure provides a Chimeric Stimulatory Receptor (CSR) to deliver CD3z primary stimulation to allogeneic T cells in the absence of an endogenous TCR (and, consequently, an endogenous CD3ζ) when stimulated using standard activation/stimulation reagents, including agonist anti-CD3 mAb.

In the absence of an endogenous TCR, Chimeric Stimulatory Receptors (CSRs) of the disclosure provide a CD3ζ stimulus to enhance activation and expansion of allogeneic T cells. In other words, in the absence of an endogenous TCR, Chimeric Stimulatory Receptors (CSRs) of the disclosure rescue the allogeneic cell from an activation-based disadvantage when compared to non-allogeneic T-cells that express an endogenous TCR. In some embodiments, CSRs of the disclosure comprise an agonist mAb epitope extracellularly and a CD3ζ stimulatory domain intracellularly and, functionally, convert an anti-CD28 or anti-CD2 binding event on the surface into a CD3z signaling event in an allogeneic T cell modified to express the CSR. In some embodiments, a CSR comprises a wild type CD28 or CD2 protein and a CD3z intracellular stimulation domain, to produce CD28z CSR and CD2z CSR, respectively. In preferred embodiments, CD28z CSR and/or CD2z CSR further express a non-naturally occurring antigen receptor and/or a therapeutic protein. In preferred embodiments, the non-naturally occurring antigen receptor comprises a Chimeric Antigen Receptor.

The data provided herein demonstrate that modified allogeneic T cells of the disclosure comprising/expressing a CSR of the disclosure improve or rescue, the expansion of allogeneic T cells that no longer express endogenous TCR when compared to those cells that do not comprise/express a CSR of the disclosure.

A wildtype/natural human CD28 protein (NCBI: CD28_HUMAN; UniProt/Swiss-Prot: P10747.1) comprises or consists of the amino acid sequence of:

(SEQ ID NO: 17096)

MLRLLLALNLFPSIQVTGNKILVKQSPMLVAYDNAVNLSCKYSYNLFSRE

FRASLHKGLDSAVEVCVVYGNYSQQLQVYSKTGFNCDGKLGNESVTFYLQ

NLYVNQTDIYFCKIEVMYPPPYLDNEKSNGTIIHVKGKHLCPSPLFPGPS

KPFWVLVVVGGVLACYSLLVTVAFIIFWVRSKRSRLLHSDYMNMTPRRPG

PTRKHYQPYAPPRDFAAYRS

A nucleotide sequence encoding wildtype/natural CD28 protein (NCBI: CCDS2361.1) comprises or consists of the nucleotide sequence of:

(SEQ ID NO: 17097)

ATGCTCAGGCTGCTCTTGGCTCTCAACTTATTCCCTTCAATTCAAGTAAC

AGGAAACAAGATTTTGGTGAAGCAGTCGCCCATGCTTGTAGCGTACGACA

ATGCGGTCAACCTTAGCTGCAAGTATTCCTACAATCTCTTCTCAAGGGAG

TTCCGGGCATCCCTTCACAAAGGACTGGATAGTGCTGTGGAAGTCTGTGT

TGTATATGGGAATTACTCCCAGCAGCTTCAGGTTTACTCAAAACGGGGTT

CAACTGTGATGGGAAATTGGGCAATGAATCAGTGACATTCTACCTCCAGA

ATTTGTATGTTAACCAAACAGATATTTACTTCTGCAAAATTGAAGTTATG

TATCCTCCTCCTTACCTAGACAATGAGAAGAGCAATGGAACCATTATCCA

TGTGAAAGGGAAACACCTTTGTCCAAGTCCCCTATTTCCCGGACCTTCTA

AGCCCTTTTGGGTGCTGGTGGTGGTTGGTGGAGTCCTGGCTTGCTATAGC

TTGCTAGTAACAGTGGCCTTTATTATTTTCTGGGTGAGGAGTAAGAGGAG

CAGGCTCCTGCACAGTGACTACATGAACATGACTCCCCGCCGCCCCGGGC

CCACCCGCAAGCATTACCAGCCCTATGCCCCACCACGCGACTTCGCAGCC

TATCGCTCCTGA

An exemplary CSR CD28z protein of the disclosure comprises or consists of the amino acid sequence of (CD28 Signal peptide, CD28 Extracellular Domain, CD28 Transmembrane domain, CD28 Cytoplasmic Domain, CD3z Intracellular Domain):

(SEQ ID NO: 17060)

MLRLLLALNLFPSIQVTG
NKILVKQSPMLVAYDNAVNLSCKYSYNLFSRE

FRASLHKGLDSAVEVCVVYGNYSQQLQVYSKTGFNCDGKLGNESVTFYLQ

NLYVNQTDIYFCKIEVMYPPPYLDNEKSNGTIIHVKGKHLCPSPLFPGPS

KP
FWVLVVVGGVLACYSLLVTVAFIIFWV
custom-character

RVKFSRSADAPAYKQGQNQLYN

ELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYS

EIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR

CD28 Signal Peptide:

(SEQ ID NO: 17098)

MLRLLLALNLFPSIQVTG

CD28 Extracellular Domain:

(SEQ ID NO: 17099)

NKILVKQSPMLVAYDNAVNLSCKYSYNLFSREFRASLHKGLDSAVEVCVV

YGNYSQQLQVYSKTGFNCDGKLGNESVTFYLQNLYVNQTDIYFCKIEVMY

PPPYLDNEKSNGTIIHVKGKHLCPSPLFPGPSKP

CD28 Transmembrane Domain:

(SEQ ID NO: 17100)

FWVLVVVGGVLACYSLLVTVAFIIFWV

CD28 Cytoplasmic Domain:

(SEQ ID NO: 17101)

RSKRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRS

CD3z Intracellular Domain:

(SEQ ID NO: 17102)

RVKFSRSADAPAYKQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPR

RKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDT

YDALHMQALPPR

An exemplary nucleotide sequence encoding a CSR CD28z protein of the disclosure comprises or consists of the nucleotide sequence of (CD28 Signal peptide, CD28 Extracellular Domain, CD28 Transmembrane domain, CD28 Cytoplasmic Domain, CD3z Intracellular Domain):

(SEQ ID NO: 17061)

ATGCTGAGACTGCTGCTGGCCCTGAATCTGTTCCCCAGCATCCAAGTGAC

CGGC
AACAAGATCCTGGTCAAGCAGAGCCCTATGCTGGTGGCCTACGACA

ACGCCGTGAACCTGAGCTGCAAGTACAGCTACAACCTGTTCAGCAGAGAG

TTCCGGGCCAGCCTGCACAAAGGACTGGATTCTGCTGTGGAAGTGTGCGT

GGTGTACGGCAACTACAGCCAGCAGCTGCAGGTCTACAGCAAGACCGGCT

TCAACTGCGACGGCAAGCTGGGCAATGAGAGCGTGACCTTCTACCTGCAA

ACCTGTACGTGAACCAGACCGACATCTATTTCTGCAAGATCGAAGTGATG

TACCCGCCTCCTTACCTGGACAACGAGAAGTCCAACGGCACCATCATCCA

CGTGAAGGGCAAGCACCTGTGTCCTTCTGGACTGTTGGCCCGACCTAGCA

AGCCT
TTCTGGGTGCTCGTTGTTGTTGGCGGCGTGCTGGCCTGTTATAGC

CTGCTGGTTACAGTGGCCTTCATCATCTTTTGGGTC

custom-character

AGAGTGAAGTTCTCCAGATCCGCCGATGCTCCCGCCTATAAGCAGGGCCA

GAACCAGCTGTACAACGAGCTGAACCTGGGGAGAAGAGAAGAGTACGATG

TGCTGGACAAGCGGAGAGGCAGAGATCCTGAGATGGGCGGCAAGCCCAGA

CGGAAGAATCCTCAAGAGGGCCTGTACAATGAACTGCAGAAAGACAAGAT

GGCCGAGGCCTACAGCGAGATCGGAATGAAGGGCGAGCGCAGAAGAGGCA

AGGGACACGATGGACTGTACCAGGCCTGAGCACCGCCACCAAGGATACCT

ATGATGCCCTGCACATGCAGGCCCTGCCTCCAAGA

CD28 Signal Peptide:

(SEQ ID NO: 17103)

ATGCTGAGACTGCTGCTGGCCCTGAATCTGTTCCCCAGCATCCAAGTGAC

CGGC

CD28 Extracellular Domain:

(SEQ ID NO: 17104)

AACAAGATCCTGGTCAAGCAGAGCCCTATGCTGGTGGCCTACGACAACGC

CGTGAACCTGAGCTGCAAGTACAGCTACAACCTGTTCAGCAGAGAGTTCC

GGGCCAGCCTGCACAAAGGACTGGATTCTGCTGTGGAAGTGTGCGTGGTG

TACGGCAACTACAGCCAGCAGCTGCAGGTCTACAGCAAGACCGGCTTCAA

CTGCGACGGCAAGCTGGGCAATGAGAGCGTGACCTTCTACCTGCAAAACC

TGTACGTGAACCAGACCGACATCTATTTCTGCAAGATCGAAGTGATGTAC

CCGCCTCCTTACCTGGACAACGAGAAGTCCAACGGCACCATCATCCACGT

GAAGGGCAAGCACCTGTGTCCTTCTCCACTGTTCCCCGGACCTAGCAAGC

CT

CD28 Transmembrane Domain:

(SEQ ID NO: 17105)

TTCTGGGTGCTCGTTCTTGTTGGCGGCCTGCTGGCCTGTTATAGCCTCCT

GCTTACAGTGGCCTTCATCATCTTTTGGGTC

CD28 Cytoplasmic Domain:

(SEQ ID NO: 17106)

CGAAGCAAGCGGAGCCGGCTGCTGCACAGCGACTACATGAACATGACCCC

TAGACGGCCCGGACCAACCAGAAAGCACTACCAGCCTTACGCTCCTCCTA

GAGACTTCGCCGCCTACCGGTCC

CD3z Intracellular Domain:

(SEQ ID NO: 17107)

AGAGTGAAGTTCTCCAGATCCGCCGATGCTCCCGCCTATAAGCAGGGCCA

GAACCAGCTGTACAACGAGCTGAACCTGGGGAGAAGAGAAGAGTACGATG

TGCTGGACAAGCGGAGAGGCAGAGATCCTGAGATGGGCGGCAAGCCCAGA

CGGAAGAATCCTCAAGAGGGCCTGTACAATGAACTGCAGAAAGACAAGAT

GGCCGAGGCCTACAGCGAGATCGGAATGAAGGGCGAGCGCAGAAGAGGCA

AGGGACACGATGGACTGTACCAGGGCCTGAGCACCGCCACCAAGGATACC

TATGATGCCCTGCACATGCAGGCCCTGCCTCCAAGA

A wildtype/natural human CD2 protein (NCBI: CD2_HUMAN; UniProt/Swiss-Prot: P06729.2) comprises or consists of the amino acid sequence of:

(SEQ ID NO: 17108)

MSFPCKFVASFLLIFNVSSKGAVSKEITNALETWGALGQDINLDIPSFQM

SDDIDDIKWEKTSDKKKIAQFRKEKETFKEKDTYKTFKNGTLKIKHLKTD

DQDIYKVSIYDTKGKNVLEKIFDLKIQERVSKPKISWTCINTTLTCEVMN

GTDPELNLYQDGKHLKLSQRVITHKWTTSLSAKFKCTAGNKVSKESSVEP

VSCPEKGLDIYLIIGICGGGSLLMVFVALLVFYITKRKKQRSRRNDEELE

TRAHRVATEERGRKPHQIPASTPQNPATSQHPPPPPPGHRSQAPSHRPPP

PGHRVQHQPQKRPPAPSGTQVHQQKGPPLPRPRVQPKPPHGAAENSLSPS

SN

A nucleotide sequence encoding wildtype/natural CD2 protein (NCBI: CCDS889.1) comprises or consists of the nucleotide sequence of:

(SEQ ID NO: 17109)

ATGAGCTTTCCATGTAAATTTGTAGCCAGCTTCCTTCTGATTTTCAATGT

TTCTTCCAAAGGTGCAGTCTCCAAAGAGATTACGAATGCCTTGGAAACCT

GGGGTGCCTTGGGTCAGGACATCAACTTGGACATTCCTAGTTTTCAAATG

AGTGATGATATTGACGATATAAAATGGGAAAAAACTTCAGACAAGAAAAA

GATTGCACAATTCAGAAAAGAGAAAGAGACTTTCAAGGAAAAAGATACAT

ATAAGCTATTTAAAAATGGAACTCTGAAAATTAAGCATCTGAAGACCGAT

GATCAGGATATCTACAAGGTATCAATATATGATACAAAAGGAAAAAATGT

GTTGGAAAAAATATTTGATTTGAAGATTCAAGAGAGGGTCTCAAAACCAA

AGATCTCCTGGACTTGTATCAACACAACCCTGACCTGTGAGGTAATGAAT

GGAACTGACCCCGAATTAAACCTGTATCAAGATGGGAAACATCTAAAACT

TTCTCAGAGGGTCATCACACACAAGTGGACCACCAGCCTGAGTGCAAAAT

TCAAGTGCACAGCAGGGAACAAAGTCAGCAAGGAATCCAGTGTCGAGCCT

GTCAGCTGTCCAGAGAAAGGTCTGGACATCTATCTCATCATTGGCATATG

TGGAGGAGGCAGCCTCTTGATGGTCTTTGTGGCACTGCTCGTTTTCTATA

TCACCAAAAGGAAAAAACAGAGGAGTCGGAGAAATGATGAGGAGCTGGAG

ACAAGAGCCCACAGAGTAGCTACTGAAGAAAGGGGCCGGAAGCCCCACCA

AATTCCAGCTTCAACCCCTCAGAATCCAGCAACTTCCCAACATCCTCCTC

CACCACCTGGTCATCGTTCCCAGGCACCTAGTCATCGTCCCCCGCCTCCT

GGACACCGTGTTCAGCACCAGCCTCAGAAGAGGCCTCCTGCTCCGTCGGG

CACACAAGTTCACCAGCAGAAAGGCCCGCCCCTCCCCAGACCTCGAGTTC

AGCCAAAACCTCCCCATGGGGCAGCAGAAAACTCATTGTCCCCTTCCTCT

AATTAA

An exemplary CSR CD2z protein of the disclosure comprises or consists of the amino acid sequence of (CD2 Signal peptide, CD2 Extracellular Domain, CD2 Transmembrane domain, CD2 Cytoplasmic Domain, CD3z Intracellular Domain):

(SEQ ID NO: 17062)

MSFPCKFVASFLLIFNVSSKGAVS
KEITNALETWGALGQDINLDIPSFQM

SDDIDDIKWEKTSDKKKIAQFRKEKETFKEKDTYKLFKNGTLKIKHLKTD

DQDIYKVSIYDTKGKNVLEKIFDLKIQERVSKPKISWTCINTTLTCEVMN

GTDPELNLYQDGKHLKLSQRVITHKWTTSLSAKFKCTAGNKVSKESSVEP

VSCPEKGLD
IYLIIGICGGGSLLMVFVALLVFYIT

custom-character

RVKFSRSADAPAYKQGQN

QLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMA

EAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR

CD2 Signal Peptide:

(SEQ ID NO: 17110)

MSFPCKEVASFLLIFNVSSKGAVS

CD2 Extracellular Domain:

(SEQ ID NO: 17111)

KEITNALETWGALGQDINLDIPSFQMSDDIDDIKTNEKTSDKIKKIAQFR

KEKETFKEKDTYKLFKNGTLKIKHLKTDDQDIYKVSIYDTKGKNVLEKIF

DLKIQERVSKPKISWTCINTTLTCEVMNGTDPELNLYQDGKHLKLSQRVI

THKWTTSLSAKFKCTAGNKVSKESSVEPVSCPEKGLD

CD2 Transmembrane Domain:

(SEQ ID NO: 17112)

IYLIIGICGGGSLLMVFVALLVFYIT

CD2 Cytoplasmic Domain:

(SEQ ID NO: 17113)

KRKKQRSRRNDEELETRAERVATEERGRKPHQIPASTPQNPATSQHPPPP

PGHRSQAPSHRPPPPGHRVQHQPQKRPPAPSGTQVHQQKGPPLPRPRVQP

KPPHGAAENSLSPSSN

CD3z Intracellular Domain:

(SEQ ID NO: 17102)

RVKFSRSADAPAYKQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPR

RKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDT

YDALHMQALPPR

The present disclosure provides a non-naturally occurring CSR CD2 protein comprising, consisting essential of, or consisting of an amino acid sequence at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical to SEQ ID NO:17062. The present disclosure provides a CD2 signal peptide comprising, consisting essential of, or consisting of an amino acid sequence at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical to SEQ ID NO:17110. The present disclosure provides a CD2 extracellular domain comprising, consisting essential of, or consisting of an amino acid sequence at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical to SEQ ID NO:17111. The present disclosure provides a CD2 transmembrande domain comprising, consisting essential of, or consisting of an amino acid sequence at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical to SEQ ID NO:17112. The present disclosure provides a CD2 cytoplasmic domain comprising, consisting essential of, or consisting of an amino acid sequence at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical to SEQ ID NO:17113. The present disclosure provides a CD3z intracellular domain comprising, consisting essential of, or consisting of an amino acid sequence at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical to SEQ ID NO:17102.

An exemplary nucleotide sequence encoding a CSR CD2z protein of the disclosure comprises or consists of the amino acid sequence of (CD2 Signal peptide, CD2 Extracellular Domain, CD2 Transmembrane domain CD2 Cytoplasmic Domain, CD3z Intracellular Domain):

(SEQ ID NO: 17063)

ATGAGCTTCCCTTGCAAGTTCGTGGCCAGCTTCCTGCTGATCTTCAACGT

GTCCTCTAAGGGCGCCGTGTCC
AAAGAGATCACAAACGCCCTGGAAACCT

GGGGAGCCCTCGGCCAGGATATTAACCTGGACATCCCCAGCTTCCAGATG

AGCGACGACATCGATGACATCAAGTGGGAGAAAACCAGCGACAAGAAGAA

GATCGCCCAGTTCCGGAAAGAGAAAGAGACATTCAAAGAGAAGGACACCT

ACAAGCTGTTCAAGAACGGCACCCTGAAGATCAAGCACCTGAAAACCGAC

GACCAGGACATCTATAAGGTGTCCATCTACGACACCAAGGGCAAGAACGT

GCTGGAAAAGATCTTCGACCTCAAGATCCAAGAGCGGGTGTCCAAGCCTA

AGATCAGCTGGACCTGCATCAACACCACACTGACCTGCGAAGTGATGAAC

GGCACAGACCCCGAGCTGAACCTGTACCAGGATGGCAAACACCTGAAGCT

GAGCCAGCGCGTGATCACCCACAAGTGGACAACAAGCCTGAGCGCCAAGT

TCAAGTGCACCGCCGGAAACAAAGTGTCTAAAGAGTCCAGCGTCGAGCCC

GTGTCTTGCCCTGAAAAAGGACTGGAC
ATCTACCTGATCATCGGCATCTG

TGGCGGCGGAAGCCTGCTGATGGTGTTTGTGGCTCTGCTGGTGTTCTACA

TCACC
custom-character

AGAGTGAAGTTCAGCCGCAGCGCCGATGCTCCTGCCTATAAGCAGGGACA

GAACCAGCTGTACAACGAGCTGAATCTGGGGCGCAGAGAAGAGTACGATG

TGCTGGACAAGCGGAGAGGCAGAGATCCTGAGATGGGCGGCAAGCCCAGA

CGGAAGAATCCTCAAGAGGGCCTGTATAATGAGCTGCAGAAAGACAAGAT

GGCCGAGGCCTACAGCGAGATCGGAATGAAGGGCGAGCGCAGAAGAGGGL

AGGGACACGATGGACTGTATCAGGGCCTGAGCACCGCCACCAAGGATACC

TATGATGCCCTGCACATGCAGGCCCTGCCTAAGA

CD2 Signal Peptide:

(SEQ ID NO: 17114)

ATGAGCTTCCCTTGCAAGTTCGTGGCCAGCTTCCTGCTGATCTTCAACGT

CTCCTCTAAGGGCGCCGTGTCC

CD2 Extracellular Domain:

(SEQ ID NO: 17115)

AAAGAGATCACAAACGCCCTGGAAACCTGGGGAGCCCTCGGCCAGGATAT

TAACCTGGACATCCCCAGCTTCCAGATGAGCGACGACATCGATGACATCA

AGTGGGAGAAAACCAGCGACAAGAAGAAGATCGCCCAGTTCCGGAAAGAG

AAAGAGACATTCAAAGAGAAGGACACCTACAAGCTGTTCAAGAACGGCAC

CCTGAACATCAAGCACCTGAAAACCGACGACCAGGACATCTATAAGGTGT

CCATCTACGACACCAAGGGCAAGAACGTGCTGGAAAAGATCTTCGACCTC

AAGATCCAAGAGCGGGTGTCCAAGCCTAAGATCAGCTGGACCTGCATCAA

CACCACACTGACCTGCGAAGTGATGAACGGCACAGACCCCGAGCTGAACC

TCTACCAGGATGGCAAACACCTGAAGCTGAGCCAGCGCGTGATCACCCAC

AAGTCGACAACAAGCCTGAGCGCCAAGTTCAAGTGCACCGCCGGAAACAA

AGTGTCTAAAGAGTCCAGCGTCGAGCCCGTGTCTTGCCCTGAAAAAGGAC

TGGAC

CD2 Transmembrane Domain:

(SEQ ID NO: 17116)

ATCTACCTGATCATCGGCATCTGTGGCGGCGGAAGCCTGCTGATGGTGTT

TGTGGCTCTGCTGGTGTTCTACATCACC

CD2 Cytoplasmic Domain:

(SEQ ID NO: 17117)

AAGCGGAAGAAGCAGCGGAGCAGACGGAACGACGAGGAACTGGAAACACG

GGCCCATAGAGTGGCCACCGAGaAAAGAGGaAaAAAGCCCCACCAGATTC

CAGCCAGCACACCCCAGAATCCTGCCACCTCTCAACACCCTCCACCTCCA

CCTGGACACAGATCTCAGGCCCCATCTCACAGACCTCCACCACCTGGTCA

TCGGGTGCAGCACCAGCCTCAAAALGACCTCCTGCTCCTAGCGGCACACA

GGTGCACCAGCAAAAAGGACCTCCACTGCCTCGGCCTAGACTGCAGCCTA

AACCTCCTCATGGCGCCGCTGACAACAGCCTGTCTCCAAGCACCAAC

CD3z Intracellular Domain:

(SEQ ID NO: 17107)

AGAGTGAAGTTCAGCCGCAGCGCCGATGCTCCTGCCTATAAGCAGGGACA

GAACCAGCTGTACAACGAGCTGAATCTGGGGCGCAGAGAAGAGTACGATG

TGCTGGACAAGCGGAGAGGCAGAGATCCTGAGATGGGCGGCAAGCCCAGA

CGGAAGAATCCTCAAGAGGGCCTGTATAATGAGCTGCAGAAAGACAAGAT

GGCCGAGGCCTACAGCGAGATCGGAATGAAGGGCGAGCGCAGAAGAGGCA

AGGGACACGATGGACTGTATCAGGGCCTGAGCACCGCCACCAAGGATACC

TATGATGCCCTGCACATGCAGGCCCTGCCTCCAAGA

An exemplary mutant CSR CD2z-D111H protein of the disclosure comprises or consists of the amino acid sequence of (CD2 Signal peptide, CD2 Extracellular domain with D111H mutation within the CD2 Extracellular domain, CD2 Transmembrane domain, CD2 Cytoplasmic domain, CD3z Intracellular domain):

(SEQ ID NO: 17118)

MSFPCKFVASFLLIFNVSSKGAVS
KEITNALETWGALGQDINLDIPSFQM

SDDIDDIKWEKTSDKKKIAQFRKEKETFKEKDTYKLFKNGTLMIKHLKTD

DQDIYKVSIY

H

TKGKNVLEKIFDLKIQERVSKPKISWTCINTTLTCEVMN

GTDPELNLYQDGKHLKLSQRVITHKWTTSLSAKFKCTAGNKYSKESSVEP

VSCPEKGLD
IYLIIGICGGGSLLMVEVALLVFYIT

custom-character

RVKFSRADAPAYKQGQNQ

LYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAE

AYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR

CD2 Signal Peptide:

(SEQ ID NO: 17110)

MSFPCKFVASFLLIFNVSSKGAVS

CD2 Extracellular domain with D111H mutation within the CD2 Extracellular domain:

(SEQ ID NO: 17119)

KEITNALETWGALGQDINLDIPSFQMSDDIDDIKWEKTSDKKKIAQFRKE

KETFKEKDTYKLFKNGTLKIKHLKTDDQDIYKVSIYHTKGKNVLEKIFDL

KIQERVSKPKISWTCINTTLTCEVMNGTDPELNLYQDGKHLKLSQRVITH

KWTTSLSAKFKCTAGNKVSKESSVEPVSCPEKGL

CD2 Transmembrane Domain:

(SEQ ID NO: 17112)

IYLIIGICGGGSLLMVFVALLVFYIT

CD2 Cytoplasmic Domain:

(SEQ ID NO: 17113)

KRKKQRSRRNDEELETRAHRVATEERGRKPHQIPASTPQNPATSQHPPPP

PGHRSQAPSHRPPPPGHRVQHQPQKRPPAPSGTQVHQQKGPPLPRPRVQP

KPPHGAAENSLSPSSN

CD3z Intracellular Domain:

(SEQ ID NO: 17102)

RVKFSRSADAPAYKQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPR

RKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDT

YDALHMQALPPR

An exemplary nucleotide sequence encoding a mutant CSR CD2z-D111H protein of the disclosure comprises or consists of the amino acid sequence of (CD2 Signal peptide, CD2 Extracellular domain with D111H mutation within the CD2 Extracellular domain, CD2 Transmembrane domain, CD2 Cytoplasmic domain, CD3z Intracellular domain):

(SEQ ID NO: 17120)

ATGAGCTTCCCTTGCAAGTTCGTGGCCAGCTTCCTGCTGATCTTCAACGT

GTCCTCTAAGGGCGCCGTGTCC
AAAGAGATCACAAACGCCCTGGAAACCT

GGGGAGCCCTCGGCCAGGATATTAACCTGGACATCCCCAGCTTCCAGATG

AGCGACGACATCGATGACATCAAGTGGGAGAAAACCAGCGACAAGAAGAA

GATCGCCCAGTTCCGGAAAGAGAAAGAGACATTCAAAGAGAAGGACACCT

ACAAGCTGTTCAAGAACGGCACCCTGAAGATCAAGCACCTGAAAACCGAC

GACCAGGACATCTATAAGGTGTCCATCTAC

CAC

ACCAAGGGCAAGAACGT

GCTGGAAAAGATCTTCGACCTCAAGATCCAAGAGCGGGTGTCCAAGCCTA

AGATCAGCTGGACCTGCATCAACACCACACTGACCTGCAAGTGATGAACG

GCACAGACCCCGAGCTGAACCTGTACCAGGATGGCAAACACCTGAAGCTG

AGCCAGCGCGTGATCACCCACAAGTGGACAACAAGCCTGAGCGCCAAGTT

CAAGTGCACCGCCGGAAACAAAGTGTCTAAAGAGTCCAGCGTCGAGCCCG

TGTCTTGCCCTGAAAAAGGACTGGAC
ATCTACCTCATCATCGCCATCTGT

CGCGCCGGAAGCCTGCTGATCGTGTTTGTGGCTCTGCTGGTGTTCTACAT

CACC
custom-character

AGAGTGAAGTTCAGCCGCAGCGCCGATGCTCCTCGCTATAAGCAGGGACA

GAACCAGCTGTACAACGAGCTGAATCTGGGGCGCAGAGAAGAGTACGATG

TGCTGGACAAGCGGAGAGGCAGAGATCCTGAGATGGGCGGCAAGCCCAGA

CGGAAGAATCCTCAAGAGGGCCTGTATAATGAGCTGCAGAAAGACAAGAT

GGCCGAGGCCTACAGCGAGATCGGAATGAAGGGCGAGCGCAGAAGAGGCA

AGGGACACGATGGACTGTATCAGGGCCTGAGCACCGCCACCAAGGATACC

TATGATCCCCTGCACATGCAGGCCCTGCCTCCAAGA

CD2 Signal Peptide:

(SEQ ID NO: 17114)

ATGAGCTTCCCTTGCAAGTTCGTGGCCAGCTTCCTGCTGATCTTCAACGT

GTCCTCTAAGGGCGCCGTGTCC

CD2 Extracellular Domain with D111H Mutation within, the CD12 Extracellular Domain:

(SEQ ID NO: 17121)

AAAGAGATCACAAACGCCCTGGAAACCTGGGGAGCCCTCGGCCAGGATAT

TAACCTGGACATCCCCAGCTTCCAGATGAGCGACGACATCGATGACATCA

AGTGGGAGAAAACCAGCGACAAGAAGAAGATCGCCCAGTTCCGGAAAGAG

AAAGAGACATTCAAAGAGAAGGACACCTACAAGCTGTTCAAGAACGGCAC

CCTGAAGATCAAGCACCTGAAAACCGACGACCAGGACATCTATAAGGTGT

CCATCTACCACACCAAGGGCAAGAACGTGCTGGAAAAGATCTTCGACCTC

AAGATCCAAGAGCGGGTGTCCAAGCCTAAGATCAGCTGGACCTGCATCAA

CACCACACTGACCTGCGAAGTGATGAACGGCACAGACCCCGAGCTGAACC

TGTACCAGGATGGCAAACACCTGAAGCTGAGCCAGCGCGTGATCACCCAC

AAGTGGACAACAAGCCTGAGCGCCAAGTTCAAGTGCACCGCCGGAAACAA

AGTGTCTAAAGAGTCCAGCGTCGAGCCCGTGTCTTGCCCTGAAAAAGGAC

TGGAC

CD2 Transmembrane Domain:

(SEQ ID NO: 17116)

ATCTACCTGATCATCGGCATCTGTGGCGGCGGAAGCCTGCTGATGGTGTT

TGTGGTCTGCTGGTGTTCTACATCACC

CD2 Cytoplasmic Domain:

(SEQ ID NO: 17117)

AAGCGGAAGAAGCAGCGGAGCAGACGGAACGACGAGGAACTGGAAACACG

GGCCCATAGAGTGGCCACCGAGGAAAGAGGCAGAAAGCCCCACCAGATTC

CAGCCAGCACACCCCAGAATCCTGCCACCTCTCAACACCCTCCACCTCCA

CCTGGACACAGATCTCAGGCCCCATCTCACAGACCTCCACCACCTGGTCA

TCGGGTGCAGCACCAGCCTCAGAAAAGACCTCCTGCTCCTAGCGGCACAC

AGGTGCACCAGCAAAAAGGACCTCCACTGCCTCGGCCTAGAGTGCAGCCT

AAACCTCCTCATGGCGCCGCTGAGAACAGCCTGTCTCCAAGCAGCAAC

CD3z Intracellular Domain:

Endogenous TCR Knock-Out

Gene editing compositions of the disclosure, including but not limited to, RNA-guided fusion proteins comprising dCas9-Clo051, may be used to target and decrease or eliminate expression of an endogenous T-cell receptor of an allogeneic cell of the disclosure. In preferred embodiments, the gene editing compositions of the disclosure target and delete a gene, a portion of a gene, or a regulatory element of a gene (such as a promoter) encoding an endogenous T-cell receptor of an allogeneic cell of the disclosure.

Nonlimiting examples of primers (including a T7 promoter, genome target sequence, and gRNA scaffold) for the generation of guide RNA (gRNA) templates for targeting and deleting TCR-alpha (TCR-α) are provided in Table 10.

TABLE 10

Target sequences underlined

Name
Sequence
SEQ ID NO:

TCRa-
TAATACGACTCACTATA GCTGGTACACGGCAGGGTCA
16821

gRNA-WT
GTTTTAGAGCTAGAAATAG

1

TCRa-
TAATACGACTCACTATA GAGAATCAAAATCGGTGAAT
16822

gRNA-WT

2

TCRa-
TAATACGACTCACTATA GTGCTAGACATGAGGTCTA
16823

gRNA--WT

4

TCRa-
TAATACGACTCACTATAG GCTGGTACACGGCAGGGTCA
16824

gRNA--WT

1-2G

TCRa-
TAATACGACTCACTATA GAGAATCAAAATCGGTGAAT
16825

gRNA-WT
GTTTTAGAGCTAGAAATAG

2

TCRa-
TAATACGACTCACTATA GGATTTAGAGTCTCTCAGC
16826

gRNA-WT
GTTTTAGAGCTAGAAATAG

3

TCRa-
TAATACGACTCACTATA GTGCTAGACATGAGGTCTA
16827

gRNA-WT
GTTTTAGAGCTAGAAATAG

4

TCRa-
TAATACGACTCACTATA GACACCTTCTTCCCCAGCCC
16828

gRNA-WT
GTTTTAGAGCTAGAAATAG

5

TCRa-
TAATACGACTCACTATA g tggaataatgctgttgttga
16829

gRNA-
GTTTTAGAGCTAGAAATAG

NG1-L

TCRa-
TAATACGACTCACTATA g catcacaggaactttctaaa
16830

gRNA-
GTTTTAGAGCTAGAAATAG

NG2-L

TCRa-
TAATACGACTCACTATA gtaaaaccaagaggccacag
16831

gRNA-
GTTTTAGAGCTAGAAATAG

NG3-L

TCRa-
TAATACGACTCACTATA g acccggccactttcaggagg
16832

gRNA-
GTTTTAGAGCTAGAAATAG

NG4-L

TCRa-
TAATACGACTCACTATA gattaaacccggccactttc
16833

gRNA-
GTTTTAGAGCTAGAAATAG

NG5-L

TCRa-
TAATACGACTCACTATA g agcccaggtaagggcagctt
16834

gRNA-
GTTTTAGAGCTAGAAATAG

NG1-R

TCRa-
TAATACGACTCACTATA g agctttgaaacaggtaagac
16835

gRNA-
GTTTTAGAGCTAGAAATAG

NG2-1-R

TCRa-
TAATACGACTCACTATA gctttgaaacaggtaagaca
16836

gRNA-
GTTTTAGAGCTAGAAATAG

NG2-2-R

TCRa-
TAATACGACTCACTATA g tttcaaaacctgtcagtgat
16837

gRNA-
GTTTTAGAGCTAGAAATAG

NG3-R

TCRa-
TAATACGACTCACTATA g ctgcggctgtggtccagctg
16838

gRNA-
GTTTTAGAGCTAGAAATAG

NG4-R

TCRa-
TAATACGACTCACTATA gctgtggtccagctgaggtg
16839

gRNA-
GTTTTAGAGCTAGAAATAG

NG5-1-R

TCRa-
TAATACGACTCACTATA g ctgtggtccagctgaggtga
16840

gRNA-
GTTTTAGAGCTAGAAATAG

NG5-2-R

TCRa-
TAATACGACTCACTATA g tgtggtccagctgaggtgag
16841

gRNA-
GTTTTAGAGCTAGAAATAG

NG5-3-R

TCRa-
TAATACGACTCACTATA gtgtggtccagctgaggtgag
16842

gRNA-
GTTTTAGAGCTAGAAATAG

NG5-3-Rb

Nonlimiting examples of primers for the generation of guide RNA (gRNA) templates for targeting and deleting TCR-beta (TCR-β) are provided in Table 11.

TABLE 11

Target sequences underlined

Name
Sequence
SEQ ID NO:

TCRb-
TAATACGACTCACTATA GGCTGCTCCTTCTAGGGGCTG
16843

gRNA-WT
GTTTTAGAGCTAGAAATAG

1

TCRb-
TAATACGACTCACTATA GGCAGTATCTGGAGTCATTG
16844

gRNA-WT
GTTTTAGAGCTAAATAG

2

TCRb-
TAATACGACTCACTATA GGCCTCGGCGCTGACGATCT
16845

gRNA-WT

3

TCRb-
TAATACGACTCACTATA GGCTCTCGGAGAATGACGAG
16846

gRNA-WT

5

TCRb-
TAATACGACTCACTATA GGCCTCGGCGCTGACGATCT
16847

gRNA-WT
GTTTTAGAGCTAGAAATAG

3

TCRb-
TAATACGACTCACTATA GGAGAATGACGAGTGGACCC
16848

gRNA-WT
GTTTTAGAGCTAGAAATAG

4

TCRb-
TAATACGACTCACTATA GGCTCTCGGAGAATGACGAG
16849

gRNA-WT
GTTTTAGAGCTAGAAATAG

5

TCRb-
TAATACGACTCACTATA G CAAACACAGCGACCTCGGGT
16850

gRNA-
GTTTTAGAGCTAGAAATAG

NC1-L

TCRb-
TAATACGACTCACTATA G TGGCTCAAACACAGCGACCT
16851

gRNA-
GTTTTAGAGCTAGAAATAG

NG2-L

TCRb-
TAATACGACTCACTATA G AGGGCGGGCTGCTCCTTGAG
16852

gRNA-
GTTTTAGAGCTAGAAATAG

NG3-L

TCRb-
TAATACGACTCACTATA GTATCTGGAGTCATTGAGGG
16853

gRNA-
GTTTAGAGCTAGAAATAG

NG4-L

TCRb-
TAATACGACTCACTATA G ACTGGACTTGACAGCGGAAG
16854

gRNA-
GTTTTAGAGCTAGAAATAG

NG5-L

TCRb-
TAATACGACTCACTATA G AGAGATCTCCCACACCCAAA
16855

gRNA-
GTTTTAGAGCTAGAAATAG

NG1-R

TCRb-
TAATACGACTCACTATA G CCACACCCAAAGGCCACAC
16856

gRNA-
GTTTTAGAGCTAGAAATAG

NG2-R

TCRb-
TAATACGACTCACTATA G ACTGCCTGAGCAGCCGCCTG
16857

gRNA-
GTTTTAGAGCTAGAAATAG

NG3-R

TCRb-
TAATACGACTCACTATA G TGAGGGTCTCGGCCACCTTC
16858

gRNA-
GTTTTAGAGCTAGAAATAG

NG4-R

TCRb-
TAATACGACTCACTATA G ATGACGAGTGGACCCAGGAT
16859

gRNA-
GTTTTAGAGCTAGAAATAG

NG5-R

TCRb-
TAATACGACTCACTATA G TGGCTCAAACACAGCGACCT
16860

gRNA-
GTTTTAGAGCTAGAAATAG

NG6-L

TCRb-
TAATACGACTCACTATA G CCACACCCAAAAGGCCACAC
16861

gRNA-
GTTTTAGAGCTAGAAATAG

NG6-R

Nonlimiting examples of primers for the generation of guide RNA (gRNA) templates for targeting and deleting beta-2-microglobulin (β2M) are provided in Table 12.

TABLE 12

Target sequences underlined

Primer

No.
Name
Sequence
SEQ ID NO:

1
B2-
TAATACGACTCACTATAG AGACAGGTGACGGTCCCTGC
16862

Prom-
GTTTTAGAGCTAGAAATAG

NG1-R

2
B2-
TAATACGACTCACTATA GCAGTGCCAGGTTAGAGAGA
16863

Prom-
GTTTTAGAGCTAGAAATAG

NG1-L

3
B2-
TAATACGACTCACTATA GAAGTTGACTTACTGAAGAA
16864

Ex2-
GTTTTAGAGCTAGAAATAG

NG-R

4
B2-
TAATACGACTCACTATA G ACCCAGACACATACAATTC
16865

Ex2-
GTTTTAGAGCTAGAAATAG

NG-L

5
B2-
TAATACGACTCACTATA G TCACGTCATCCAGCAGAGAA
16866

Ex2-
GTTTTAGAGCTAGAAATAG

NG2-R

6
B2-
TAATACGACTCACTATA gatattcctcagGTACTCCA
16867

Ex2-
GTTTTAGAGCTAGAAATAG

NG2-L

7
b2MEx1
TAATACGACTCACTATA GGCCACGGAGCGAGACATCT
16868

NG-
GTTTTAGAGCTAGAAATAG

left

8
b2MEH1
TAATACGACTCACTATAG ACTCTCTCTTTCTGGCCTGG
16869

NG-
GTTTTAGAGCTAGAAATAG

right

9
b2M-
TAATACGACTCACTATAG GAGAGAGAATTGAAAAAG
16870

gRNA
GTTTTAGAGCTAGAAATAG

WT Ex2

Endogenous MHC Knock-Out

Gene editing compositions of the disclosure, including but not limited to, RNA-guided fusion proteins comprising dCas9-Clo051, may be used to target and decrease or eliminate expression of an endogenous MHCI, MHCIL, or MHC activator of an allogeneic cell of the disclosure. In preferred embodiments, the gene editing compositions of the disclosure target and delete a gene, a portion of a gene, or a regulatory element of a gene (such as a promoter) encoding one or more components of an endogenous MHCI, MHCII, or MHC activator of an allogeneic cell of the disclosure.

Nonlimiting examples of guide RNAs (gRNAs) for targeting and deleting MHC activators are provided in Tables 13 and 14.

TABLE 13

Reagent/

SEQ ID
Right Target
SEQ ID

Gene
Type
Left Target Sequence
NO:
Sequence
NO

C2TA
C2TA
CATCGCTGTTAAaAAGCTCC
16871
CTACCACTTCTATGACCAGA
16880

exon 4

NG

C2TA
GGCCCTCCAGCTGGGAGTCC
16872
CAGTAAGTTTGTGGTGGGTG
16881

exon6

NG

RFXANK
RFXANK
GGGTCTGCTGGGTCTGGATG
16873
GGACCCTGAAGACCCCGGAG
16882

exon1

NG1

RFXANK
GTTCTGAGGCAGGGGTCTGC
16874
CCCGGAGAGGAGGCTGCAGA
16883

exon1

NG2

RFXAP
RFXAP
CCCGCCCCAACGCTGCCCCC
16875
CTGTGCGAAGGGGCCGGGGA
16884

Exon 1

NG1

RFXAP
CCTTCGCACAGGTACCTAAG
16876
AGAGGAGGCTGGGGAGGACG
16885

Exon 1

NG2

RFX5
RFX5
GTCTTGGGGCTCTTAGCATC
16877
CCCAGGTGGTGCTGAGGCTG
16886

exon 1

NG1

RFX5
ACGGCCTTGCTGTGGGGAAG
16878
GGGATCCTGGTAAGTGTGTT
16887

exon 2

NG2

RFX5
TCTGATGATCTTGCCAAAGT
16879
ATCAAAGCTCGAAGGCTTGG
16888

exon5

NG3

TABLE 14

SEQ

SEQ

SEQ

Reagent/
Exon or
NG-Left Target
ID
NG-Right Target
ID
Target sequence
ID

Gene
Type
region
Sequence
NO:
Sequence
NO.
(if WT crispr)
NO

Beta2-
B2-
promoter
GCAGTGCCAGGTTAGAGAGA
16889
AGACAGGTGACGGTCCCTGC
16913

MG
Promoter-

NG1

B2-
promoter
CAAGCCAGCGACGCAGTGCC
16890
CCTGCGGGCCTTGTCCTGAT
16914

Promoter-

NG2

B2-
promoter
CCAATCAGGACAAGGCCCGC
15891
TATAAGTGGAGGCGTCGCGC
16915

Promoter-

NG3

B2-Ex2-
exon 2
ACCCAGACACATAGCAATTC
16892
GAAGTTGACTTACTGAAGAA
16916

NG

B2-Ex2-
exon 2

gatattcctcagGTACTCCA

16893

TCACGTCATCCAGCAGAGAA

16917

NG2

B2-Ex1-NG
exon 1
GGCCACGGAGCGAGACATCT
16894
ACTCTCTCTTTCTGGCCTGG
16918

WT-B2MG-

GGAGAGAGAATTGAAAAAG
16937

exon2

WT-B3MC-
cuts in

GGGCCTTGTCCTGATGGC
16938

promoter-
promoter

4
region Y

WT-B2MG-
cuts in

GGCACTGCGTCGCTGGCT
16939

promoter-
promoter

5
region

C2TA
C2TA
exon 4
CATCGCTGTTAAGAAGCTCC
16895
CTACCACTTCTATGACCAGA
16919

exon 4

NG

C2TA
exon 4

GGTCCATCTGGTCATAGAAG

16896

AGATTGAGCTCTACTCAGGT

16920

exon4

NG2

C2TA
exon6
GGCCCTCCAGCTGGGAGTCC
16897
CAGTAAGTTTGTGGTGGGTG
16921

exon6

NG

C2TA
exon 4

GGTCCATCTGGTCATAGAAG
16940

exon4-

WT

C2TA
exon 6

GGAGTCCTGGAAGACATAC
16941

exon6-

WT

C2TA
exon 6

CCTTGCTCAGGCCCTCCAGC

16898

TGTGGTGGGTGGGGAGGTCT

16922

exon6

NG2

RFXANK
RFXANK
exon 1
GGGTCTGCTGGGTCTGGATG
16899
GGACCCTGAAGACCCCGGAG
16923

exon1

NG1

RFXANK
exon 1
GTTCTGAGGaAGGGGTCTGC
16900
CCCGGAGAGGAGGCTGCAGA
16924

exon1

NG2

RFXANK
exon 2

TGAGAGTGGTGGAGTGCTTC

16901

GAACGAGGTGTCAGCTCTGC

16925

exon2

NG1

RFXANK
exon 2

CTCGTTCCCTCGCTGCCGGT

16902

GGCCACCCTAGACTGTGAGT

16926

Exon2

NG2

RFXANK-
exon 1

GGTCCCaAAGTTCTGAGGC
16942

WT-

exon1-3

RFXANK-
exon 1

GGCAGGGGTCTGCTGGGTC
16943

WT-

exon1-4

RFXAP
RFXAP
exon 1
CCCGCCCCAACGCTGCCCCC
16903
CTGTGCGAAGGGGCCGGGGA
16927

Exon 1

NG1

RFXAP
exon 1
CCTTCGCACAGGTACCTAAC
16904
AGAGGAGGCTGGGGAGGACG
16928

Exon 1

NG2

RFXAP
exon 1

CAGCCGGGGCTAGGGCCGCG

16905

CTTGGCGCCAGCCTCGGTGG

16929

Exon1

NG3

RFXAP
exon 1

GCCGCGGCCGCCACCGAGGC

16906

CTAGTGATGCAACCCTGTGC

16930

Exon1

NG4

RFXAP
exon 1

GCCGCGCTCTCGCCTCCCCC

16907

GAGGACGAGGAGACTCACTC

16931

Exon1

NG5

WT-
exon 1

GGCCCCCGGGGGCAGCGTT
16944

RFXAP-

ex1-3

WT-
exon 1

GGTACCTGTGCGAAGGGGC
16945

RFXAP-

ex1-4

RFX5
RFX5
exon 1
GTCTTGGGGCTCTTAGCATC
16908
CCCAGGTGGTGCTGAGGCTG
16932

exon1

NG1

RFX5
exon 2
ACGGCCTTGCTGTGGGGAAG
16909
GGGATCCTGGTAAGTGTGTT
16933

exon2

NG2

RFX5
exon 5
TCTGATGATCTTGCCAAAGT
16910
ATCAAAGCTCGAAGGCTTGG
16934

exon5

NG3

RFX5

GTCTTGGGGCTCTTAGCATC

16911

CCCCAGGTGGTGCTGAGGCT

16935

exon1

NG2

RFX5

AGGCTCATCTTCTGCCATCC

16912

ACTGGGGGAAGGGCCCCCCC

16936

exon1

NG3

WT-
Exon 1

GGGAAGGGCCCCCCCAGG
16946

RFX5-

ex1-4

WT-
Exon 5

GCCTTCGAGCTTTGATGTC
16947

RFX5-

ex5-5

Engineered HLA-E Compositions

MHCI knockout (KO) renders cells resistant to killing by T cells, but also makes them susceptible to natural killer (NK) cell-mediated cytotoxicity (“Missing-self hypothesis”) (see FIG. 30). It is hypothesized that NK rejection would reduce the in vivo efficacy and/or persistence of these KO cells in a therapeutic setting, such as allogeneic (allo) CAR-T therapy. Retention of MHCI on the surface of allo CAR-T cells would render them susceptible to killing by host T cells, as observed in the classic mixed lymphocyte reaction (MLR) experiment. It is estimated that up to 10% of a person's T cells are specific to foreign MHC, which would mediate the rejection of foreign cells and tissues. A targeted KO of MHCI, specifically HLA-A. B and C, which can be achieved by targeted KO of B2M, results in a loss of additional HLA molecules including HLA-E. Loss of HLA-E, for example, renders the KO cells more susceptible to NK cell-mediated cytotoxicity due to the “Missing-self Hypothesis”. NK-mediated cytotoxicity against missing-self cells is a defense mechanism against pathogens that downregulate MHC on the surface of infected cells to evade detection and killing by cells of the adaptive immune system.

Two strategies are contemplated by the disclosure for engineering allo (MHCI-neg) T cells (including CAR-T cells) more resistant to NK cell-mediated cytotoxicity. In some embodiments, a sequence encoding a molecule (such as single-chain HLA-E) that reduces or prevents NK killing is introduced or delivered to an allogeneic cell. Alternatively, or in addition, gene editing methods of the disclosure retain certain endogenous HLA molecules (such as endogenous HLA-E). For example, the first approach involves piggyBac® (PB) delivery of a single-chain (sc)HLA-E molecule to B2M KO T cells.

The second approach uses a gene editing composition with guide RNAs selective for HLA-A, HLA-B and HLA-C, but not, for example, HLA-E or other molecules that are protective against natural-killer cell mediated cytotoxicity for MHCI KO cells.

Alternative or additional molecules to HLA-E that are protective against NK cell-mediated cytotoxicity include, but are not limited to, CD47, interferon alpha/beta receptor 1 (IFNAR1), human IFNAR1, interferon alpha/beta receptor 2 (IFNAR2), human IFNAR2, HLA-G1, HLA-G2, HLA-G3. HLA-G4, HLA-G5, HLA-G6, HLA-G7, human carcino embryonic antigen-related cell adhesion molecule 1 (CEACAM1), viral hemoagglutinins, CD48, LLT1 (also referred to as C-type lectin domain family 2 member (CLC2D)), ULBP2, ULBP3, and sMICA or a variant thereof.

An exemplary CD47 protein of the disclosure comprises or consists of the amino acid sequence of (Signal peptide, Extracellular, TM, Cytoplasmic):

(SEQ ID NO: 17016)

MWPINAALLGSACCGSAQLLFNKTKSVEFTFCNDTVVIPCFVTNMEAQNT

TEVYVKWKFKGRDIYTFDGALNKSTVPTDFSSAKIEVSQLLKGDASIKMD

KSDAVSHTGNYTCEVTELTREGETIIELKYRVVSWFSPNENI

custom-character

KFVAS

NQKTIQPPRKAVEEPLNAFKESKGMMNDE

An exemplary INFAR1 protein of the disclosure comprises or consists of the amino acid sequence of (Signal peptide. Extracellular, TM, Cytoplasmic):

(SEQ ID NO: 17017)

MMVVLLGATTLVLVAVAPWVLSAAAGGKNLKSPQKVEVDIIDDNFILRWN

RSDESVGNVTFSFDYQKTGMDNWIKLSGCQNITSTKCNFSSLKLNVYEEI

KLRIRAEKENTSSWYEVDSFTPFRKAQIGPPEVHLEAEDKAIVIHISPGT

KDSVMWALDGLSFTYSLVIWKNSSGVEERIENIYSRHKIYKLSPETTYCL

KVKAALLTSWKIGVSPVHCIKTTVENELPPPENIEVSVQNQNYVLKWDYT

YANMTFQVQWLHAFLKRNPGNHLYKWQIPDCENVKTTQCVFPQNVFQKGI

YLLRVQASDGNNTSFWSEEIKFDTEIQAFLLPPVFNIRSLSDSFHIYIGA

PKQSGNTPVIQDYPLIYEIIFWENTSNAEKRIIEKKTDVTVPNLKPLTVY

CVKARAHTMDEKLNKSSVFSDAVCEKTKPGNTSK

custom-character

KVFLRCINYVFFPSLKPSSSIDEYFSEQ

PLKNLLSTSEEQIEKCFIIENISTIATVEETNQTDEDHKKYSSQTSQDSG

NYSNEDESESKTSEELQQDFV.

An exemplary INFAR2 protein of the disclosure comprises or consists of the amino acid sequence of (Signal peptide, Extracellular, TM, Cytoplasmic):

(SEQ ID NO: 17018)

MLLSQNAFIERSLNLVLMVYISLVEGISYDSPDYTDESCTFKISLRNFRS

ILSWELKNHSIVPTHYTLLYTIMSKPEDLKVVKNCANTTRSFCDLTDEWR

STHEAYVTVLEGFSGNTTLFSCSHNEWLAIDMSFEPPEFEIVGFTNHINV

MVKFTSIVEEELQFDLSLVIEEQSEGIVKKHKPEIKGNMSGNFTYIIDKL

IPNTNYCVSVYLEHSDEQAVIKSPLKCTLLPPGQESESAESAK

custom-character

KWIGYICLRNSLPKVLNFHNFLAWPFPN

LPPLEAMDMVEVIYINRKKKVWDYNYDDESDSDTEAAPRTSGGGYTMHGL

TVRPLGQASATSTESQLIDPESEEEPDLPEVDVELPTMPKDSPQQLELLS

GPCERRKSPLQDPFPEEDYSSTEGSGGRITFNVDLNSVFLRVLDDEDSDD

LEAPLMLSSHLEEMVDPEDPDNVQSNHLLASGEGTQPTFTSPSSEGLWSE

DAPSDQSDTSESDVDLGDGYIMR.

An exemplary HLA-G1 protein of the disclosure comprises or consists of the amino acid sequence of (Alpha chain 1, Alpha chain 2, Alpha chain 3):

(SEQ ID NO: 17019)

MVVMAPRTLFLLLSGALTLTETWAGSHSMRYFSAAVSRPGRGEPRFIAMG

YVDDTQFVRFDSDSACPRMEPRAPWVEQEGPEYWEEETRNTKAHAQTDRM

NLQTLRGYYNQSEA
SSHTLQWMIGCDLGSDGRLLRGYEQYAYDGKDYLAL

NEDLRSWTAADTAAQISKRKCEAANVAEQRRAYLEGTCVEWLHRYLENGK

EMLQRA
DPPKTHVTHHPVFDYEATLRCWALGFYPAEIILTWQRDGEDQTQ

DVELVETRPAGDGTFQKWAAVVVPSGEEQRYTCHVQHEGLPEPLMLRWKQ

SSLPTIPIMGIVAGLVVLAAVVTGAAVAAVLWRKKSSD.

An exemplary HLA-G2 protein of the disclosure comprises or consists of the amino acid sequence of (Alpha chain 1, Alpha chain 2, Alpha chain 3):

(SEQ ID NO: 17020)

MVVMAPRTLFLLLSGALTLTETWAGSHSMRYFSAAVSRPGRGEPRFIAMG

YVDDTQFVRFDSDSACPRMEPRAPWVEQEGPEYWEEETRNTKAHAQTDRM

NLQTLRGYYNQSEA
DPPKTHVTHHPVFDYEATLRCWALGFYPAEIILTWQ

RDGEDQTQDVELVETRPAGDGTFQKWAAVVVPSGEEQRYTCHVQHEGLPE

PLMLRWKQSSLPTIPIMGIVAGLVVLAAVVTGAAVAAVLWRKKSSD.

An exemplary HLA-G3 protein of the disclosure comprises or consists of the amino acid sequence of (Alpha chain 1, Alpha chain 2, Alpha chain 3):

(SEQ ID NO: 17021)

MVVMAPRTLFLLLSGALTLTETWAGSHSMRYFSAAVSRPGRGEPRFIAMG

YVDDTQFVRFDSDSACPRMEPRAPWVEQEGPEYWEEETRNTKAHAQTDRM

NLQTLRGYYNQSEAKQSSLPTIPIMGIVAGLVVLAAVVTGAAVAAVLWRK

KSSD.

An exemplary HLA-G4 protein of the disclosure comprises or consists of the amino acid sequence of (Alpha chain 1, Alpha chain 2, Alpha chain 3):

(SEQ ID NO: 17022)

MVVMAPRTLFLLSGALTLTETWAGSHSMRYFSAAVSRPGRGEPRFIAMGY

VDDTQFVRFDSDSACPRMEPRAPWVEQEGPEYWEEETRNTKAHAQTDRMN

LQTLRGYYNQSEA
SSHTLQWMIGCDLGSDGRLLRGYEQYAYDGKDLALNE

DLRSWTAADTAAQISKRKCEAANVAEQRRAYLEGTCVEWLHRYLENGKEM

LQRAKQSSLPTIPIMGIVAGLVVLAAVVTGAAVAAVLWRKKSSD.

An exemplary HLA-G5 protein of the disclosure comprises or consists of the amino acid sequence of (Alpha chain 1, Alpha chain 2, Alpha chain 3, intron 4):

(SEQ ID NO: 17023)

MVVMAPRTLFLLSGALTLTETWAGSHSMRYFSAAVSRPGRGEPRFIAMGY

VDDTQFVRFDSDSACPRMEPRAPWVEQEGPEYWEEETRNTKAHAQTDRMN

LQTLRGYYNQSEA
SSHTLQWMIGCDLGSDGRLLRGYEQYAYDGKDLALNE

DLRSWTAADTAAQISKRKCEAANVAEQRRAYLEGTCVEWLHRYLENGKEM

LQRA
DPPKTHVTHHPVFDYEATLRCWALGFYPAEIILTWQRDGEDQTQDV

ELVETRPAGDGTFQKWAAVVVPSGEEQRYTCHVQHEGLPEPLMLRW

custom-character

An exemplary HLA-G5 protein of the disclosure comprises or consists of the amino acid sequence of (Alpha chain 1, Alpha chain 2. Alpha chain 3, intron 4):

(SEQ ID NO: 17024)

MVVMAPRTLFLLLSGALTLTETWAGSHSMRYFSAAVSRPGRGEPRFIAMG

YVDDTQFVRFDSDSACPRMEPRAPWVEQEGPEYWEEETRNTKAHAQTDRM

NLQTLRGYYNQSEA
DPPKTHVTHHPVFDYEATLRCWALGFYPAEIILTWQ

RDGEDQTQDVELVETRPAGDGTFQKWAAVVVPSGEEQRYTCHVQHEGLPE

PLMLRW
custom-character

An exemplary HLA-G5 protein of the disclosure comprises or consists of the amino acid sequence of (Alpha chain 1, Alpha chain 2, Alpha chain 3, intron 2):

(SEQ ID NO: 17025)

MVVMAPRTLFLLLSGALTLTETWAGSHSMRYFSAAVSRPGRGEPRFIAMG

YVDDTQFVRFDSDSACPRMEPRAPWVEQEGPEYWEEETRNTKAHAQTDRM

NLQTLRGYYNQSEA
custom-character

An exemplary CEACAM1 protein of the disclosure comprises or consists of the amino acid sequence of (Extracellular, TM, Cytoplasmic):

(SEQ ID NO: 17026)

MGHLSAPLHRVRVPWQGLLLTASLLTFWNPPTTAQLTTESMPFNVAEGKE

VLLLVHNLPQQLFGYSWYKGERVDGNRQIVGYAIGTQQATPGPANSGRET

IYPNASLLIQNVTQNDTGFYTLQVIKSDLVNEEATGQFHVYPELPKPSIS

SNNSNPVEDKDAVAFTCEPETQDTTYLWWINNQSLPVSPRLQLSNGNRTL

TLLSVTRNDTGPYECEIQNPVSANRSDPVTLNVTYGPDTPTISPSDTYYR

PGANLSLSCYAASNPPAQYSWLINGTFQQSTQELFIPNITVNNSGSYTCH

ANNSVTGCNRTTVKTIIVTELSPVVAKPQIKASKTTVTGDKDSVNLTCST

NDTGISIRWFFKNQSLPSSERMKLSQGNTTLSINPVKREDAGTYWCEVFN

PISKNQSKPIMLNVNYNALPQENGLSPG
AIAGIVIGVVALVALIAVALAC

FL
HFGKTGRASDQRDLTEHKPSVSNHTQDHSNDPPNKMNEVTYSTLNFEA

QQPTQPTSASPSLTATEIIYSEVKKQ.

An exemplary viral hemagglutinin protein of the disclosure comprises or consists of the amino acid sequence of (HA for Influenza A virus (A/NewCaledonia/20/1999(H1N1): TM):

(SEQ ID NO: 17027)

MKAKLLVLLCTFTATYADTICIGYHANNSTDTVDTVLEKNVTVIHSVNLL

EDSHNGKLCLLKGIAPLQLGNCSVAGWILGNPECELLISKESWSYIVETP

NPENGTCYPGYFADYEELREQLSSVSSFERFEIFPKESSWPNHTVTGVSA

SCSHNGKSSFYRNLLWLTGKNGLYPNLSKSYVNNKEKEVLVLWGVHHPPN

IGNQRALYHTENAYVSVVSSHYSRRFTPEIAKRPKVRDQEGRINYYWTLL

EPGDTIIFEANGNLIAPWYAFALSRGFGSGIITSNAPMDECDAKCQTPQG

AINSSLPFQNVHPVTIFECPKYVRSAKLRMVTGLRNIPSIQSRGLFGAIA

GFIEGGWTGMVDGWYGYHHQNEQGSGYAADQKSTQNAINGITNKVNSVIE

KMNTQFTAVGKEFNKLERRMENLNKKVDDGFLDIQTYNAELLVLLENERT

LDFHDSNVKNLYEKVKSQLKNNAKEIGNGCFEFYHKCNNECMESVKNGTY

DYPKYSEESKLNREKIDGVKLESMGVYQILAIYSTVASSLVLLVSLAGIS

FWMCSNGSLQCRICI.

An exemplary CD48 protein of the disclosure comprises or consists of the amino acid sequence of (Signal peptide, Chain, Pro peptide removed in mature form):

(SEQ ID NO: 17028)

MCSRGWDSCLALELLLLPLSLLVTSI
QGHLVHMTVVSGSNVTLNISESLP

ENYKQLTWFYTFDQKIVEWDSRKSKYFESKFKGRVRLDPQSGALYISKVQ

KEDNSTYIMRVLKKTGNEQEWKIKLQVLDPVPKPVIKIEKIEDMDDNCYL

KLSCVIPGESVNYTWYGDKRPFPKELQNSVLETTLMPHNYSRCYTCQVSN

SVSSKNGTVCLSPPCTLARS
FGVEWIASWLVVTVPTILGLLLT.

An exemplary LLT1 protein of the disclosure comprises or consists of the amino acid sequence of (Cytoplasmic, TM. Extracellular):

(SEQ ID NO: 17029)

MHDSNNVEKDITPSELPANPGCLHSKEHSIKATLIWRL
FFLIMFLTIIVC

GMVAALSAI
RANCHQEPSVCLQAACPESWIGFQRKCFYFSDDTKNWTSSQ

RFCDSQDADLAQVESFQELNFLLRYKGPSDHWIGISREQGQPWKWINGTE

WTRQFPILGAGECAYLNDKGASSARHYTERKWICSKSDIHV.

An exemplary ULBP2 protein of the disclosure comprises or consists of the amino acid sequence of (also known as NKG2D ligand; Genbank ACCESSION No. AAQ89028):

(SEQ ID NO: 17030)

1 maaaaatkil lclpllllls gwsragradp hslcyditvi pkfrpgprwc avqgqvdekt

61 flhydcgnkt vtpvsplgkk invttawkaq npvlrevvdi lteqlrdiql enytpkeplt

121 lqarmsceqk aeghssgswq fsfdggifll fdsekrmwtt vhpgarkmke kewndkvvam

181 sfhyfsmgdc igwledflmg mdstlepsag aplamssgtt qlratattli lcclliilpc

241 filpgi.

An exemplary ULBP3 protein of the disclosure comprises or consists of the amino acid sequence of (also known as NKG2D ligand; Genbank ACCESSION No. NP 078794):

(SEQ ID NO: 17031)

1 maaaaspail prlailpyll fdwsgtgrad ahslwynfti ihlprhgqqw cevqsqvdqk

61 nfisydcgsd kvlsmghlee qlyatdawgk qlemlrevgq rlrleladte ledftpsgpl

121 tlqvrmscec eadgyirgsw qfsfdgrkfl lfdsnnrkwt vvhagarrmk ekwekdsglt

181 tffkmvsmrd ckswlrdflm hrkkrlepta pptmapglaq kpaiattlsp wsfliilcfi

241 lpgi.

An exemplary sMICA protein of the disclosure comprises or consists of the amino acid sequence of (Signal Peptide Portion of Extracellular domain, TM and cytoplasmic domain) (Genbank Accession No. Q29983):

(SEQ ID NO: 17032)

1 mglgpvflll agifpfappg aaaephslry nltvlswdgs vqsgfltevh ldgqpflrcd

61 rqkcrakpqg qwaedvlgnk twdretrdlt gngkdlrmtl ahikdqkegl hslqeirvce

121 ihednstrss qhfvydgelf isqnletkew tmpqssraqt iamnvrnflk edamktkthy

181 hamhadclqe irrylksgvv lrrtvppmvn vtrseasegn itvtcrasgf ypwnitlswr

241 qdgvslshdt qqwgdvlpdg ngtyqtwvat ricqgeeqrf tcymehsgnh sthpvpsgkv

301 lvlqshwqtf hvsavaaaai fviiifyvrc ckkktsaaeg pelvslqvld qhpvgtsdhr

361 datglgfqpl msdlgstgst ega.

An exemplary sMICA protein of the disclosure comprises or consists of the amino acid sequence of (Alpha-1 Alpha-2, Alpha-3):

(SEQ ID NO: 17033)

1 mglgpvflll agifpfappg aaaephslry nltvlswdgs vqsgfltevh ldgqpflrcd

61 rqkcrakpqg qwaedvlgnk twdretrdlt gngkdlrmtl ahikdqke
gl hslqeirvce

121 ihednstrss qhfvydgelf isqnletkew tmpqssraqt iamnvrnflk edamktkthy

181 hamhadclqe irrylksgvv lrrtvppmvn vtrseasegn itvtcrasgf ypwnitlswr

241 qdgvslshdt qqwgdvlpdg ngtyqtwvat ricqgeeqrf tcymehsgnh sthpvpsgkv

301 lvlqshwqtf hvsavaaaai fviiifyvrc ckkktsaaeg pelvslqvld qhpvgtsdhr

361 datglgfqpl msdlgstgst ega.

An exemplary sMICA protein of the disclosure comprises or consists of the amino acid sequence of (Signal peptide; Alpha-1. Alpha-2, Alpha-3):

(SEQ ID NO: 170734)

custom-character

ephsiry nltvlswdgs vqsqfltevh ldgqpflrcd

61 rqkcrakpqq qwaedvignk twdretrdlt gngkdlrmtl ahikdqke
gl hslqeirvce

121 ihednstrss qhfyydgelf lsqnletkew tmpqssraqt l thy

181 hamhadclqe lrrylksgvv lrrtvppmvn vtrseasegn itvtcrasgt ypwnitlswr

241 qdgvslshdt qqwgdvlpdg ngtyqtwvat ricqgeeqrf tcymehsgnh sthpvpsgkv

301 lvlqshw.

An exemplary sMICA protein of the disclosure comprises or consists of the amino acid sequence of (Signal peptide):

(SEQ ID NO: 17035)

custom-character

EPHSLRYNLTVLSWDGSVQSGFL

TEVHLDGQPFLRCDRQKCRAKPQGQWAEDVLGNKTWDRETRDLTGNGKL

DLRMTLAHIKLDQKEGLHSLQEIRVCEIHEDNSTRSSQHFYYNGELFLS

QNLETKEWTMPQSSRAQTLTHYHAMHADCLQELRRYLKSGVVLRRTVPP

MVDVTRSEASEGNITVTCRASGFYPWNITLSWRQDGVSLSHDTQQWGDV

LPDGNGTYQTWVATRICQGEEQRFTCYMEHSGNHSTHPVPSGKVLVLQS

HW.

An exemplary bGBE Trimer (270G and 484S) protein of the disclosure comprises or consists of the amino acid sequence of:

(SEQ ID NO: 16972)

MSRSVALAVLALLSLSGLEAVMAPRTLILGGGGSGGGGSGGGGSIQRTP

KIQVYSRHPAENGKSNFLNCYVSGFHPSDIEVDLLKNGERIEKVEHSDL

SFSKDWSFYLLYYTEFTPTEKDEYACRVNHVTLSUKIVKWDRDMGGGGS

GGGGSGGGGSGGGGSGSHSLKYFHTSVSRPGRGEPRFISVGYVDDTQFV

RFDNDAASPRMVPRAPWMEQEGSEYWDRETRSARDTAQIFRVNLRTLRG

YYNQSEAGSHTLQWMHGCELGPDGRFLRGYEQFAYDGKDYLTLNEDLRS

WTAVDTAAQISEQKSNDASEAEHQRAYLEDTCVEWLHKYLEKGKETLLH

LEPPKTHVTHHPISDHEATLRCWALGFYPAETILTWQQDGEGHTQDTEL

VETRPAGDGTFQKWAAVVVPSGEEQRYTCHVQHEGLPEPVTLRWKPASQ

PTIPIVGIIAGLVLLGSVVSGAVVAAVIWRKKSSGGKGGSY custom-character

KAEWSDS

AQGSESHSL*.

An exemplary bGBE Trimer (270G and 484S) protein of the disclosure comprises or consists of the nucleic acid sequence of;

(SEQ ID NO: 16973)

atgtctcgcagcgtggccctggccgtgctggccctgctgtccctgtctggc

ctggaggccgtgatggccccccggaccctgatcctgggaggaggaggcagc

ggcggaggaggctccggaggcggcggctctatccagcgcacacctaagatc

caggtgtattctcggcacccagccgagaacggcaagagcaacttcctgaat

tgctacgtgagcggctttcacccttccgacatcgaggtggatctgctgaag

aatggcgagagaatcgagaaggtggagcactccgacctgagcttctccaag

gattggtctttttatctgctgtactataccgagtttacccctacagagaag

gacgagtacgcctgtcgcgtgaaccacgtgacactgtcccagccaaagatc

gtgaagtgggaccgggatatgggcggcggcggctctggcggcggcggcagc

ggcggcggcggctccggaggaggcggctctggcagccactccctgaagtat

ttccacacctctgtgagccggccaggcagaggagagccacggttcatctct

gtgggctacgtggacgatacacagttcgtgaggtttgacaatgatgccgcc

agcccaagaatggtgcctagggccccatggatggagcaggagggcagcgag

tattgggacagggagacccggagcgccagagacacagcacagattttccgg

gtgaacctgagaaccctgaggggctactataatcagtccgaggccggctct

cacacactccagtggatgcacggatgcgagctgggaccagatggccgcttc

ctgcggggctacgagcagtttgcctatgacggcaaggattacctgaccctg

aacgaggacctgagatcctggaccgccgtggatacagccgcccagatcagc

gagcagaagtccaatgacgcatctgaggcagagcaccagagggcatatctg

gaggatacctgcgtggagtggctgcacaagtacctggagaagggcaaggag

acactgctgcacctggagccccctaagacccacgtgacacaccacccaatc

agcgaccacgaggccaccctgaggtgttgggcactgggcttctatcccgcc

gagatcaccctgacatggcagcaggacggagagggacacacccaggataca

gagctggtggagaccaggcccgccggcgatggcacatttcagaagtgggcc

gccgtggtggtgccttccggagaggagcagagatacacctgtcacgtgcag

cacgagggactgccagagccagtgaccctgaggtggaagcctgccagccag

cccacaatccctatcgtgggaatcatcgcaggcctggtgctgctgggctct

gtggtgagcggagcagtggtggccgccgtgatctggcggaagaagagcagc

ggaggcaagggaggctcctact custom-character

caaggcagagtggagcgactccgcccag

ggctctgagagccactccctgtga.

An exemplary bGBE Trimer (270R and 484S) protein of the disclosure comprises or consists of the amino acid sequence of:

(SEQ ID NO: 16974)

MSRSVALAVLALLSLSGLEAVMAPRILILGGGGSGGGGSGGGGSIQRTPKI

QVYSRHPAENGKSNFLNCYVSGFHPSDIEVDLLKNGERIEKVEHSDLSFSK

DWSFYLLYYTEFTPTEKDEYACRVNHVTLSQPKIVKWDRDMGGGGSGGGGS

GGGGSGGGGSGSHSLKYFHTSVSRPGRGEPRFISVGYVDDTQFVREDNDAA

SPRMVPRAPWMEQEGSEYWDRETRSARDTAQIFRVNLRTLRGYYNQSEAGS

HTLQWMHGCELGPDRRELRGYEQFAYDGKDYLTLNEDLRSWTAVDTAAQIS

EQKSNDASEAEHQRAYLEDTCVEWLHKYLEKGKETLLHLEPPKTHVTHHPI

SDHEATLRCWALGFYPAEITLTWQQDGEGHTQDTELVETRPAGDGTFQKWA

AVVVPSGEEQRYTCHVQHEGLPEPVTLRWKPASQPTIPIVGIIAGLVLLGS

VVSGAVVAAVIWRKKSSGGKGGSY custom-character

KAEWSDSAQGSESHSL*.

An exemplary bGBE Trimer (270R and 484S) protein of the disclosure comprises or consists of the nucleic acid sequence of:

(SEQ ID NO: 16975)

atgtctcgcagcgtggccctggccgtgctggccctgctgtccctgtctggc

ctggaggccgtgatggccccccggaccctgatcctgggaggaggaggcagc

ggcggaggaggctccggaggcggcggctctatccagcgcacacctaagatc

caggtgtattctcggcacccagccgagaacggcaagagcaacttcctgaat

tgctacgtgagcggctttcacccttccgacatcgaggtggatctgctgaag

aatggcgagagaatcgagaaggtggagcactccgacctgagcttctccaag

gattggtctttttatctgctgtactataccgagtttacccctacagagaag

gacgagtacgcctgtcgcgtgaaccacgtgacactgtcccagccaaagatc

gtgaagtgggaccaggatatgggcggcggcggctctggcggcggcggcagc

ggcggcggcggctccggaggaggcggctctggcagccactccctgaagtat

ttccacacctctgtgagccggccaggcagaggagagccacggttcatctct

gtgggctacgtggacgatacacagttcgtgaggtttgacaatgatgccgcc

agcccaagaatggtgcctagggccccatggatggagcaggagggcagcgag

tattgggacagggagacccggagcgccagagacacagcacagattttccgg

gtgaacctgagaaccctgaggggctactataatcagtccgaggccggctct

cacacactccagtggatgcacggatgcgagctgggaccagatcgccgcttc

ctgcggggctacgagcagtttgcctatgacggcaaggattacctgaccctg

aacgaggacctgagatcctggaccgccgtggatdcagccgcccagatcagc

gagcagaagtccaatgacgcatctgaggcagagcaccagagggcatatctg

gaggatacctgcgtggagtggctgcacaagtacctggagaagggcaaggag

acactgctgcacctggagccccctaagacccacgtgacacaccacccaatc

agcgaccacgaggccaccctgaggtgttgggcactgggcttctatcccgcc

gagatcaccctgacatggcagcaggacggagagggacacacccaggataca

gagctggtggagaccaggcccgccggcgatggcacatttcagaagtgggcc

gccgtggtggtgccttccggagaggagcagagatacacctgtcacgtgcag

cacgagggactgccagagccagtgaccctgaggtggaagcctgccagccag

cccacaatccctatcgtgggaatcatcgcaggcctggtgctgctgggctct

gtggtgagcggagcagtggtggccgccgtgatctggcggaagaagagcagc

ggaggcaagggaggctcctact custom-character

caaggcagagtggagcgactccgcccag

ggctctgagagccactccctgtga.

An exemplary gBE Dimer (R and S) protein of the disclosure comprises or consists of the amino acid sequence of:

(SEQ ID NO: 16976)

MSRSVALAVLALLSLSGLEAIQRTPKIQVYSRHPAENGKSNFLNCYVSGFH

PSDIEVDLLKNGERIEKVEHSDLSFSKDWSFYLLYYTEFTPTEKDEYACRV

NHVTLSQPKIVKWDRDMGGGGSGGGGSGGGGSGGGGSGSHSLKYFHTSVSR

PGRGEPRFISVGYVDDTQFVRFDNDAASPRMVPRAPWMEQESGEYWDRETR

SARDTAQIFRVNLRTLRGYYNQSEAGSHTLQWMHGCELGPDRRFLRGYEQF

AYDGKDYLTLNEDLRSWTAVDTAAQISEQKSNDASEAEHQRAYLEDTCVEW

LHKYLEKGKETLLHLEPPKTHVTHHPISDHEATLRCWALGFYPAEITLTWQ

QDGEGHTQDTELVETRPAGDGTFQKWAAVVVPSGEEQRYTCHVQHEGLPEP

VTLRWKPASQPTIPIVGIIAGLVLLGSVVSGAVVAAVIWRKKSSGGKGGSY

custom-character

KAEWSDSAQGSESHSL.

An exemplary gBE Dimer (R and S) protein of the disclosure comprises or consists of the nucleic acid sequence of:

(SEQ ID NO: 16977)

ATGAGCAGATCTCTGGCCCTGGCTGTTCTGGCTCTGCTCTCTCTCTCTGCC

CTCGAAGCCATCCAGCGGACCCCTAAGATCCAGGTGTACAGCAGACACCCC

GCCGAGAACGGCAAGAGCAACTTCCTGAACTGCTACGTGTCCGGCTTTCAC

CCCAGCGACATTGAGGTGGACCTGCTCAAGAACGGCGAGCGGATCGAGAAG

GTGGAACACACCGATCTGAGCTTCAGCAAGGACTGGTCCTTCTACCTGCTG

TACTACACCGAGTTCACCCCTACCGAGAAGGACGAGTACGCCTGCAGAGTG

AACCACGTGACACTGAGCCAGCCTAAGATCCTGAAGTGGGACAGAGATATG

GGCGGAGGCGCATCTGGTGGCGGAGGAAGTGGCGGCGGAGGATCTGGCGGT

GGTGGTTCTGGATCTCACAGCCTGAAGTACTTTCACACCTCCGTGTCCAGA

CCTGGCAGAGGCGAGCCTAGATTCATCAGCGTGGGCTACGTGGACGACACC

CAGTTCGTCAGATTCGACAACGACGCCGCCTCTCCTCGGATCGTTCCTAGA

GCACCCTGGATGGAACAAGAGGGCAGCGAGTACTGGGATCGCGAGACAAGA

AGCGCCAGAGACACACCCCAGATCTTCCGCGTGAACCTGAGAACCCTGCGG

GGCTACTACAATCAGTCTGAGGCCGGCTCTCACACCCTGCAGTGGATGCAT

CGATGTGAACTGGGCCCCGACAGACGGTTCCTGAGAGGGTATGAGCAGTTC

GCCTACGACGGCAAGGACTACCTGACACTGAACGAGGACCTGAGAAGCTGG

ACCGGCGTGGATACAGCCGCTCAGATCAGCGAGCAGAAGTCTAACGACGCC

AGCGAGGCCGAACACCAGAGAGCCTATCTGGAAGATACCTGCGTGGAATGG

CTGCACAAGTACCTGGAAAAGGGCAAAGAGACACTGCTGCACCTGGAACCT

CCAAAGACACATGTGACCCACCATCCTATCAGCGACCACGAGGCCACACTG

AGATGTTGGGCCCTGGGCTTTTACCCTGCCGAGATCACACTGACATGGCAG

GAGGATGGCGAGGGCCACACACAGGATACAGAGCTGGTGGAAACAAGACCT

GCCGGCGACGGCACCTTCCAGAAATGGGCTGCTGTGGTTGTGCCCAGCGGC

GAGGAACAGAGATACACCTGTCACGTGCAGCACGAGGGACTGCCTGAACCT

GTCACTCTGAGATGGAAGCCTGCCAGCCAGCCAACAATCCCCATCGTGGGA

ATCATTGCCGGCCTGGTGCTGCTGGGATCTGTGGTTTCTGGTGCTCTGGTG

GCCGCCGTGATTTGGAGAAAGAAGTCCTCTGGCGGCAAAGGCGGCTCCTAC

custom-character

AAGGCCGAGTGGAGCGATTCTGCCCAGGGCTCTGAAAGCCACAGCCTG

TAGATAA.

An exemplary gBE Dimer (G and S) protein of the disclosure comprises or consists of the amino acid sequence of:

(SEQ ID NO: 16978)

DLLKNGERIEKVEHSDLSFSKDWSFYLLYYTEFTPTEKDEYACRVNHVTLS

QPKIVKWDRDMGGGGSGGGGSGGGGSGGGGSGSHSLKYFHTSVSRPGRGEP

RFISVGYVDDTQFVRFDNDAASPRMVPRAPWMEQESGEYWDRETRSARDTA

QIFRVNLRTLRGYYNQSEAGSHTLQWMHGCELGPDGRFLRGYEQFAYDGKD

YLTLNEDLRSWTAVDTAAQISEQKSNDASEAEHQRAYLEDTCVEWLHKYLE

KGKETLLHLEPPKTHVTHHPISDHEATLRCWALGFYPAEITLTWQQDGEGH

TQDTELVETRPAGDGTFQKWAAVVVPSGEEQRYTCHVQHEGLPEPVTLRWK

PASQPTIPIVGIIAGLVLLGSVVSGAVVAAVIWRKKSSGGKGGSY custom-character

KAEWS

DSAQGSESHSL

An exemplary gBE Dimer (G and S) protein of the disclosure comprises or consists of the amino acid sequence of:

(SEQ ID NO: 16979)

ATGAGCAGATCTCTGGCCCTGGCTGTTCTGGCTCTGCTCTCTCTCTCTGCC

CTCGAAGCCATCCAGCGGACCCCTAAGATCCAGGTGTACAGCAGACACCCC

GCCGAGAACGGCAAGAGCAACTTCCTGAACTGCTACGTGTCCGGCTTTCAC

CCCAGCGACATTGAGGTGGACCTGCTCAAGAACGGCGAGCGGATCGAGAAG

GTGGAACACACCGATCTGAGCTTCAGCAAGGACTGGTCCTTCTACCTGCTG

TACTACACCGAGTTCACCCCTACCGAGAAGGACGAGTACGCCTGCAGAGTG

AACCACGTGACACTGAGCCAGCCTAAGATCCTGAAGTGGGACAGAGATATG

GGCGGAGGCGCATCTGGTGGCGGAGGAAGTGGCGGCGGAGGATCTGGCGGT

GGTGGTTCTGGATCTCACAGCCTGAAGTACTTTCACACCTCCGTGTCCAGA

CCTGGCAGAGGCGAGCCTAGATTCATCAGCGTGGGCTACGTGGACGACACC

CAGTTCGTCAGATTCGACAACGACGCCGCCTCTCCTCGGATCGTTCCTAGA

GCACCCTGGATGGAACAAGAGGGCAGCGAGTACTGGGATCGCGAGACAAGA

AGCGCCAGAGACACACCCCAGATCTTCCGCGTGAACCTGAGAACCCTGCGG

GGCTACTACAATCAGTCTGAGGCCGGCTCTCACACCCTGCAGTGGATGCAT

GGATGTGAACTGGGCCCCGACAGACAGTTCCTGAGAGGGTATGAGCAGTTC

GCCTACGACGGCAAGGACTACCTGACACTGAACGAGGACCTGAGAAGCTGG

ACCGGCGTGGATACAGCCGCTCAGATCAGCGAGCAGAAGTCTAACGACGCC

AGCGAGGCCGAACACCAGAGAGCCTATCTGGAAGATACCTGCGTGGAATGG

CTGCACAAGTACCTGGAAAAGGGCAAAGAGACACTGCTGCACCTGGAACCT

CCAAAGACACATGTGACCCACCATCCTATCAGCGACCACGAGGCCACACTG

AGATGTTGGGCCCTGGGCTTTTACCCTGCCGAGATCACACTGACATGGCAG

GAGGATGGCGAGGGCCACACACAGGATACAGAGCTGGTGGAAACAAGACCT

GCCGGCGACGGCACCTTCCAGAAATGGGCTGCTGTGGTTGTGCCCAGCGGC

GAGGAACAGAGATACACCTGTCACGTGCAGCACGAGGGACTGCCTGAACCT

GTCACTCTGAGATGGAAGCCTGCCAGCCAGCCAACAATCCCCATCGTGGGA

ATCATTGCCGGCCTGGTGCTGCTGGGATCTGTGGTTTCTGGTGCTCTGGTG

GCCGCCGTGATTTGGAGAAAGAAGTCCTCTGGCGGCAAAGGCGGCTCCTAC

custom-character

AAGGCCGAGTGGAGCGATTCTGCCCAGGGCTCTGAAAGCCACAGCCTG

TAGATAA.

A wildtype/natural human HLA-E protein (NCBI: HLAE_HUMAN; UniProt/Swiss-Prot: P13747.4) comprises or consists of the amino acid sequence of:

(SEQ ID NO: 17122)

MVDGTLLLLLSEALALTQTWAGSHSLFYFHTSVSRPGRGEPRFISVGYVDD

TQFVRFDNDAASPRMVPRAPWMEQEGSEYWDRETRSARDTAQIFRVNLRTL

RGYYNQSEAGSHTLQWMHGCELGPDGRFLRGYEQFAYDGKDYLTLNEDLRS

WTAVDTAAQISEQKSNDASEAEHQRAYLEDTCVEWLHKLEKGKETLLHLEP

PKTHVTHHPISDHEATLRCWALGFYPAEITLTWQQDGEGHTQDTELVETRP

AGDGTFQKWAAVVVPSGEEQRYTCHVQHEGLPEPVTLRWKPASQPTIPIVG

IIAGLVLLGSVVSGAVVAAVIWRKKSSGGKGGSYSKAEWSDSAQGSESHSL

A nucleotide sequence encoding wildtype/natural HLA-E protein (NCBI: CCDS34379.1) comprises or consists of the nucleotide sequence of:

(SEQ ID NO: 17123)

ATGGTAGATGGAACCCTCCTTTTACTCCTCTCGGAGGCCCTGGCCCTTACC

CAGACCTGGGCGGGCTCCCACTCCTTGAAGTATTTCCACACTTCCGTGTCC

CGGCCCGGCCGCGGGGAGCCCCGCTTCATCTCTGTGGGCTACGTGGACGAC

ACCCAGTTCGTGCGCTTCGACAACGACGCCGCGAGTCCGAGGATGGTGCCG

CGGGCGCCGTGGATGGAGCAGGAGGGGTCAGAGTATTGGGACCGGGAGACA

CGGAGCGCCAGGGACACCGCACAGATTTTCCGAGTGAATCTGCGGACGCTG

CGCGGCTACTACAATCAGAGCGAGGCCGGGTCTCACACCCTGCAGTGGATG

CATGGCTGCGAGCTGGGGCCCGACGGGCGCTTCCTCCGCGGGTATGAACAG

TTCGCCTACGACGGCAAGGATTATCTCACCCTGAATGAGGACCTGCGCTCC

TGGACCGCGGTGGACACGGCGGCTCAGATCTCCGAGCAAAAGTCAAATGAT

GCCTCTGAGGCGGAGCACCAGACACCCTACCTGGAAGACACATGCGTGGAG

TGGCTCCACAAATACCTGGAGAAGGGGAAGGAGACGCTGCTTCACCTGGAG

CCCCCAAAGACACACGTGACTCACCACCCCATCTCTGACCATGAGGCCACC

CTGAGGTGCTGGGCCCTGGGCTTCTACCCTGCGGAGATCACACTGACCTGG

CAGCAGCATGGGGAGGGCCATACCCAGGACACGGAGCTCGTGGAGACCAGG

CCTGCAGGGGATGGAACCTTCCAGAAGTGGGCAGCTGTGGTGGTGCCTTCT

GGAGAGGAGCAGAGATACACGTGCCATGTGCAGCATGAGGGGCTACCCGAG

CCCGTCACCCTGAGATGGAAGCCGGCTTCCCAGCCCACCATCCCCATCGTG

GGCATCATTGCTGGCCTGGTTCTCCTTGGATCTGTGGTCTCTGGAGCTGTG

GTTGCTGCTGTGATATGGAGGAAGAAGAGCTCAGGTGGAAAAGGAGGGAGC

TACTCTAAGGCTGAGTGGAGCGACAGTGCCCAGGGGTCTGAGTCTCACAGC

TTGTAA

An exemplary WT HLA-E Monomer (R and S) protein of the disclosure comprises or consists of the amino acid sequence of.

(SEQ ID NO: 16980)

MSRSVALAVLALLSLSGLEAGSHSLKYFHTSVSRPGRGEPRFISVGYVDDT

QFVRFDNDAASPRMVPRAPWMEQEGSEYWDRETRSARDTAQIFRVNLRTLR

GYYNQSEAGSHTLQWHGCELGPDRRFLRGYEQFAYDGKDYLTLNEDLRSWT

AVDTAAQISEQKSNDASEAEHQRAYLEDTCVEWLHKYLEKGKETLLHLEPP

KTHVTHHPISDHEATLRCWALGFYPAEITLTWQQDGEGHTQDTELVETRPA

GDGTFQKWAAVVVPSGEEQRYTCHVQHEGLPEPVTLRWKPASQPTIPTVGI

IAGLVLLGSVVSGAVVAAVIWRKKSSGGKGGSY custom-character

KAEWSDSAQGSESHSL

An exemplary WT HLA-E Monomer (R and S) protein of the disclosure comprises or consists of the nucleic acid sequence of:

(SEQ ID NO: 16981)

ATGAGCAGATCTGTGGCCCTGGCTGTTCTGGCTCTGCTGTCTCTGTCTGGA

CTGGAAGCCGGCAGCCACAGCCTGAAGTACTTTCACACCAGCGTGTCCAGA

CCTGGCAGAGGCGAGCCTAGATTCATCAGCGTGGGCTACGTGGACGACACC

CAGTTCGTCAGATTCGACAACGACGCCGCCTCTCCTCGGATGGTTCCTAGA

GCACCCTGGATGGAACAAGAGGGCAGCGAGTACTGGGACAGAGAGACAAGA

AGCGCCAGAGACACAGCCCAGATCTTCAGAGTGAACCTGCGGACCCTGCGG

GGCTACTACAATCAGTCTGAAGCCGGCTCTCACACCCTGCAGTGGATGCAC

GGATGTGAACTGGGCCCCGACAGAAGATTCCTGAGAGGCTACGAGCAGTTC

GCCTACGACGGCAAGGACTACCTGACACTGAACGAGGACCTGAGAAGCTGG

ACCGCCGTGGATACAGCCGCTCAGATCAGCGAGCAGAAGTCTAACGACGCC

TCTGAGGCCGAACACCAGAGAGCCTACCTGGAAGATACCTGCGTGGAATGG

CTGCACAAGTACCTGGAAAAGGGCAAAGAGACACTGCTGCACCTGGAACCT

CCAAAGACACACGTGACCCACCATCCTATCAGCGACCACGAGGCCACACTG

AGATGTTGGGCCCTGGGCTTTTACCCCGCCGAGATCACACTGACATGGCAG

CAGGATGGCGAGGGCCACACACAGGATACAGAGCTGGTGGAAACAAGACCT

GCCGGCGACGGCACCTTCCAGAAATGGGCTGCTGTGGTGGTTCCCAGCGGC

GAGGAACAGAGATACACCTGTCACGTGCAGCACGAGGGACTGCCTGAACCT

GTGACACTGAGGTGGAAGCCTGCCAGCCAGCCTACAATCCCCATCGTGGGA

ATCATTGCCGGCCTGGTGCTGCTGGGATCTGTGGTTTCTGGTGCAGTGGTG

GCCGCCGTGATCTGGCGGAAAAAAAGCTCAGGCGGCAAAGGCGGCTCCTAC

custom-character

AAAGCCGAGTGGAGCGATTCTGCCCAGGGCTCTGAAAGCCACTCTCTG

TAGATAA.

An exemplary WT HLA-E Monomer (G and S) protein of the disclosure comprises or consists of the nucleic acid sequence of:

(SEQ ID NO: 16982)

MSRSVALAVLALLSLSGLEAGSHSLKYFHTSVSRPGRGEPRFISVGYVDDT

QFVRFDNDAASPRKVPRAPWMFQEGSEYWDRETRSARDTAQIFRVNLRTLR

GYYNQSEAGSHTLQWMHGCELGPDGRFLRGYEQFAYDGKDYLTLNEDLRSW

TAVDTAAQISEQKSNDASEAEHQRAYLEDICVEWLHKYLEKGKETLLHLEP

PKTHVTHHPISDHEATLRCWALGFYPAEITLTWQQDGEGHTQDTELVETRP

AGDGTFQKWAAVVVPSGEEQRYTCHVQHEGLPEPVTLRWKPASQPTTPIVG

IIAGLVLLGSVVSGAVVAAVIWRKKSSGGKGGSY custom-character

KAEWSDSAQGSESHS

L.

An exemplary WT HLA-E Monomer (G and S) protein of the disclosure comprises or consists of the nucleic acid sequence of:

(SEQ ID NO: 16983)

ATGAGCAGATCTGTGGCCCTGGCTGTTCTGGCTCTGCTGTCTCTGTCTGGA

CTGGAAGCCGGCAGCCACAGCCTGAAGTACTTTCACACCAGCGTGTCCAGA

CCTGGCAGAGGCGAGCCTAGATTCATCAGCGTGGGCTACGTGGACGACACC

CAGTTCGTCAGATTCGACAACGACGCCGCCTCTCCTCGGATGGTTCCTAGA

GCACCCTGGATGGAACAAGAGGGCAGCGAGTACTGGGACAGAGAGACAAGA

AGCGCCAGAGACACAGCCCAGATCTTCAGAGTGAACCTGCGGACCCTGCGG

GGCTACTACAATCAGTCTGAAGCCGGCTCTCACACCCTGCAGTGGATGCAC

GGATGTGAACTGGGCCCCGACGGAAGATTCCTGAGAGGCTACGAGCAGTTC

GCCTACGACGGCAAGGACTACCTGACACTGAACGAGGACCTGAGAAGCTGG

ACCGCCGTGGATACAGCCGCTCAGATCAGCGAGCAGAAGTCTAACGACGCC

TCTGAGGCCGAACACCAGAGAGCCTACCTGGAAGATACCTGCGTGGAATGG

CTGCACAAGTACCTGGAAAAGGGCAAAGAGACACTGCTGCACCTGGAACCT

CCAAAGACACACGTGACCCACCATCCTATCAGCGACCACGAGGCCACACTG

AGATGTTGGGCCCTGGGCTTTTACCCCGCCGAGATCACACTGACATGGCAG

GAGGATGGCGAGGGCCACACACAGGATACAGAGCTGGTGGAAACAAGACCT

GCCGGCGACGGCACCTTCCAGAAATGGGCTGCTGTGGTGGTTCCCAGCGGC

GAGGAACAGAGATACACCTGTCACGTGCAGCACGAGGGACTGCCTGAACCT

GTGACACTGAGGTGGAAGCCTGCCAGCCAGCCTACAATCCCCATCGTGGGA

ATCATTGCCGGCCTGGTGCTGCTGGGATCTGTGGTTTCTGGTGCAGTGGTG

GCCGCCGTGATCTGGCGGAAAAAAAGCTCAGGCGGCAAAGGCGGCTCCTAC

custom-character

AAAGCCGAGTGGAGCGATTCTGCCCAGGGCTCTGAAAGCCACTCTCTG

TAGATAA.

A wildtype/natural human B2M protein (NCBI: B2MG_HUMAN; UniProt/Swiss-Prot: P61769.1) comprises or consists of the amino acid sequence of:

(SEQ ID NO: 17124)

MSRSVALAVLALLSLSGLEAIQRTPKIQVYSRHPAENGKSNFLNCYVSGFH

PSDIEVDLLKNGERIEKVEHSDLSFSKDWSFYLLYYTEFTPTEKDEYACRV

NHVILSQPKIVKWDRDM

A nucleotide sequence encoding wildtype/natural B2M protein (NCBI: CCDS10113.1) comprises or consists of the nucleotide sequence of:

(SEQ ID NO: 17125)

ATGTCTCGCTCCGTGGCCTTAGCTGTGCTCGCGCTACTCTCTCTTTCTGGC

CTGGAGGCTATCCAGCGTACTCCAAAGATTCAGGTTTACTCACGTCATCCA

GCAGAGAATGGAAAGTCAAATTTCCTGAATTGCTATGTGTCTGGGTTTCAT

CCATCCGACATTGAAGTTGACTTACTGAAGAATGGAGAGAGAATTGAAAAA

GTGGAGCATTCAGACTTGTCTTTCAGCAAGGACTGGTCTTTCTATCTCTTG

TACTACACTGAATTCACCCCCACTGAAAAAGATGAGTATGCCTGCCGTGTG

AACCATGTGACTTTGTCACAGCCCAAGATAGTTAAGTGGGATCGAGACATG

TAA

An exemplary HLA-bGBE (Single Chain Trimer) protein of the disclosure comprises or consists of the amino acid sequence of (B2M Signal peptide, peptide, Linker, B2M domain, Linker, HLA-E peptide):

(SEQ ID NO: 17064)

MSRSVALAVLALLSLSGLEA
VMAPRTLIL
GGGGSGGGGS

custom-character

GGGGSGGGGSGGGGSGGGGSGSHSLKYFHT

SVSRPGRGEPRFISVGYVDDTQFVREDNDAASPRMVPRAPWMEQEGSEY

WDRETRSARDTAQIFRVNLRTLRGYYNQSEAGSHTLQWMHGCELGPDGR

FLRGYEQFAYDGKDYLTLNEDLRSWTAVDTAAQISEQKSNDASEAEHQR

AYLEDTCVEWLHKYLEKGKETLLHLEPPKTHVTHHPISDHEATLRCWAL

GFYPAEITLTWQQDGEGHTQDTELVETRPAGDGTFQKWAAVVVPSGEEQV

RYTCHVQHEGLPEPVTLRWKPASQPTIPTVGIIAGLVLLGSVVSGAVVA

AVIWRKKSSGGKGGSYSKAEWSDSAQGSESHSL

B2M Signal Peptide

(SEQ ID NO: 17126)

MSRSVALAVLALLSLSGLEA

Peptide:

(SEQ ID NO: 17127)

VMAPRTLIL

Linker:

(SEQ ID NO: 17128)

GGGGSGGGGSGGGGS

B2M Domain:

(SEQ ID NO: 17129)

IQRTPKIQVYSRHPAENGKSNFLNCYVSGFHPSDIEVDLLKNGERIEKVE

HSDLSFSKDWSFYLLYYTEFTPTEKDEYACRVNHVTLSQPKIVKWDRDM

Linker:

(SEQ ID NO: 17130)

GGGGSGGGGSGGGGSGGGGS

HLA-E Peptide:

(SEQ ID NO: 17131)

GSHSLKYFHTSVSRPGRGEPRFISVGYVDDTQFVRFDNDAASPRMVPRAP

WMEQEGSEYWDRETRSARDTAQIFRVNLRTLRGYYNQSEAGSHTLQWMHG

CELGPDGRFLRGYEQFAYDGKDYLTLNEDLRSWTAVDTAAQISEQKSNDA

SEAEHQRAYLEDTCVEWLHKYLEKGKETLLHLEPPKTHVTHHPISDHEAT

LRCWALGFYPAEITLTWQQDGEGHTQDTELVETRPAGDGTFQKWAAVVVP

SGEEQRYTCHVQHEGLPEPVTLRWKPASQPTIPIVGIIAGLVLLGSVVSG

AVVAAVIWRKKSSGGKGGSYSKAEWSDSAQGSESHSL

An exemplary nucleotide sequence encoding a HLA-bGBE (Single Chain Trimer) protein of the disclosure comprises or consists of the nucleotide sequence of (B2M Signal peptide, peptide, Linker, B2M domain, Linker, HLA-E peptide):

(SEQ ID NO: 17065)

ATGTCTCGCAGCGTGGCCCTGGCCGTGCTGGCCCTGCTGTCCCTGTCTGG

CCTGGAGGCC
GTGATGGCCCCCCGGACCCTGATCCTG
GGAGGAGGAGGCA

GCCCCGGAGGAGGCTCCGGAGGCGGCGGCTCT

custom-character

GGCGGCGGCGGCTCTGGCGGCGGCGGCAGCGGCG

GCGGCGGCTCCGGAGGAGGCGGCTCTGGCAGCCACTCCCTGAAGTATTTC

CACACCTCTGTGAGCCGGCCAGGCAGAGGAGAGCCACGGTTCATCTCTGT

GGGCTACGTGGACGATACACAGTTCGTGAGGTTTGACAATGATGCCGCCA

GCCCAAGAATGGTGCCTAGGGCCCCATGGATGGAGCAGGAGGGCAGCGAG

TATTGGGACAGGGAGACCCGGAGCGCCAGAGACACAGCACAGATTTTCCG

GGTGAACCTGAGAACCCTGAGGGGOTACTATAATCAGTCCGAGGCCGGCT

CTCACACACTCCAGTGGATGCACGGATGCGAGCTGGGACCAGATGGCCGC

TTCCTGCGGGGCTACGAGCAGTTTGCCTATGACGGCAAGGATTACCTGAC

CCTGAACGAGGACCTGAGATOCTGGACCGCCGTGGATACAGCCGCCCAGA

TCAGCGAGCAGAAGTCCAATGACGCATCTGAGGCAGAGCACCAGAGGGCA

TATCTGGAGGATACCTGCGTGGAGTGGCTGCACAAGTACCTGGAGAAGGG

CAAGGAGACACTGOTGCACCTGGAGCCCCCTAAGACCCACGTGACACACC

ACCCAATCAGCGACCACGAGGCCACCCTGAGGTGTTGGGCACTGGGCTTC

TATCCCGCCGAGATCACCCTGACATGGCAGCAGGACGGAGAGGGACACAC

CCAGGATACAGAGCTGGTGGAGACCAGGCCCGCCGGCGATGGCACATTTC

AGAAGTGGGCCGCCGTGGTGGTGCCTTCCGGAGAGGAGCAGAGATACACC

TGTCACGTGCAGCACGAGGGACTGOCAGAGCCAGTGACCCTGAGGTGGAA

GCCTGCCAGCCAGCCCACAATCCCTATCGTGGGAATCATCGCAGGCCTGG

TGCTGCTGGGCTCTGTGGTGAGCGGAGCAGTGGTGGCCGCCGTGATCTGG

CGGAAGAAGAGCAGCGGAGGCAAGGGAGGCTCCTACTCCAAGGCAGAGTG

GAGCGACTCCGCCCAGGGCTCTGAGAGCCACTCCCTGTGA

B2M Signal Peptide:

(SEQ ID NO: 17132)

ATGTCTCGCAGCGTGGCCCTGGCCGTGCTGGCCCTGCTGTCCCTGTCTGG

CCTGGAGGCC

Peptide:

(SEQ ID NO: 17133)

GTGATGGCCCCCCGGACCCTGATCCTG

Linker:

(SEQ ID NO: 17134)

GGAGGAGGAGGCAGCGGCGGAGGAGGCTCCGGAGGCGGCGGCTCT

B2M Domain:

(SEQ ID NO: 17135)

ATCCAGCGCACACCTAAGATCCAGGTGTATTCTCGGCACCCAGCCGAGAA

CGGCAAGAGCAACTTCCTGAATTGCTACGTGAGCGCCTTTCACCCTTCCG

ACATCGAGGTGGATCTGCTGAAGAATGGCGAGAGAATCGAGAAGGTGGAG

CACTCCGACCTCAGCTTCTCCAAGGATTCGTCTTTTTATCTGCTGTACTA

TACCGAGTTTACCCCTACAGAGAAGGACGAGTACGCCTGTCGCGTGAACC

ACGTGACACTGTCCCAGCCAAAGATCGTGAAGTGGGACCGGGATATG

Linker:

(SEQ ID NO: 17136)

GGCGGCGGCGGCTCTGGCGGCGGCGGCAGCGGCGGCGGCGGCTCCGGAGG

AGGCGGCTCT

HLA-A Peptide:

(SEQ ID NO: 17137)

GGCAGCCACTCCCTGAAGTATTTCCACACCTCTGTGAGCCGGCCAGGCAG

AGGAGAGCCACGGTTCATCTCTGTGGGCTACGTGGACGATACACAGTTCG

TGAGGTTTGACAATGATGCCGCCAGCCCAAGAATGGTGCCTAGGGCCCCA

TGGATGGAGCAGGAGGGCAGCGAGTATTGGGACAGGGAGACCCGGAGCGC

CAGAGACACAGCACAGATTTTCCGGGTGAACCTGAGAACCCTGAGGGGCT

ACTATAATCAGTCCGAGGCCGGCTCTCACACACTCCAGTGGATGCACGGA

TGCGAGCTGGGACCAGATGGCCGCTTCCTGCGGGGCTACGAGCAGTTTGC

CTATGACGGCAAGGATTACCTGACCCTGAACGAGGACCTGAGATCCTGGA

CCGCCGTGGATACAGCCGCCCAGATCAGCGAGCAGAAGTCCAATGACGCA

TCTGAGGCAGAGCACCAGAGGGCATATCTGGAGGATACCTGCGTGGAGTG

GCTGCACAAGTACCTGGAGAAGGGCAAGGAGACACTGCTGCACCTGGAGC

CCCCTAAGACCCACGTGACACACCACCCAATCAGCGACCACGAGGCCACC

CTGAGGTGTTGGGCACTGGGCTTCTATCCCGCCGAGATCACCCTGACATG

GCAGCAGGACGGAGAGGGACACACCCAGGATACAGAGCTGGTGGAGACCA

GGCCCGCCGGCGATGGCACATTTCAGAAGTGGGCCGCCGTGGTGGTGCCT

TCCGGAGAGGAGCAGAGATACACCTGTCACGTGCAGCACGAGGGACTGCC

AGAGCCAGTGACCCTGAGGTGGAAGCCTGCCAGCCAGCCCACAATCCCTA

TCGTGGGAATCATCGCAGGCCTGGTGCTGCTGGGCTCTGTGGTGAGCGGA

GCAGTGGTGGCCGCCGTGATCTGGCGGAAGAAGAGCAGCGGAGGCAAGGG

AGGCTCCTACTCCAAGGCAGAGTGGAGCGACTCCGCCCAGGGCTCTGAGA

GCCACTCCCTGTGA

An exemplary HLA-gBE (Single Chain Dimer) protein of the disclosure comprises or consists of the amino acid sequence of (B2M Signal peptide, B2M domain, Linker, HLA-E peptide):

(SEQ ID NO: 17066)

MSRSVALAVLALLSLSGLEA
IQRTPKIQVYSRHPAENGKSNFLNCYVSGF

HPSDIEVDLLKNGERIEKVEHSDLSFSKDWSFYLLYYTEFTPTEKDEYAC

RVNHVTLSQPKIVKWDRDM
GGGGSGGGGSGGGGSGGGGSGSHSLKYFHTS

VSRTGRGEPRFISVGYVDDTQFVREDNDAASPRMVPRAPWMEQEGSEYWD

RETRSARDTAQIFRVNLRTLRGYYNQSEAGSHTLQWMHGCELGPDRRFLR

GYEQFAYDGKDYLTLNEDLRSWTAVDTAAQISEQKSNDASEAEHQRAYLE

DTCVEWLHKYLEKGKETLLHLEPPKTHVTHHPISDHEATLRCWALGFYPA

EITLTWQQDGEGHTQDTELVETRPAGDGTFQKWAAVVVPSGEEQRYTCHV

QHEGLPEPVTLRWKPASQPTIPIVGIIAGLVLLGSVVSGAVVAAVIWRKK

SSGGKGGSYYKAEWSDSAQGSESHSL

B2M Signal Peptide

(SEQ ID NO: 17126)

MSRSVALAVLALLSLSGLEA

B2M Domain:

(SEQ ID NO: 17129)

IQRTPKIQVYSRHPAENGKSNFLNCYVSGFHPSDIEVDLLKNGERIEKVE

HSDLSFSKDWSFYLLYYTEFTPTEKDEYACRVNHVTLSQPKIVKWDRDM

Linker:

(SEQ ID NO: 17130)

GGGGSGGGGSGGGGSGGGGS

HLA-E Peptide:

(SEQ ID NO: 17131)

GSHSLKYFHTSVSRPGRGEPRFISVGYVDDTQFVRFDNDAASPRMVPRAP

WMEQEGSEYWDRETRSARDTAQIFRVNLRTLRGYYNQSEAGSHTLQWMHG

CELGPDRRFLRGYEQFAYDGKDYLTLNEDLRSWTAVDTAAQISEQNSNDA

SEAEHQRAYLEDTCVEWLHKYLEKGKETLLHLEPPKTHVTHHPISDHEAT

LRCWALGFYPAEITLTWQQDGEGHTQDTELVETRPAGDGTFQKWAAVVVP

SGEEQRYTCHVQHEGLPEPVTLRWKPASQPTIPIVGIIAGLVLLGSVVSG

AVVAAVIWRKKSSGGKGGSYYKAEWSDSAQGSESHSL

An exemplary nucleotide sequence encoding a HLA-gBE (Single Chain Dimer) protein of the disclosure comprises or consists of the nucleotide sequence of (B2M Signal peptide, B2M domain, Linker, HLA-E peptide):

(SEQ ID NO: 17067)

ATGAGCAGATCTGTGGCCCTGGCTGTTCTGGCTCTGCTGTCTCTGTCTGG

CCTGGAAGCC
ATCCAGCGGACCCCTAAGATCCAGGTGTACAGCAGACACC

CCGCCGAGAACGGCAAGAGCAACTTCCTGAACTGCTACGTGTCCGGCTTT

CACCCCAGCGACATTGAGGTGGACCTGCTGAAGAACGGCGAGCGGATCGA

GAAGGTGGAACACAGCGATCTGAGCTTCAGCAAGGACTGGTCCTTCTACC

TGCTGTACTACACCGAGTTCACCCCTACCGAGAAGGACGAGTACGCCTGC

AGAGTGAACCACGTGACACTGAGCCAGCCTAAGATCGTGAAGTGGGACAG

AGATATG
GGCGGAGGCGGATCTGGTGGCGGAGGAAGTGGCGGCGGAGGAT

CTGGCGGTGGTGGTTCTGGATCTCACAGCCTGAAGTACTTTCACACCTCC

GTGTCCAGACCTGGCAGAGGCGAGCCTAGATTCATCAGCGTGGGCTACGT

GGACGACACCCAGTTCGTCAGATTCGACAACGACGCCGCCTCTCCTCGGA

TGGTTCCTAGAGCACCCTGGATGGAACAAGAGGGCAGCGAGTACTGGGAT

CGCGAGACAAGAAGCGCCAGAGACACAGCCCAGATCTTCCGCGTGAACCT

GAGAACCCTGCGGGGCTACTACAATCAGTCTGAGGCCGGCTCTCACACCC

TGCAGTGGATGCATGGATGTGAACTGGGCCCCGACAGACGGTTCCTGAGA

GGCTATGAGCAGTTCGCCTACGACGGCAAGGACTACCTGACACTGAACGA

GGACCTGAGAAGCTGGACCGCCGTGGATACAGCCGCTCAGATCAGCGAGC

AGAAGTCTAACGACGCCAGCGAGGCCGAACACCAGAGAGCCTATCTGGAA

GATACCTGCGTGGAATGGCTGCACAAGTACCTGGAAAAGGGCAAAGAGAC

ACTGCTGCACCTGGAACCTCCAAAGACACATGTGACCCACCATCCTATCA

GCGACCACGAGGCCACACTGAGATGTTGGGCCCTGGGCTTTTACCCTGCC

GAGATCACACTGACATGGCAGCAGGATGGCGAGGGCCACACACAGGATAC

AGAGCTGGTGGAAACAAGACCTGCCGGCGACGGCACCTTCCAGAAATGGG

CTGCTGTGGTTGTGCCCAGCGGCGAGGAACAGAGATACACCTGTCACGTG

CAGCACGAGGGACTGCCTGAACCTGTGACTCTGAGATGGAAGCCTGCCAG

CCAGCCAACAATCCCCATCGTGGGAATCATTGCCGGCCTGGTGCTGCTGG

GATCTGTGGTTTCTGGTGCTGTGGTGGCCGCCGTGATTTGGAGAAAGAAG

TCCTCTGGCGGCAAAGGCGGCTCCTACTATAAGGCCGAGTGGAGCGATTC

TGCCCAGGGCTCTGAAAGCCACAGCCTGTGA

B2M Signal Peptide:

(SEQ ID NO: 17132)

ATGAGCAGATCTGTGGCCCTGGCTGTTCTGGCTCTGCTGTCTCTGTCTGG

CCTGGAAGCC

B2M Domain:

(SEQ ID NO: 17135)

ATCCAGCGGACCCCTAAGATCCAGGTGTACAGCAGACACCCCGCCGAGAA

CGGCAAGAGCAACTTCCTGAACTGCTACGTGTCCGGCTTTCACCCCAGCG

ACATTGAGGTGGACCTGCTGAAGAACGGCGAGCGGATCGAGAAGGTGGAA

CACAGCGATCTGAGCTTCAGCAAGGACTGGTCCTTCTACCTGCTGTACTA

CACCGAGTTCACCCCTACCGAGAAGGACGAGTACGCCTGCAGAGTGAACC

ACGTGACACTGAGCCAGCCTAAGATCGTGAAGTGGGACAGAGATATG

Linker:

(SEQ ID NO: 17136)

GGCGGAGGCGGATCTGGTGGCGGAGGAAGTGGCGGCGGAGGATCTGGCGG

TGGTGGTTCT

HLA-E Peptide:

(SEQ ID NO: 17137)

GGATCTCACAGCCTGAAGTACTTTCACACCTCCGTGTCCAGACCTGGCAG

AGGCGAGCCTAGATTCATCAGCGTGGGCTACGTGGACGACACCCAGTTCG

TCAGATTCGACAACGACGCCGCCTCTCCTCGGATGGTTCCTAGAGCACCC

TGGATGGAACAAGAGGGCAGCGAGTACTGGGATCGCGAGACAAGAAGCGC

CAGAGACACAGCCCAGATCTTCCGCGTGAACCTGAGAACCCTGCGGGGCT

ACTACAATCAGTCTGAGGCCGGCTCTCACACCCTGCAGTGGATGCATGGA

TGTGAACTGGGCCCCGACAGACGGTTCCTGAGAGGCTATGAGCAGTTCGC

CTACGACGGCAAGGACTACCTGACACTGAACGAGGACCTGAGAAGCTGGA

CCGCCGTGGATACAGCCGCTCAGATCAGCGAGCAGAAGTCTAACGACGCC

AGCGAGGCCGAACACCAGAGAGCCTATCTGGAAGATACCTGCGTGGAATG

GCTGCACAAGTACCTGGAAAAGGGCAAAGAGACACTGCTGCACCTGGAAC

CTCCAAAGACACATGTGACCCACCATCCTATCAGCGACCACGAGGCCACA

CTGAGATGTTGGGCCCTGGGCTTTTACCCTGCCGAGATCACACTGACATG

GCAGCAGGATGGCGAGGGCCACACACAGGATACAGAGCTGGTGGAAACAA

GACCTGCCGGCGACGGCACCTTCCAGAAATGGGCTGCTGTGGTTGTGCCC

AGCGGCGAGGAACAGAGATACACCTGTCACGTGCAGCACGAGGGACTGCC

TGAACCTGTGACTCTGAGATGGAAGCCTGCCAGCCAGCCAACAATCCCCA

TCGTGGGAATCATTGCCGGCCTGGTGCTGCTGGGATCTGTGGTTTCTGGT

GCTGTGGTGGCCGCCGTGATTTGGAGAAAGAAGTCCTCTGGCGGCAAAGG

CGGCTCCTACTATAAGGCCGAGTGGAGCGATTCTGCCCAGGGCTCTGAAA

GCCACAGCCTGTGA

An exemplary HLA-bE (Monomer) protein of the disclosure comprises or consists of the amino acid sequence of (B2M Signal peptide, HLA-E peptide):

(SEQ ID NO: 17068)

MSRSVALAVLALLSLSGLEAGSHSLKYFHTSVSRPGRGEPRFISVGYVDD

TQFVRFDNDAASPRMVPRAPWMEQEGSEYWDRETRSARDTAQIFRVNLRT

LRGYYNQSEAGSHTLQWMHGCELGPDRRFLRGYEQFAYDGKDYLTLNEDL

RSWTAVDTAAQISEQKSNDASEAEHQRAYLEDTCVEWLHKYLEKGKETLL

HLEPPKTHVTHHPISDHEATLRCWALGFYPAEITLTWQQDGEGHTQDTEL

VETRPAGDGTFQKWAAVVVPSGEEQRYTCHVQHEGLPEPVTLRWKPASQP

TIPIVGIIAGLVLLGSWSGAWAAVIWRKKSSGGKGGSYYKAEWSDSAQGS

ESHSL

B2M Signal Peptide:

(SEQ ID NO: 17126)

MSRSVALAVLALLSLSGLEA

HLA-E Peptide:

(SEQ ID NO: 17131)

GSHSLKYFHTSVSRPGRGEPRFISVGYVDDTQFVRFDNDAASPRMVPRAP

WMEQEGSEYWDRETRSARDTAQIFRVNLRTLRGYYNQSEAGSHTLQWMHG

CELGPDRRFLRGYEQFAYDGKDYLTLNEDLRSWTAVDTAAQISEQKSNDA

SEAEHQRAYLEDTCVEWLHKYLEKGKETLLHLEPPKTHVTHHPISDHEAT

LRCWALGFYPAEITLTWQQDGEGHTQDTELVETRPAGDGTFQKWAAVVVP

SGEEQRYTCHVQHEGLPEPVTLRWKPASQPTIPIVGIIAGLVLLGSVVSG

AVVAAVIWRKKSSGGKGGSYYKAEWSDSAQGSESHSL

An exemplary nucleotide sequence encoding a HLA-bE (Monomer) protein of the disclosure comprises or consists of the nucleotide sequence of (B2M Signal peptide, HLA-E peptide):

(SEQ ID NO: 17069)

ATGTCTCGCAGCGTGGCCCTGGCCGTGCTGGCCCTGCTGTCCCTGTCTGG

CCTGGAGGCCGGCAGCCACTCCCTGAAGTATTTCCACACCTCTGTGAGCC

GGCCAGGCAGAGGAGAGCCACGGTTCATCTCTGTGGGCTACGTGGACGAT

ACACAGTTCGTGAGGTTTGACAATGATGCCGCCAGCCCAAGAATGGTGCC

TAGGGCCCCATGGATGGAGCAGGAGGGCAGCGAGTATTGGGACAGGGAGA

CCCGGAGCGCCAGAGACACAGCACAGATTTTCCGGGTGAACCTGAGAACC

CTGAGGGGCTACTATAATCAGTCCGAGGCCGGCTCTCACACACTCCAGTG

GATGCACGGATGCGAGCTGGGACCAGATCGCCGCTTCCTGCGGGGCTACG

AGCAGTTTGCCTATGACGGCAAGGATTACCTGACCCTGAACGAGGACCTG

AGATCCTGGACCGCCGTGGATACAGCCGCCCAGATCAGCGAGCAGAAGTC

CAATGACGCATCTGAGGCAGAGCACCAGAGGGCATATCTGGAGGATACCT

GCGTGGAGTGGCTGCACAAGTACCTGGAGAAGGGCAAGGAGACACTGCTG

CACCTGGAGCCCCCTAAGACCCACGTGACACACCACCCAATCAGCGACCA

CGAGGCCACCCTGAGGTGTTGGGCACTGGGCTTCTATCCCGCCGAGATCA

CCCTGACATGGCAGCAGGACGGAGAGGGACACACCCAGGATACAGAGCTG

GTGGAGACCAGGCCCGCCGGCGATGGCACATTTCAGAAGTGGGCCGCCGT

GGTGGTGCCTTCCGGAGAGGAGCAGAGATACACCTGTCACGTGCAGCACG

AGGGACTGCCAGAGCCAGTGACCCTGAGGTGGAAGCCTGCCAGCCAGCCC

ACAATCCCTATCGTGGGAATCATCGCAGGCCTGGTGCTGCTGGGCTCTGT

GGTGAGCGGAGCAGTGGTGGCCGCCGTGATCTGGCGGAAGAAGAGCAGCG

GAGGCAAGGGAGGCTCCTACTATAAGGCAGAGTGGAGCGACTCCGCCCAG

GGCTCTGA

B2M Signal Peptide:

(SEQ ID NO: 17132)

ATGTCTCGCAGCGTGGCCCTGGCCGTGCTGGCCCTGCTGTCCCTGTCTGG

CCTGGAGGCC

HLA-E Peptide:

(SEQ ID NO: 17137)

GGCAGCCACTCCCTGAAGTATTTCCACACCTCTGTGAGCCGGCCAGGCAG

AGGAGAGCCACGGTTCATCTCTGTGGGCTACGTGGACGATACACAGTTCG

TGAGGTTTGACAATGATGCCGCCAGCCCAAGAATGGTGCCTAGGGCCCCA

TGGATGGAGCAGGAGGGCAGCGAGTATTGGGACAGGGAGACCCGGAGCGC

CAGAGACACAGCACAGATTTTCCGGGTGAACCTGAGAACCCTGAGGGGCT

ACTATAATCAGTCCGAGGCCGGCTCTCACACACTCCAGTGGATGCACGGA

TGCGAGCTGGGACCAGATCGCCGCTTCCTGCGGGGCTACGAGCAGTTTGC

CTATGACGGCAAGGATTACCTGACCCTGAACGAGGACCTGAGATCCTGGA

CCGCCGTGGATACAGCCGCCCAGATCAGCGAGCAGAAGTCCAATGACGCA

TCTGAGGCAGAGCACCAGAGGGCATATCTGGAGGATACCTGCGTGGAGTG

GCTGCACAAGTACCTGGAGAAGGGCAAGGAGACACTGCTGCACCTGGAGC

CCCCTAAGACCCACGTGACACACCACCCAATCAGCGACCACGAGGCCACC

CTGAGGTGTTGGGCACTGGGCTTCTATCCCGCCGAGATCACCCTGACATG

GCAGCAGGACGGAGAGGGACACACCCAGGATACAGAGCTGGTGGAGACCA

GGCCCGCCGGCGATGGCACATTTCAGAAGTGGGCCGCCGTGGTGGTGCCT

TCCGGAGAGGAGCAGAGATACACCTGTCACGTGCAGCACGAGGGACTGCC

AGAGCCAGTGACCCTGAGGTGGAAGCCTGCCAGCCAGCCCACAATCCCTA

TCGTGGGAATCATCGCAGGCCTGGTGCTGCTGGGCTCTGTGGTGAGCGGA

GCAGTGGTGGCCGCCGTGATCTGGCGGAAGAAGAGCAGCGGAGGCAAGGG

AGGCTCCTACTATAAGGCAGAGTGGAGCGACTCCGCCCAGGGCTCTGA

Immune and Immune Precursor Cells

In certain embodiments, immune cells of the disclosure comprise lymphoid progenitor cells, natural killer (NK) cells, T lymphocytes (T-cell), stem memory T cells (T_SCMcells), central memory T cells (T_CM), stem cell-like T cells, B lymphocytes (B-cells), myeloid progenitor cells, neutrophils, basophils, eosinophils, monocytes, macrophages, platelets, erythrocytes, red blood cells (RBCs), megakaryocytes or osteoclasts.

In certain embodiments, immune precursor cells comprise any cells which can differentiate into one or more types of immune cells. In certain embodiments, immune precursor cells comprise multipotent stem cells that can self renew and develop into immune cells. In certain embodiments, immune precursor cells comprise hematopoietic stem cells (HSCs) or descendants thereof. In certain embodiments, immune precursor cells comprise precursor cells that can develop into immune cells. In certain embodiments, the immune precursor cells comprise hematopoietic progenitor cells (HPCs).

Hematopoietic Stem Cells (HSCs)

Hematopoietic stem cells (HSCs) are multipotent, self-renewing cells. All differentiated blood cells from the lymphoid and myeloid lineages arise from HSCs. HSCs can be found in adult bone marrow, peripheral blood, mobilized peripheral blood, peritoneal dialysis effluent and umbilical cord blood.

HSCs of the disclosure may be isolated or derived from a primary or cultured stem cell. HSCs of the disclosure may be isolated or derived from an embryonic stem cell, a multipotent stem cell, a pluripotent stem cell, an adult stem cell, or an induced pluripotent stem cell (iPSC).

Immune precursor cells of the disclosure may comprise an HSC or an HSC descendent cell. Exemplary HSC descendent cells of the disclosure include, but are not limited to, multipotent stem cells, lymphoid progenitor cells, natural killer (NK) cells, T lymphocyte cells (T-cells), B lymphocyte cells (B-cells), myeloid progenitor cells, neutrophils, basophils, eosinophils, monocytes, and macrophages.

HSCs produced by the methods of the disclosure may retain features of “primitive” stem cells that, while isolated or derived from an adult stem cell and while committed to a single lineage, share characteristics of embryonic stem cells. For example, the “primitive” HSCs produced by the methods of the disclosure retain their “sternness” following division and do not differentiate. Consequently, as an adoptive cell therapy, the “primitive” HSCs produced by the methods of the disclosure not only replenish their numbers, but expand in vivo. “Primitive” HSCs produced by the methods of the disclosure may be therapeutically-effective when administered as a single dose. In some embodiments, primitive HSCs of the disclosure are CD34+. In some embodiments, primitive HSCs of the disclosure are CD34+ and CD38−. In some embodiments, primitive HSCs of the disclosure are CD34+, CD38− and CD90+. In some embodiments, primitive HSCs of the disclosure are CD34+, CD38−, CD90+ and CD45RA−. In some embodiments, primitive HSCs of the disclosure are CD34+, CD38−, CD90+, CD45RA−, and CD49f+. In some embodiments, the most primitive HSCs of the disclosure are CD34+, CD38−, CD90+, CD45RA−, and CD49f+.

In some embodiments of the disclosure, primitive HSCs, HSCs, and/or HSC descendent cells may be modified according to the methods of the disclosure to express an exogenous sequence (e.g. a chimeric antigen receptor or therapeutic protein). In some embodiments of the disclosure, modified primitive HSCs, modified HSCs, and/or modified HSC descendent cells may be forward differentiated to produce a modified immune cell including, but not limited to, a modified T cell, a modified natural killer cell and/or a modified B-cell of the disclosure.

T Cells

Modified T cells of the disclosure may be derived from modified hematopoietic stem and progenitor cells (HSPCs) or modified HSCs.

Unlike traditional biologics and chemotherapeutics, modified-T cells of the disclosure possess the capacity to rapidly reproduce upon antigen recognition, thereby potentially obviating the need for repeat treatments. To achieve this, in some embodiments, modified-T cells of the disclosure not only drive an initial response, but also persist in the patient as a stable population of viable memory T cells to prevent potential relapses. Alternatively, in some embodiments, when it is not desired, modified-T cells of the disclosure do not persist in the patient.

Intensive efforts have been focused on the development of antigen receptor molecules that do not cause T cell exhaustion through antigen-independent (tonic) signaling, as well as of a modified-T cell product containing early memory T cells, especially stem cell memory (T_SCM) or stem cell-like T cells. Stem cell-like modified-T cells of the disclosure exhibit the greatest capacity for self-renewal and multipotent capacity to derive central memory (T_CM) T cells or T_CMlike cells, effector memory (T_EM) and effector T cells (T_E), thereby producing better tumor eradication and long-term modified-T cell engraftment. A linear pathway of differentiation may be responsible for generating these cells: Naïve T cells (T_N)>T_SCM>T_CM>T_EM>T_E>T_TE, whereby T_Nis the parent precursor cell that directly gives rise to T_SCM, which then, in turn, directly gives rise to T_CM, etc. Compositions of T cells of the disclosure may comprise one or more of each parental T cell subset with T_SCMcells being the most abundant (e.g. T_SCM>T_CM>T_EM>T_E>T_TE).

In some embodiments of the methods of the disclosure, the immune cell precursor is differentiated into or is capable of differentiating into an early memory T cell, a stem cell like T-cell, a Naïve T cells (T_N), a T_SCM, a T_CM, a T_EM, a T_E, or a T_TEIn some embodiments, the immune cell precursor is a primitive HSC, an HSC, or a HSC descendent cell of the disclosure.

In some embodiments of the methods of the disclosure, the immune cell is an early memory T cell, a stem cell like T-cell, a Naïve T cells (T_N), a T_SCM, a T_CM, a T_EM, a T_E, or a T_TE.

In some embodiments of the methods of the disclosure, the immune cell is an early memory T cell.

In some embodiments of the methods of the disclosure, the immune cell is a stem cell like T-cell.

In some embodiments of the methods of the disclosure, the immune cell is a T_SCM.

In some embodiments of the methods of the disclosure, the immune cell is a T_CM.

In some embodiments of the methods of the disclosure, the methods modify and/or the methods produce a plurality of modified T cells, wherein at least 2%, 5%, 10%, 15%, 20%, 25%, 309%, 35%, 40%, 45%, 50%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% or any percentage in between of the plurality of modified T cells expresses one or more cell-surface marker(s) of an early memory T cell. In certain embodiments, the plurality of modified early memory T cells comprises at least one modified stem cell-like T cell. In certain embodiments, the plurality of modified early memory T cells comprises at least one modified T_SCM. In certain embodiments, the plurality of modified early memory T cells comprises at least one modified T_CM.

In some embodiments of the methods of the disclosure, the methods modify and/or the methods produce a plurality of modified T cells, wherein at least 2%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% or any percentage in between of the plurality of modified T cells expresses one or more cell-surface marker(s) of a stem cell-like T cell. In certain embodiments, the plurality of modified stem cell-like T cells comprises at least one modified T_SCM. In certain embodiments, the plurality of modified stem cell-like T cells comprises at least one modified T_CM.

In some embodiments of the methods of the disclosure, the methods modify and/or the methods produce a plurality of modified T cells, wherein at least 2%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% or any percentage in between of the plurality of modified T cells expresses one or more cell-surface marker(s) of a stem memory T cell (T_SCM). In certain embodiments, the cell-surface markers comprise CD62L and CD45RA. In certain embodiments, the cell-surface markers comprise one or more of CD62L, CD45RA, CD28. CCR7, CD127, CD45RO, CD95, CD95 and IL-2Rβ. In certain embodiments, the cell-surface markers comprise one or more of CD45RA, CD95, IL-2Rβ, CCR7, and CD62L.

In some embodiments of the methods of the disclosure, the methods modify and/or the methods produce a plurality of modified T cells, wherein at least 2%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% or any percentage in between of the plurality of modified T cells expresses one or more cell-surface marker(s) of a central memory T cell (T_CM). In certain embodiments, the cell-surface markers comprise one or more of CD45RO, CD95, IL-2Rβ, CCR7, and CD62L.

In some embodiments of the methods of the disclosure, a buffer comprises the immune cell or precursor thereof. The buffer maintains or enhances a level of cell viability and/or a stem-like phenotype of the immune cell or precursor thereof, including T-cells. In certain embodiments, the buffer maintains or enhances a level of cell viability and/or a stem-like phenotype of the primary human T cells prior to the nucleofection. In certain embodiments, the buffer maintains or enhances a level of cell viability and/or a stem-like phenotype of the primary human T cells during the nucleofection. In certain embodiments, the buffer maintains or enhances a level of cell viability and/or a stem-like phenotype of the primary human T cells following the nucleofection. In certain embodiments, the buffer comprises one or more of KCl, MgCl₂, ClNa, Glucose and Ca(NO₃)₂in any absolute or relative abundance or concentration, and, optionally, the buffer further comprises a supplement selected from the group consisting of HEPES, Tris/HCl, and a phosphate buffer. In certain embodiments, the buffer comprises 5 mM KCl, 15 mM MgCl₂, 90 mM ClNa, 10 mM Glucose and 0.4 mM Ca(NO₃)₂. In certain embodiments, the buffer comprises 5 mM KCl, 15 mM MgCl₂, 90 mM ClNa, 10 mM Glucose and 0.4 mM Ca(NO₃)₂and a supplement comprising 20 mM HEPES and 75 mM Tris/ICI. In certain embodiments, the buffer comprises 5 mM KCl, 15 mM MgCl₂, 90 mM ClNa, 10 mM Glucose and 0.4 mM Ca(NO₃)₂and a supplement comprising 40 mM Na₂HPO₄/NaH₂PO₄at pH 7.2. In certain embodiments, the composition comprising primary human T cells comprises 100 μl of the buffer and between 5×10⁶and 25×10⁶cells. In certain embodiments, the composition comprises a scalable ratio of 250×10⁶primary human T cells per milliliter of buffer or other media during the introduction step.

In some embodiments of the methods of the disclosure, the methods comprise contacting an immune cell of the disclosure, including a T cell of the disclosure, and a T-cell expansion composition. In some embodiments of the methods of the disclosure, the step of introducing a transposon and/or transposase of the disclosure into an immune cell of the disclosure may further comprise contacting the immune cell and a T-cell expansion composition. In some embodiments, including those in which the introducing step of the methods comprises an electroporation or a nucleofection step, the electroporation or a nucleofection step may be performed with the immune cell contacting T-cell expansion composition of the disclosure.

In some embodiments of the methods of the disclosure, the T-cell expansion composition comprises, consists essentially of or consists of phosphorus; one or more of an octanoic acid, a palmitic acid, a linoleic acid, and an oleic acid; a sterol; and an alkane.

In certain embodiments of the methods of producing a modified T cell of the disclosure, the expansion supplement comprises one or more cytokine(s). The one or more cytokine(s) may comprise any cytokine, including but not limited to, lymphokines. Exemplary lympokines include, but are not limited to, interleukin-2 (IL-2), interleukin-3 (IL-3), interleukin-4 (IL-4), interleukin-5 (IL-5), interleukin-6 (IL-6), interleukin-7 (IL-7), interleukin-15 (IL-15), interleukin-21 (IL-21), granulocyte-macrophage colony-stimulating factor (GM-CSF) and interferon-gamma (INFγ). The one or more cytokine(s) may comprise IL-2.

In some embodiments of the methods of the disclosure, the T-cell expansion composition comprises human serum albumin, recombinant human insulin, human transferrin, 2-Mercaptoethanol, and an expansion supplement. In certain embodiments of this method, the T-cell expansion composition further comprises one or more of octanoic acid, nicotinamide, 2,4,7,9-tetramethyl-5-decyn-4,7-diol (TMDD), diisopropyl adipate (DIPA), n-butyl-benzenesulfonamide, 1,2-benzenedicarboxylic acid, bis(2-methylpropyl) ester, palmitic acid, linoleic acid, oleic acid, stearic acid hydrazide, oleamide, a sterol and an alkane. In certain embodiments of this method, the T-cell expansion composition further comprises one or more of octanoic acid, palmitic acid, linoleic acid, oleic acid and a sterol. In certain embodiments of this method, the T-cell expansion composition further comprises one or more of octanoic acid at a concentration of between 0.9 mg/kg to 90 mg/kg, inclusive of the endpoints; palmitic acid at a concentration of between 0.2 mg/kg to 20 mg/kg, inclusive of the endpoints; linoleic acid at a concentration of between 0.2 mg/kg to 20 mg/kg, inclusive of the endpoints; oleic acid at a concentration of 0.2 mg/kg to 20 mg/kg, inclusive of the endpoints; and a sterol at a concentration of about 0.1 mg/kg to 10 mg/kg, inclusive of the endpoints. In certain embodiments of this method, the T-cell expansion composition further comprises one or more of octanoic acid at a concentration of about 9 mg/kg, palmitic acid at a concentration of about 2 mg/kg, linoleic acid at a concentration of about 2 mg/kg, oleic acid at a concentration of about 2 mg/kg and a sterol at a concentration of about 1 mg/kg. In certain embodiments of this method, the T-cell expansion composition further comprises one or more of octanoic acid at a concentration of between 6.4 μmol/kg and 640 μmol/kg, inclusive of the endpoints; palmitic acid at a concentration of between 0.7 μmol/kg and 70 μmol/kg, inclusive of the endpoints; linoleic acid at a concentration of between 0.75 μmol/kg and 75 μmol/kg, inclusive of the endpoints; oleic acid at a concentration of between 0.75 μmol/kg and 75 μmol/kg, inclusive of the endpoints; and a sterol at a concentration of between 0.25 μmol/kg and 25 μmol/kg, inclusive of the endpoints. In certain embodiments of this method, the T-cell expansion composition further comprises one or more of octanoic acid at a concentration of about 64 μmol/kg, palmitic acid at a concentration of about 7 μmol/kg, linoleic acid at a concentration of about 7.5 μmol/kg, oleic acid at a concentration of about 7.5 μmol/kg and a sterol at a concentration of about 2.5 μmol/kg.

In certain embodiments, the T-cell expansion composition comprises one or more of human serum albumin, recombinant human insulin, human transferrin, 2-Mercaptoethanol, and an expansion supplement to produce a plurality of expanded modified T-cells, wherein at least 2% of the plurality of modified T-cells expresses one or more cell-surface marker(s) of an early memory T cell, a stem cell-like T cell, a stem memory T cell (T_SCM) and/or a central memory T cell (T_CM). In certain embodiments, the T-cell expansion composition comprises or further comprises one or more of octanoic acid, nicotinamide, 2,4,7,9-tetramethyl-5-decyn-4,7-diol (TMDD), diisopropyl adipate (DIPA), n-butyl-benzenesulfonamide, 1,2-benzenedicarboxylic acid, bis(2-methylpropyl) ester, palmitic acid, linoleic acid, oleic acid, stearic acid hydrazide, oleamide, a sterol and an alkane. In certain embodiments, the T-cell expansion composition comprises one or more of octanoic acid, palmitic acid, linoleic acid, oleic acid and a sterol (e.g. cholesterol). In certain embodiments, the T-cell expansion composition comprises one or more of octanoic acid at a concentration of between 0.9 mg/kg to 90 mg/kg, inclusive of the endpoints; palmitic acid at a concentration of between 0.2 mg/kg to 20 mg/kg, inclusive of the endpoints; linoleic acid at a concentration of between 0.2 mg/kg to 20 mg/kg, inclusive of the endpoints; oleic acid at a concentration of 0.2 mg/kg to 20 mg/kg, inclusive of the endpoints; and a sterol at a concentration of about 0.1 mg/kg to 10 mg/kg, inclusive of the endpoints (wherein mg/kg=parts per million). In certain embodiments, the T-cell expansion composition comprises one or more of octanoic acid at a concentration of about 9 mg/kg, palmitic acid at a concentration of about 2 mg/kg, linoleic acid at a concentration of about 2 mg/kg, oleic acid at a concentration of about 2 mg/kg, and a sterol at a concentration of about 1 mg/kg (wherein mg/kg=parts per million). In certain embodiments, the T-cell expansion composition comprises one or more of octanoic acid at a concentration of 9.19 mg/kg, palmitic acid at a concentration of 1.86 mg/kg, linoleic acid at a concentration of about 2.12 mg/kg, oleic acid at a concentration of about 2.13 mg/kg, and a sterol at a concentration of about 1.01 mg/kg (wherein mg/kg=parts per million). In certain embodiments, the T-cell expansion composition comprises octanoic acid at a concentration of 9.19 mg/kg, palmitic acid at a concentration of 1.86 mg/kg, linoleic acid at a concentration of 2.12 mg/kg, oleic acid at a concentration of about 2.13 mg/kg, and a sterol at a concentration of 1.01 mg/kg (wherein mg/kg=parts per million). In certain embodiments, the T-cell expansion composition comprises one or more of octanoic acid at a concentration of between 6.4 μmol/kg and 640 μmol/kg, inclusive of the endpoints; palmitic acid at a concentration of between 0.7 μmol/kg and 70 μmol/kg, inclusive of the endpoints; linoleic acid at a concentration of between 0.75 μmol/kg and 75 μmol/kg, inclusive of the endpoints; oleic acid at a concentration of between 0.75 μmol/kg and 75 μmol/kg, inclusive of the endpoints; and a sterol at a concentration of between 0.25 μmol/kg and 25 μmol/kg, inclusive of the endpoints. In certain embodiments, the T-cell expansion composition comprises one or more of octanoic acid at a concentration of about 64 μmol/kg, palmitic acid at a concentration of about 7 μmol/kg, linoleic acid at a concentration of about 7.5 μmol/kg, oleic acid at a concentration of about 7.5 μmol/kg and a sterol at a concentration of about 2.5 μmol/kg. In certain embodiments, the T-cell expansion composition comprises one or more of octanoic acid at a concentration of about 63.75 μmol/kg, palmitic acid at a concentration of about 7.27 μmol/kg, linoleic acid at a concentration of about 7.57 μmol/kg, oleic acid at a concentration of about 7.56 μmol/kg and a sterol at a concentration of about 2.61 μmol/kg. In certain embodiments, the T-cell expansion composition comprises octanoic acid at a concentration of about 63.75 μmol/kg, palmitic acid at a concentration of about 7.27 μmol/kg, linoleic acid at a concentration of about 7.57 μmol/kg, oleic acid at a concentration of 7.56 μmol/kg and a sterol at a concentration of 2.61 μmol/kg.

As used herein, the terms “supplemented T-cell expansion composition” or “T-cell expansion composition” may be used interchangeably with a media comprising one or more of human serum albumin, recombinant human insulin, human transferrin, 2-Mercaptoethanol, and an expansion supplement at 37° C. Alternatively, or in addition, the terms “supplemented T-cell expansion composition” or “T-cell expansion composition” may be used interchangeably with a media comprising one or more of the following components: octanoic acid (CAS No. 124-07-2), nicotinamide (CAS No. 98-92-0), 2,4,7,9-tetramethyl-5-decyn-4,7-diol (TMDD) (CAS No. 126-86-3), diisopropyl adipate (DIPA) (CAS No. 6938-94-9), n-butyl-benzenesulfonamide (CAS No. 3622-84-2), 1,2-benzenedicarboxylic acid, bis(2-methylpropyl) ester (CAS No. 84-69-5), palmitic acid (CAS No. 57-10-3), linoleic acid (CAS No. 60-33-3), oleic acid (CAS No. 112-80-1), stearic acid hydrazide (CAS No. 4130-54-5), oleamide (CAS No. 3322-62-1), sterol (e.g., cholesterol) (CAS No. 57-88-5), and alkanes (e.g., nonadecane) (CAS No. 629-92-5). In certain embodiments, the terms “supplemented T-cell expansion composition” or “T-cell expansion composition” may be used interchangeably with a media comprising one or more of the following components: octanoic acid (CAS No. 124-07-2), nicotinamide (CAS No. 98-92-0), 2,4,7,9-tetramethyl-5-decyn-4,7-diol (TMDD) (CAS No. 126-86-3), diisopropyl adipate (DIPA) (CAS No. 6938-94-9), n-butyl-benzenesulfonamide (CAS No. 3622-84-2), 1,2-benzenedicarboxylic acid, bis(2-methylpropyl) ester (CAS No. 84-69-5), palmitic acid (CAS No. 57-10-3), linoleic acid (CAS No. 60-33-3), oleic acid (CAS No. 112-80-1), stearic acid hydrazide (CAS No. 4130-54-5), oleamide (CAS No. 3322-62-1), sterol (e.g., cholesterol) (CAS No. 57-88-5), alkanes (e.g., nonadecane) (CAS No. 629-92-5), and phenol red (CAS No. 143-74-8). In certain embodiments, the terms “supplemented T-cell expansion composition” or “T-cell expansion composition” may be used interchangeably with a media comprising one or more of the following components: octanoic acid (CAS No. 124-07-2), nicotinamide (CAS No. 98-92-0), 2,4,7,9-tetramethyl-5-decyn-4,7-diol (TMDD) (CAS No. 126-86-3), diisopropyl adipate (DIPA) (CAS No. 6938-94-9), n-butyl-benzenesulfonamide (CAS No. 3622-84-2), 1,2-benzenedicarboxylic acid, bis(2-methylpropyl) ester (CAS No. 84-69-5), palmitic acid (CAS No. 57-10-3), linoleic acid (CAS No. 60-33-3), oleic acid (CAS No. 112-80-1), stearic acid hydrazide (CAS No. 4130-54-5), oleamide (CAS No. 3322-62-1), phenol red (CAS No. 143-74-8) and lanolin alcohol.

In certain embodiments, the terms “supplemented T-cell expansion composition” or “T-cell expansion composition” may be used interchangeably with a media comprising one or more of human serum albumin, recombinant human insulin, human transferrin, 2-Mercaptoethanol, and an expansion supplement at 37° C. Alternatively, or in addition, the terms “supplemented T-cell expansion composition” or “T-cell expansion composition” may be used interchangeably with a media comprising one or more of the following ions: sodium, ammonium, potassium, magnesium, calcium, chloride, sulfate and phosphate.

As used herein, the terms “supplemented T-cell expansion composition” or “T-cell expansion composition” may be used interchangeably with a media comprising one or more of human serum albumin, recombinant human insulin, human transferrin, 2-Mercaptoethanol, and an expansion supplement at 37° C. Alternatively, or in addition, the terms “supplemented T-cell expansion composition” or “T-cell expansion composition” may be used interchangeably with a media comprising one or more of the following free amino acids: histidine, asparagine, serine, glutamate, arginine, glycine, aspartic acid, glutamic acid, threonine, alanine, proline, cysteine, lysine, tyrosine, methionine, valine, isoleucine, leucine, phenylalanine and tryptophan. In certain embodiments, the terms “supplemented T-cell expansion composition” or “T-cell expansion composition” may be used interchangeably with a media comprising one or more of the following free amino acids in the corresponding average mole percentages: histidine (about 1%), asparagine (about 0.5%), serine (about 1.5%), glutamine (about 67%), arginine (about 1.5%), glycine (about 1.5%), aspartic acid (about 1%), glutamic acid (about 2%), threonine (about 2%), alanine (about 1%), proline (about 1.5%), cysteine (about 1.5%), lysine (about 3%), tyrosine (about 1.5%), methionine (about 1%), valine (about 3.5%), isoleucine (about 3%), leucine (about 3.5%), phenylalanine (about 1.5%) and tryptophan (about 0.5%). In certain embodiments, the terms “supplemented T-cell expansion composition” or “T-cell expansion composition” may be used interchangeably with a media comprising one or more of the following free amino acids in the corresponding average mole percentages: histidine (about 0.78%), asparagine (about 0.4%), serine (about 1.6%), glutamine (about 67.01%), arginine (about 1.67%), glycine (about 1.72%), aspartic acid (about 1.00%), glutamic acid (about 1.93%), threonine (about 2.38%), alanine (about 1.11%), proline (about 1.49%), cysteine (about 1.65%), lysine (about 2.84%), tyrosine (about 1.62%), methionine (about 0.85%), valine (about 3.45%), isoleucine (about 3.14%), leucine (about 3.3%), phenylalanine (about 1.64%) and tryptophan (about 0.37%).

In certain embodiments, the terms “supplemented T-cell expansion composition” or “T-cell expansion composition” may be used interchangeably with a media comprising one or more of octanoic acid, palmitic acid, linoleic acid, oleic acid and a sterol (e.g. cholesterol). In certain embodiments, the terms “supplemented T-cell expansion composition” or “T-cell expansion composition” may be used interchangeably with a media comprising one or more of octanoic acid at a concentration of between 0.9 mg/kg to 90 mg/kg, inclusive of the endpoints; palmitic acid at a concentration of between 0.2 mg/kg to 20 mg/kg, inclusive of the endpoints; linoleic acid at a concentration of between 0.2 mg/kg to 20 mg/kg, inclusive of the endpoints; oleic acid at a concentration of 0.2 mg/kg to 20 mg/kg, inclusive of the endpoints; and a sterol at a concentration of about 0.1 mg/kg to 10 mg/kg, inclusive of the endpoints (wherein mg/kg=parts per million). In certain embodiments, the terms “supplemented T-cell expansion composition” or “T-cell expansion composition” may be used interchangeably with a media comprising one or more of octanoic acid at a concentration of about 9 mg/kg, palmitic acid at a concentration of about 2 mg/kg, linoleic acid at a concentration of about 2 mg/kg, oleic acid at a concentration of about 2 mg/kg, and a sterol at a concentration of about 1 mg/kg (wherein mg/kg=parts per million). In certain embodiments, the terms “supplemented T-cell expansion composition” or “T-cell expansion composition” may be used interchangeably with a media comprising one or more of octanoic acid at a concentration of 9.19 mg/kg, palmitic acid at a concentration of 1.86 mg/kg, linoleic acid at a concentration of about 2.12 mg/kg, oleic acid at a concentration of about 2.13 mg/kg, and a sterol at a concentration of about 1.01 mg/kg (wherein mg/kg=parts per million). In certain embodiments, the terms “supplemented T-cell expansion composition” or “T-cell expansion composition” may be used interchangeably with a media comprising one or more of octanoic acid at a concentration of 9.19 mg/kg, palmitic acid at a concentration of 1.86 mg/kg, linoleic acid at a concentration of 2.12 mg/kg, oleic acid at a concentration of about 2.13 mg/kg, and a sterol at a concentration of 1.01 mg/kg (wherein mg/kg=parts per million). In certain embodiments, the terms “supplemented T-cell expansion composition” or “T-cell expansion composition” may be used interchangeably with a media comprising one or more of octanoic acid at a concentration of between 6.4 μmol/kg and 640 μmol/kg, inclusive of the endpoints; palmitic acid at a concentration of between 0.7 μmol/kg and 70 μmol/kg, inclusive of the endpoints; linoleic acid at a concentration of between 0.75 μmol/kg and 75 μmol/kg, inclusive of the endpoints; oleic acid at a concentration of between 0.75 μmol/kg and 75 μmol/kg, inclusive of the endpoints; and a sterol at a concentration of between 0.25 μmol/kg and 25 μmol/kg, inclusive of the endpoints. In certain embodiments, the terms “supplemented T-cell expansion composition” or “T-cell expansion composition” may be used interchangeably with a media comprising one or more of octanoic acid at a concentration of about 64 μmol/kg, palmitic acid at a concentration of about 7 μmol/kg, linoleic acid at a concentration of about 7.5 μmol/kg, oleic acid at a concentration of about 7.5 μmol/kg and a sterol at a concentration of about 2.5 μmol/kg.

In certain embodiments, the terms “supplemented T-cell expansion composition” or “T-cell expansion composition” may be used interchangeably with a media comprising one or more of octanoic acid at a concentration of about 63.75 μmol/kg, palmitic acid at a concentration of about 7.27 μmol/kg, linoleic acid at a concentration of about 7.57 μmol/kg, oleic acid at a concentration of about 7.56 μmol/kg and a sterol at a concentration of about 2.61 μmol/kg. In certain embodiments, the terms “supplemented T-cell expansion composition” or “T-cell expansion composition” may be used interchangeably with a media comprising one or more of octanoic acid at a concentration of about 63.75 μmol/kg, palmitic acid at a concentration of about 7.27 μmol/kg, linoleic acid at a concentration of about 7.57 μmol/kg, oleic acid at a concentration of 7.56 μmol/kg and a sterol at a concentration of 2.61 μmol/kg.

In certain embodiments of the methods of producing a modified T cell (e.g. a stem cell-like T cell, a T_SCMand/or a T_CM) of the disclosure, the method comprises contacting a modified T cell and an inhibitor of the PI3K-Akt-mTOR pathway. Modified T-cells of the disclosure, including modified stem cell-like T cells, T_SCMand/or Tem of the disclosure, may be incubated, cultured, grown, stored, or otherwise, combined at any step in the methods of the procedure with a growth medium comprising one or more inhibitors a component of a PI3K pathway. Exemplary inhibitors a component of a PI3K pathway include, but are not limited to, an inhibitor of GSK3β such as TWS119 (also known as GSK 3B inhibitor XII; CAS Number 601514-19-6 having a chemical formula C₁₈H₁₄N₄O₂). Exemplary inhibitors of a component of a PI3K pathway include, but are not limited to, bb007 (BLUEBIRDBIO™). Additional Exemplary inhibitors of a component of a PI3K pathway include, but are not limited to, an allosteric Akt inhibitor VIII (also referred to as Akti-1/2 having Compound number 10196499), ATP competitive inhibitors (Orthosteric inhibitors targeting the ATP-binding pocket of the protein kinase B (Akt)), Isoquinoline-5-sulfonamides (H-8, H-89, and NL-71-101), Azepane derivatives (A series of structures derived from (−)-balanol), Aminofurazans (GSK690693), Heterocyclic rings (7-azaindole, 6-phenylpurine derivatives, pyrrolo[2,3-d]pyrimidine derivatives, CCT128930, 3-aminopyrrolidine, anilinotriazole derivatives, spiroindoline derivatives. AZD5363, ipatasertib (GDC-0068, RG7440), A-674563, and A-443654), Phenylpyrazole derivatives (AT7867 and AT13148), Thiophenecarboxamide derivatives (Afuresertib (GSK2110183), 2-pyrimidyl-5-amidothiophene derivative (DC120), uprosertib (GSK2141795)), Allosteric inhibitors (Superior to orthosteric inhibitors providing greater specificity, reduced side-effects and less toxicity), 2,3-diphenylquinoxaline analogues (2,3-diphenylquinoxaline derivatives, triazolo[3,4-f][1,6]naphthyridin-3(2H)-one derivative (MK-2206)), Alkylphospholipids (Edelfosine (1-O-octadecyl-2-O-methyl-rac-glycero-3-phosphocholine, ET-18-OCH₃) ilmofosine (BM 41.440), miltefosine (hexadecylphosphocholine, HePC), perifosine (D-21266), erucylphosphocholine (ErPC), erufosine (ErPC3, erucylphosphohomocholine), Indole-3-carbinol analogues (Indole-3-carbinol, 3-chloroacetylindole, diindolylmethane, diethyl 6-methoxy-5,7-dihydroindolo[2,3-b]carbazole-2,10-dicarboxylate (SR13668), OSU-A9), Sulfonamide derivatives (PH-316 and PHT-427), Thiourea derivatives (PIT-1, PIT-2, DM-PIT-1, N-[(1-methyl-1H-pyrazol-4-yl)carbonyl]-N′-(3-bromophenyl)-thiourea), Purine derivatives (Triciribine (TCN, NSC 154020), triciribine mono-phosphate active analogue (TCN-P), 4-amino-pyrido[2,3-d]pyrimidine derivative API-1, 3-phenyl-3H-imidazo[4,5-b]pyridine derivatives, ARQ 092), BAY 1125976, 3-methyl-xanthine, quinoline-4-carboxamide and 2-[4-(cyclohexa-1,3-dien-1-yl)-1H-pyrazol-3-yl]phenol, 3-oxo-tirucallic acid, 3α- and 3β-acetoxy-tirucallic acids, acetoxy-tirucallic acid, and irreversible inhibitors (antibiotics, Lactoquinomycin, Frenolicin B, kalafungin, medermycin, Boc-Phe-vinyl ketone, 4-hydroxynonenal (4-HNE), 1,6-naphthyridinone derivatives, and imidazo-1,2-pyridine derivatives).

In certain embodiments of the methods of producing a modified T cell (e.g. a stem cell-like T cell, a T_SCMand/or a T_CM) of the disclosure, the method comprises contacting a modified T cell and an inhibitor of T cell effector differentiation. Exemplary inhibitors of T cell effector differentiation include, but are not limited to, a BET inhibitor (e.g. JQ1, a hienotriazolodiazepine) and/or an inhibitor of the BET family of proteins (e.g. BRD2, BRD3, BRD4, and BRDT).

In certain embodiments of the methods of producing a modified T cell (e.g. a stem cell-like T cell, a T_SCMand/or a T_CM) of the disclosure, the method comprises contacting a modified T cell and an agent that reduces nucleo-cytoplasmic Acetyl-CoA. Exemplary agents that reduce nucleo-cytoplasmic Acetyl-CoA include, but are not limited to, 2-hydroxy-citrate (2-HC) as well as agents that increase expression of Acss1.

In certain embodiments of the methods of producing a modified T cell (e.g. a stem cell-like T cell, a TSCM and/or a TCM) of the disclosure, the method comprises contacting a modified T cell and a composition comprising a histone deacetylase (HDAC) inhibitor. In some embodiments, the composition comprising an HDAC inhibitor comprises or consists of valproic acid, Sodium Phenylbutyrate (NaPB) or a combination thereof. In some embodiments, the composition comprising an HDAC inhibitor comprises or consists of valproic acid. In some embodiments, the composition comprising an HDAC inhibitor comprises or consists of Sodium Phenylbutyrate (NaPB).

In certain embodiments of the methods of producing a modified T cell (e.g. a stem cell-like T cell, a T_SCMand/or a T_CM) of the disclosure, the activation supplement may comprise one or more cytokine(s). The one or more cytokine(s) may comprise any cytokine, including but not limited to, lymphokines. Exemplary lympokines include, but are not limited to, interleukin-2 (IL-2), interleukin-3 (IL-3), interleukin-4 (IL-4), interleukin-5 (IL-5), interleukin-6 (IL-6), interleukin-7 (IL-7), interleukin-15 (IL-15), interleukin-21 (IL-21), granulocyte-macrophage colony-stimulating factor (GM-CSF) and interferon-gamma (INFγ). The one or more cytokine(s) may comprise IL-2.

In certain embodiments of the methods of producing a modified T cell (e.g. a stem cell-like T cell, a T_SCMand/or a T_CM) of the disclosure, the activation supplement may comprise one or more activator complexes. Exemplary and nonlimiting activator complexes may comprise a monomeric, dimeric, trimeric or tetrameric antibody complex that binds one or more of CD3, CD28, and CD2. In some embodiments, the activation supplement comprises or consists of an activator complex that comprises a human, a humanized or a recombinant or a chimeric antibody. In some embodiments, the activation supplement comprises or consists of an activator complex that binds CD3 and CD28. In some embodiments, the activation supplement comprises or consists of an activator complex that binds CD3, CD28 and CD2.

Natural Killer (NK) Cells

In certain embodiments, the modified immune or immune precursor cells of the disclosure are natural killer (NK) cells. In certain embodiments, NK cells are cytotoxic lymphocytes that differentiate from lymphoid progenitor cells.

Modified NK cells of the disclosure may be derived from modified hematopoietic stem and progenitor cells (HSPCs) or modified HSCs.

In certain embodiments, non-activated NK cells are derived from CD3-depleted leukopheresis (containing CD14/CD19/CD56+ cells).

In certain embodiments, NK cells are electroporated using a Lonza 4D nucleofector or BTX ECM 830 (500V, 700 usec pulse length, 0.2 mm electrode gap, one pulse). All Lonza 4D nucleofector programs are contemplated as within the scope of the methods of the disclosure.

In certain embodiments, 5×10E6 cells were electroporated per electroporation in 100 μL P3 buffer in cuvettes. However, this ratio of cells per volume is scalable for commercial manufacturing methods.

In certain embodiments. NK cells were stimulated by co-culture with an additional cell line. In certain embodiments, the additional cell line comprises artificial antigen presenting cells (aAPCs). In certain embodiments, stimulation occurs at day 1, 2, 3, 4, 5, 6, or 7 following electroporation. In certain embodiments, stimulation occurs at day 2 following electroporation.

In certain embodiments, NK cells express CD56.

B Cells

In certain embodiments, the modified immune or immune precursor cells of the disclosure are B cells. B cells are a type of lymphocyte that express B cell receptors on the cell surface. B cell receptors bind to specific antigens.

Modified B cells of the disclosure may be derived from modified hematopoietic stem and progenitor cells (HSPCs) or modified HSCs.

In certain embodiments, HSPCs are modified using the methods of the disclosure, and then primed for B cell differentiation in presence of human IL-3, Flt3L, TPO, SCF, and G-CSF for at least 3 days, at least 4 days, at least 5 days, at least 6 days or at least 7 days. In certain embodiments, HSPCs are modified using the methods of the disclosure, and then primed for B cell differentiation in presence of human IL-3, Flt3L, TPO, SCF, and G-CSF for 5 days.

In certain embodiments, following priming, modified HSPC cells are transferred to a layer of feeder cells and fed bi-weekly, along with transfer to a fresh layer of feeders once per week. In certain embodiments, the feeder cells are MS-5 feeder cells.

In certain embodiments, modified HSPC cells are cultured with MS-5 feeder cells for at least 7, 14, 21, 28, 30, 33, 35, 42 or 48 days. In certain embodiments, modified HSPC cells were cultured with MS-5 feeder cells for 33 days.

Inducible Proapoptotic Polypeptides

Inducible proapoptotic polypeptides of the disclosure are superior to existing inducible polypeptides because the inducible proapoptotic polypeptides of the disclosure are far less immunogenic. While inducible proapoptotic polypeptides of the disclosure are recombinant polypeptides, and, therefore, non-naturally occurring, the sequences that are recombined to produce the inducible proapoptotic polypeptides of the disclosure do not comprise non-human sequences that the host human immune system could recognize as “non-self” and, consequently, induce an immune response in the subject receiving an inducible proapoptotic polypeptide of the disclosure, a cell comprising the inducible proapoptotic polypeptide or a composition comprising the inducible proapoptotic polypeptide or the cell comprising the inducible proapoptotic polypeptide.

The disclosure provides inducible proapoptotic polypeptides comprising a ligand binding region, a linker, and a proapoptotic peptide, wherein the inducible proapoptotic polypeptide does not comprise a non-human sequence. In certain embodiments, the non-human sequence comprises a restriction site. In certain embodiments, the proapoptotic peptide is a caspase polypeptide. In certain embodiments, the caspase polypeptide is a caspase 9 polypeptide. In certain embodiments, the caspase 9 polypeptide is a truncated caspase 9 polypeptide. Inducible proapoptotic polypeptides of the disclosure may be non-naturally occurring.

Caspase polypeptides of the disclosure include, but are not limited to, caspase 1, caspase 2, caspase 3, caspase 4, caspase 5, caspase 6, caspase 7, caspase 8, caspase 9, caspase 10, caspase 11, caspase 12, and caspase 14. Caspase polypeptides of the disclosure include, but are not limited to, those caspase polypeptides associated with apoptosis including caspase 2, caspase 3, caspase 6, caspase 7, caspase 8, caspase 9, and caspase 10. Caspase polypeptides of the disclosure include, but are not limited to, those caspase polypeptides that initiate apoptosis, including caspase 2, caspase 8, caspase 9, and caspase 10. Caspase polypeptides of the disclosure include, but are not limited to, those caspase polypeptides that execute apoptosis, including caspase 3, caspase 6, and caspase 7.

Caspase polypeptides of the disclosure may be encoded by an amino acid or a nucleic acid sequence having one or more modifications compared to a wild type amino acid or a nucleic acid sequence. The nucleic acid sequence encoding a caspase polypeptide of the disclosure may be codon optimized. The one or more modifications to an amino acid and/or nucleic acid sequence of a caspase polypeptide of the disclosure may increase an interaction, a cross-linking, a cross-activation, or an activation of the caspase polypeptide of the disclosure compared to a wild type amino acid or a nucleic acid sequence. Alternatively, or in addition, the one or more modifications to an amino acid and/or nucleic acid sequence of a caspase polypeptide of the disclosure may decrease the immunogenicity of the caspase polypeptide of the disclosure compared to a wild type amino acid or a nucleic acid sequence.

Caspase polypeptides of the disclosure may be truncated compared to a wild type caspase polypeptide. For example, a caspase polypeptide may be truncated to eliminate a sequence encoding a Caspase Activation and Recruitment Domain (CARD) to eliminate or minimize the possibility of activating a local inflammatory response in addition to initiating apoptosis in the cell comprising an inducible caspase polypeptide of the disclosure. The nucleic acid sequence encoding a caspase polypeptide of the disclosure may be spliced to form a variant amino acid sequence of the caspase polypeptide of the disclosure compared to a wild type caspase polypeptide. Caspase polypeptides of the disclosure may be encoded by recombinant and/or chimeric sequences. Recombinant and/or chimeric caspase polypeptides of the disclosure may include sequences from one or more different caspase polypeptides. Alternatively, or in addition, recombinant and/or chimeric caspase polypeptides of the disclosure may include sequences from one or more species (e.g. a human sequence and a non-human sequence). Caspase polypeptides of the disclosure may be non-naturally occurring.

The ligand binding region of an inducible proapoptotic polypeptide of the disclosure may include any polypeptide sequence that facilitates or promotes the dimerization of a first inducible proapoptotic polypeptide of the disclosure with a second inducible proapoptotic polypeptide of the disclosure, the dimerization of which activates or induces cross-linking of the proapoptotic polypeptides and initiation of apoptosis in the cell.

The ligand-binding (“dimerization”) region may comprise any polypeptide or functional domain thereof that will allow for induction using an endogenous or non-naturally occurring ligand (i.e. and induction agent), for example, a non-naturally occurring synthetic ligand. The ligand-binding region may be internal or external to the cellular membrane, depending upon the nature of the inducible proapoptotic polypeptide and the choice of ligand (i.e. induction agent). A wide variety of ligand-binding polypeptides and functional domains thereof, including receptors, are known. Ligand-binding regions of the disclosure may include one or more sequences from a receptor. Of particular interest are ligand-binding regions for which ligands (for example, small organic ligands) are known or may be readily produced. These ligand-binding regions or receptors may include, but are not limited to, the FKBPs and cyclophilin receptors, the steroid receptors, the tetracycline receptor, and the like, as well as “non-naturally occurring” receptors, which can be obtained from antibodies, particularly the heavy or light chain subunit, mutated sequences thereof, random amino acid sequences obtained by stochastic procedures, combinatorial syntheses, and the like. In certain embodiments, the ligand-binding region is selected from the group consisting of a FKBP ligand-binding region, a cyclophilin receptor ligand-binding region, a steroid receptor ligand-binding region, a cyclophilin receptors ligand-binding region, and a tetracycline receptor ligand-binding region.

The ligand-binding regions comprising one or more receptor domain(s) may be at least about 50 amino acids, and fewer than about 350 amino acids, usually fewer than 200 amino acids, either as the endogenous domain or truncated active portion thereof. The binding region may, for example, be small (<25 kDa, to allow efficient transfection in viral vectors), monomeric, nonimmunogenic, have synthetically accessible, cell permeable, nontoxic ligands that can be configured for dimerization.

The ligand-binding regions comprising one or more receptor domain(s) may be intracellular or extracellular depending upon the design of the inducible proapoptotic polypeptide and the availability of an appropriate ligand (i.e. induction agent). For hydrophobic ligands, the binding region can be on either side of the membrane, but for hydrophilic ligands, particularly protein ligands, the binding region will usually be external to the cell membrane, unless there is a transport system for internalizing the ligand in a form in which it is available for binding. For an intracellular receptor, the inducible proapoptotic polypeptide or a transposon or vector comprising the inducible proapoptotic polypeptide may encode a signal peptide and transmembrane domain 5′ or 3′ of the receptor domain sequence or may have a lipid attachment signal sequence 5′ of the receptor domain sequence. Where the receptor domain is between the signal peptide and the transmembrane domain, the receptor domain will be extracellular.

Antibodies and antibody subunits, e.g., heavy or light chain, particularly fragments, more particularly all or part of the variable region, or fusions of heavy and light chain to create high-affinity binding, can be used as a ligand binding region of the disclosure. Antibodies that are contemplated include ones that are an ectopically expressed human product, such as an extracellular domain that would not trigger an immune response and generally not expressed in the periphery (i.e., outside the CNS/brain area). Such examples, include, but are not limited to low affinity nerve growth factor receptor (LNGFR), and embryonic surface proteins (i.e., carcinoembryonic antigen). Yet further, antibodies can be prepared against haptenic molecules, which are physiologically acceptable, and the individual antibody subunits screened for binding affinity. The cDNA encoding the subunits can be isolated and modified by deletion of the constant region, portions of the variable region, mutagenesis of the variable region, or the like, to obtain a binding protein domain that has the appropriate affinity for the ligand. In this way, almost any physiologically acceptable haptenic compound can be employed as the ligand or to provide an epitope for the ligand. Instead of antibody units, endogenous receptors can be employed, where the binding region or domain is known and there is a useful or known ligand for binding.

For multimerizing the receptor, the ligand for the ligand-binding region/receptor domains of the inducible proapoptotic polypeptides may be multimeric in the sense that the ligand can have at least two binding sites, with each of the binding sites capable of binding to a ligand receptor region (i.e. a ligand having a first binding site capable of binding the ligand-binding region of a first inducible proapoptotic polypeptide and a second binding site capable of binding the ligand-binding region of a second inducible proapoptotic polypeptide, wherein the ligand-binding regions of the first and the second inducible proapoptotic polypeptides are either identical or distinct). Thus, as used herein, the term “multimeric ligand binding region” refers to a ligand-binding region of an inducible proapoptotic polypeptide of the disclosure that binds to a multimeric ligand. Multimeric ligands of the disclosure include dimeric ligands. A dimeric ligand of the disclosure may have two binding sites capable of binding to the ligand receptor domain. In certain embodiments, multimeric ligands of the disclosure are a dimer or higher order oligomer, usually not greater than about tetrameric, of small synthetic organic molecules, the individual molecules typically being at least about 150 Da and less than about 5 kDa, usually less than about 3 kDa. A variety of pairs of synthetic ligands and receptors can be employed. For example, in embodiments involving endogenous receptors, dimeric FK506 can be used with an FKBP12 receptor, dimerized cyclosporin A can be used with the cyclophilin receptor, dimerized estrogen with an estrogen receptor, dimerized glucocorticoids with a glucocorticoid receptor, dimerized tetracycline with the tetracycline receptor, dimerized vitamin D with the vitamin D receptor, and the like. Alternatively, higher orders of the ligands, e.g., trimeric can be used. For embodiments involving non-naturally occurring receptors, e.g., antibody subunits, modified antibody subunits, single chain antibodies comprised of heavy and light chain variable regions in tandem, separated by a flexible linker, or modified receptors, and mutated sequences thereof, and the like, any of a large variety of compounds can be used. A significant characteristic of the units comprising a multimeric ligand of the disclosure is that each binding site is able to bind the receptor with high affinity, and preferably, that they are able to be dimerized chemically. Also, methods are available to balance the hydrophobicity, hydrophilicity of the ligands so that they are able to dissolve in serum at functional levels, yet diffuse across plasma membranes for most applications.

Activation of inducible proapoptotic polypeptides of the disclosure may be accomplished through, for example, chemically induced dimerization (CID) mediated by an induction agent to produce a conditionally controlled protein or polypeptide. Proapoptotic polypeptides of the disclosure not only inducible, but the induction of these polypeptides is also reversible, due to the degradation of the labile dimerizing agent or administration of a monomeric competitive inhibitor.

In certain embodiments, the ligand binding region comprises a FK506 binding protein 12 (FKBP12) polypeptide. In certain embodiments, the ligand binding region comprises a FKBP12 polypeptide having a substitution of valine (V) for phenylalanine (F) at position 36 (F36V). In certain embodiments, in which the ligand binding region comprises a FKBP12 polypeptide having a substitution of valine (V) for phenylalanine (F) at position 36 (F36V), the induction agent may comprise AP1903, a synthetic drug (CAS Index Name: 2-Piperidinecarboxylic acid, 1-[(2S)-1-oxo-2-(3,4,5-trimethoxyphenyl)butyl]-, 1,2-ethanediylbis[imino(2-oxo-2,1-ethanediyl)oxy-3,1-phenylene[(1R)-3-(3,4-dimethoxyphenyl)propylidene]]ester, [2S-[1(R*),2R*[S*[S*[1(R*),2R*]]]]]-(9Cl) CAS Registry Number: 195514-63-7; Molecular Formula: C78H98N4O20; Molecular Weight: 1411.65)). In certain embodiments, in which the ligand binding region comprises a FKBP12 polypeptide having a substitution of valine (V) for phenylalanine (F) at position 36 (F36V), the induction agent may comprise AP20187 (CAS Registry Number: 195514-80-8 and Molecular Formula: C82H107N5O20). In certain embodiments, the induction agent is an AP20187 analog, such as, for example, AP1510. As used herein, the induction agents AP20187, AP1903 and AP1510 may be used interchangeably.

AP1903 API is manufactured by Alphora Research Inc. and AP1903 Drug Product for Injection is made by Formatech Inc. It is formulated as a 5 mg/mL solution of AP1903 in a 25% solution of the non-ionic solubilizer Solutol HS 15 (250 mg/mL, BASF). At room temperature, this formulation is a clear, slightly yellow solution. Upon refrigeration, this formulation undergoes a reversible phase transition, resulting in a milky solution. This phase transition is reversed upon re-warming to room temperature. The fill is 2.33 mL in a 3 mL glass vial (approximately 10 mg AP1903 for Injection total per vial). Upon determining a need to administer AP1903, patients may be, for example, administered a single fixed dose of AP1903 for Injection (0.4 mg/kg) via IV infusion over 2 hours, using a non-DEHP, non-ethylene oxide sterilized infusion set. The dose of AP1903 is calculated individually for all patients, and is not be recalculated unless body weight fluctuates by ≥10%. The calculated dose is diluted in 100 mL in 0.9% normal saline before infusion. In a previous Phase I study of AP1903, 24 healthy volunteers were treated with single doses of AP1903 for Injection at dose levels of 0.01, 0.05, 0.1, 0.5 and 1.0 mg/kg infused IV over 2 hours. AP1903 plasma levels were directly proportional to dose, with mean Cmax values ranging from approximately 10-1275 ng/mL over the 0.01-1.0 mg/kg dose range. Following the initial infusion period, blood concentrations demonstrated a rapid distribution phase, with plasma levels reduced to approximately 18, 7, and 1% of maximal concentration at 0.5, 2 and 10 hours post-dose, respectively. AP1903 for Injection was shown to be safe and well tolerated at all dose levels and demonstrated a favorable pharmacokinetic profile. Iuliucci J D. et al., J Clin Pharmacol. 41: 870-9, 2001.

The fixed dose of AP1903 for injection used, for example, may be 0.4 mg/kg intravenously infused over 2 hours. The amount of AP1903 needed in vitro for effective signaling of cells is 10-100 nM (1600 Da MW). This equates to 16-160 μg/L or ^˜0.016-1.6 μg/kg (1.6-160 μg/kg). Doses up to 1 mg/kg were well-tolerated in the Phase I study of AP1903 described above. Therefore, 0.4 mg/kg may be a safe and effective dose of AP1903 for this Phase I study in combination with the therapeutic cells.

The amino acid and/or nucleic acid sequence encoding ligand binding of the disclosure may contain sequence one or more modifications compared to a wild type amino acid or nucleic acid sequence. For example, the amino acid and/or nucleic acid sequence encoding ligand binding region of the disclosure may be a codon-optimized sequence. The one or more modifications may increase the binding affinity of a ligand (e.g. an induction agent) for the ligand binding region of the disclosure compared to a wild type polypeptide. Alternatively, or in addition, the one or more modifications may decrease the immunogenicity of the ligand binding region of the disclosure compared to a wild type polypeptide. Ligand binding regions of the disclosure and/or induction agents of the disclosure may be non-naturally occurring.

Modified cells, transposons and/or vectors of the disclosure may comprise an inducible proapoptotic polypeptide comprising (a) a ligand binding region, (b) a linker, and (c) a proapoptotic polypeptide, wherein the inducible proapoptotic polypeptide does not comprise a non-human sequence. In certain embodiments, the non-human sequence comprises a restriction site. In certain embodiments, the ligand binding region may be a multimeric ligand binding region. Inducible proapoptotic polypeptides of the disclosure may also be referred to as an “iC9 safety switch”. In certain embodiments, modified cells and/or transposons of the disclosure may comprise an inducible caspase polypeptide comprising (a) a ligand binding region, (b) a linker, and (c) a caspase polypeptide, wherein the inducible proapoptotic polypeptide does not comprise a non-human sequence. In certain embodiments, modified cells and/or transposons of the disclosure may comprise an inducible caspase polypeptide comprising (a) a ligand binding region, (b) a linker, and (c) a caspase polypeptide, wherein the inducible proapoptotic polypeptide does not comprise a non-human sequence. In certain embodiments, transposons of the disclosure may comprise an inducible caspase polypeptide comprising (a) a ligand binding region, (b) a linker, and (c) a truncated caspase 9 polypeptide, wherein the inducible proapoptotic polypeptide does not comprise a non-human sequence. In certain embodiments of the inducible proapoptotic polypeptides, inducible caspase polypeptides or truncated caspase 9 polypeptides of the disclosure, the ligand binding region may comprise a FK506 binding protein 12 (FKBP12) polypeptide. In certain embodiments, the amino acid sequence of the ligand binding region that comprise a FK506 binding protein 12 (FKBP12) polypeptide may comprise a modification at position 36 of the sequence. The modification may be a substitution of valine (V) for phenylalanine (F) at position 36 (F36V).

In certain embodiments, the FKBP12 polypeptide is encoded by an amino acid sequence comprising

(SEQ ID NO: 14635)

GVQVETISPGDGRTFPKRGQTCVVHYTGMLEDGKKVDSSRDRNKPFKFMLG

KQEVIRGWEEGVAQMSVGQRAKILTISPDYAYGATGHPGIIPPHATLVFDV

ELLKLE.

In certain embodiments, the FKBP12 polypeptide is encoded by a nucleic acid sequence comprising GGGGTCCAGGTCGAGACTATTTCACCAGGGGATGGGCGAACATTCCAAAAAGG GGCCAGACTTGCGTCGTGCATTACACCGGGATGCTGGAGGACGGGAAGAAAGTG GACAGCTCCAGGGATCGCAACAAGCCCTTCAAGTTCATGCTGGGAAAGCAGGAA GTGATCCGAGGATGGGAGGAAGGCGTGGCACAGATGTCAGTCGGCCAGCGGGC CAAACTGACCATTAGCCCTGACTACGCTTATGGAGCAACAGGCCACCCAGGGAT CATTCCCCCTCATGCCACCCTGGTCTTCGAT GTGGAACTGCTGAAGCTGGAG (SEQ ID NO: 14636). In certain embodiments, the induction agent specific for the ligand binding region may comprise a FK506 binding protein 12 (FKBP12) polypeptide having a substitution of valine (V) for phenylalanine (F) at position 36 (F36V) comprises AP20187 and/or API903, both synthetic drugs.

In certain embodiments of the inducible proapoptotic polypeptides, inducible caspase polypeptides or truncated caspase 9 polypeptides of the disclosure, the linker region is encoded by an amino acid comprising GGGGS (SEQ ID NO: 14637) or a nucleic acid sequence comprising GGAGGAGGAGGATCC (SEQ ID NO: 14638). In certain embodiments, the nucleic acid sequence encoding the linker does not comprise a restriction site.

In certain embodiments of the truncated caspase 9 polypeptides of the disclosure, the truncated caspase 9 polypeptide is encoded by an amino acid sequence that does not comprise an arginine (R) at position 87 of the sequence. Alternatively. or in addition, in certain embodiments of the inducible proapoptotic polypeptides, inducible caspase polypeptides or truncated caspase 9 polypeptides of the disclosure, the truncated caspase 9 polypeptide is encoded by an amino acid sequence that does not comprise an alanine (A) at position 282 the sequence. In certain embodiments of the inducible proapoptotic polypeptides, inducible caspase polypeptides or truncated caspase 9 polypeptides of the disclosure, the truncated caspase 9 polypeptide is encoded by an amino acid comprising GFGDVGALESLRGNADLAYILSMEPCGHCLIINNVNFCRESGLRTRTGSNIDCEKLRR RFSSLHFMVEVKGDLTAKKMVLALLELAQQDHGALDCCVVVILSHGCQASHLQFPG AVYGTDGCPVSVEKIVNIFNGTSCPSLGGKPKLFFIQACGGEQKDHGFEVASTSPEDE SPGSNPEPDATPFQEGLRTFDQLDAISSLPTPSDIFVSYSTFPGFVSWRDPKSGSWYVE TLDDIFEQWAHSEDLQSLLLRVANAVSVKGIYKQMPGCFNFLRKKLFFKTS (SEQ ID NO: 14639) or a nucleic acid sequence comprising

(SEQ ID NO: 14640)

TTTGGGGACGTGGGGGCCCTGGAGTCTCTGCGAGGAAATGCCGATCTGGCT

TACATCCTGAGCATGGAACCCTGCGGCCACTGTCTGATCATTAACAATGTG

AACTTCTGCAGAGAAAGCGGACTGCGAACACGGACTGGCTCCAATATTGAC

TGTGAGAAGCTGCGGAGAAGGTTCTCTAGTCTGCACTTTATGGTCGAAGTG

AAAGGGGATCTGACCGCCAAGAAAATGGTGCTGGCCCTGCTGGAGCTGGCT

CAGCAGGACCATGGAGCTCTGGATTGCTGCGTGGTCGTGATCCTGTCCCAC

GGGTGCCAGGCTTCTCATCTGCAGTTCCCCGGAGCAGTGTACGGAACAGAC

GGCTGTCCTGTCAGCGTGGAGAAGATCGTCAACATCTTCAACGGCACTTCT

TGCCCTAGTCTGGGGGGAAAGCCAAAACTGTTCTTTATCCAGGCCTGTGGC

GGGGAACAGAAAGATCACGGCTTCGAGGTGGCCAGCACCAGCCCTGAGGAC

GAATCACCAGGGAGCAACCCTGAACCAGATGCAACTCCATTCCAGGAGGGA

CTGAGGACCTTTGACCAGCTGGATGCTATCTCAAGCCTGCCCACTCCTAGT

GACATTTTCGTGTCTTACAGTACCTTCCCAGGCTTTGTCTCATGGCGCGAT

CCCAAGTCAGGGAGCTGGTACGTGGAGACACTGGACGACATCTTTGAACAG

TGGGCCCATTCAGAGGACCTGCAGAGCCTGCTGCTGCGAGTGGCAAACGCT

GTCTCTGTGAAGGGCATCTACAAACAGATGCCCGGGTGCTTCAATTTTCTG

AGAAAGAAACTGTTCTTTAAGACTTCC.

In certain embodiments of the inducible proapoptotic polypeptides, wherein the polypeptide comprises a truncated caspase 9 polypeptide, the inducible proapoptotic polypeptide is encoded by an amino acid sequence comprising GVQVETISPGDGRTFPKRGQTCVVHYTGMLEDGKKVDSSRDRNKPFKFMLGKQEVI RGWEEGVAQMSVGQRAKLTISPDYAYGATGHPGIIPPHATLVFDVELLKLEGGGGS GFGDVGALESLRGNADLAYILSMEPCGHCLIINNVNFCRESGLRTRTGSNIDCEKLRR RFSSLHFMVEVKGDLTAKKMVLALLELAQQDHGALDCCVVVILSHGCQASHLQFPG AVYGTDGCPVSVEKIVNIFNGTSCPSLGGKPKLFFIQACGGEQKDHGFEVASTSPEDE SPGSNPEPDATPFQEGLRTFDQLDAISSLPTPSDIFVSYSTFPGFVSWRDPKSGSWYVE TLDDIFEQWAHSEDLQSLLLRVANAVSVKGIYKQMPGCFNFLRKKLFFKTS (SEQ ID NO: 14641) or the nucleic acid sequence comprising

(SEQ ID NO: 14642)

ggggtccaggtcgagactatttcaccaggggatgggcgaacatttccaaaa

aggggccagacttgcgtcgtgcattacaccgggatgctggaggacgggaag

aaagtggacagctccagggatcgcaacaagcccttcaagacatgctgggaa

agcaggaagtgatccgaggatgggaggaaggcgtggcacagatgtcagtcg

gccagcgggccaaactgaccattagccctgactacgcttatggagcaacag

gccacccagggatcattccccctcatgccaccctggtcttcgatgtggaac

tgctgaagctggagggaggaggaggatccggatttggggacgtgggggccc

tggagtctctgcgaggaaatgccgatctggcttacatcctgagcatggaac

cctgcggccactgtctgatcattaacaatatgaacactgcagagaaagcag

actgcgaacacggactgactccaatattgactgtgagaagagcggagaagg

actctagtctgcactttatggtcgaagtgaaaggggatctgaccgccaaga

aaatggtgctggccctgctggagctggctcagcaggaccatggagctctgg

attgctgcgtggtcgtgatcctgtcccacgggtgccaggcttctcatctgc

agttccccggagcagtgtacggaacagacggctgtcctgtcagcgtggaga

agatcgtcaacatatcaacggcacttcttgccctagtctggggggaaagcc

aaaactgttctttaccaggcctgtagcggggaacagaaagatcacggcttc

gaggtggccagcaccagccagaggacgaatcaccagggagcaaccctgaac

cagatgcaactccattccaggagggactgaggacctttgaccagctggatg

ctatctcaagcctgcccactcctagtgacattttcgtgtcttacagtacca

cccaggctttgtctcatggcgcgatcccaagtcagggagctggtacgtgga

gacactggacgacatctttgaacagtgggcccattcagaggacctgcagag

cctgctgagcgagtggcaaacgctatctctgtgaagggcatctacaaacag

atgcccgggtgcttcaattttctgagaaagaaactgttcataagacttcc.

Inducible proapoptotic polypeptides of the disclosure may be expressed in a cell under the transcriptional regulation of any promoter capable of initiating and/or regulating the expression of an inducible proapoptotic polypeptide of the disclosure in that cell. The term “promoter” as used herein refers to a promoter that acts as the initial binding site for RNA polymerase to transcribe a gene. For example, inducible proapoptotic polypeptides of the disclosure may be expressed in a mammalian cell under the transcriptional regulation of any promoter capable of initiating and/or regulating the expression of an inducible proapoptotic polypeptide of the disclosure in a mammalian cell, including, but not limited to native, endogenous, exogenous, and heterologous promoters. Preferred mammalian cells include human cells. Thus, inducible proapoptotic polypeptides of the disclosure may be expressed in a human cell under the transcriptional regulation of any promoter capable of initiating and/or regulating the expression of an inducible proapoptotic polypeptide of the disclosure in a human cell, including, but not limited to, a human promoter or a viral promoter. Exemplary promoters for expression in human cells include, but are not limited to, a human cytomegalovirus (CMV) immediate early gene promoter, a SV40 early promoter, a Rous sarcoma virus long terminal repeat, β-actin promoter, a rat insulin promoter and a glyceraldehyde-3-phosphate dehydrogenase promoter, each of which may be used to obtain high-level expression of an inducible proapoptotic polypeptide of the disclosure. The use of other viral or mammalian cellular or bacterial phage promoters which are well known in the art to achieve expression of an inducible proapoptotic polypeptide of the disclosure is contemplated as well, provided that the levels of expression are sufficient for initiating apoptosis in a cell. By employing a promoter with well-known properties, the level and pattern of expression of the protein of interest following transfection or transformation can be optimized.

Selection of a promoter that is regulated in response to specific physiologic or synthetic signals can permit inducible expression of the inducible proapoptotic polypeptide of the disclosure. The ecdysone system (Invitrogen, Carlsbad, Calif.) is one such system. This system is designed to allow regulated expression of a gene of interest in mammalian cells. It consists of a tightly regulated expression mechanism that allows virtually no basal level expression of a transgene, but over 200-fold inducibility. The system is based on the heterodimeric ecdysone receptor of Drosophila, and when ecdysone or an analog such as muristerone A binds to the receptor, the receptor activates a promoter to turn on expression of the downstream transgene high levels of mRNA transcripts are attained. In this system, both monomers of the heterodimeric receptor are constitutively expressed from one vector, whereas the ecdysone-responsive promoter, which drives expression of the gene of interest, is on another plasmid. Engineering of this type of system into a vector of interest may therefore be useful. Another inducible system that may be useful is the Tet-Off™ or Tet-On™ system (Clontech, Palo Alto, Calif.) originally developed by Gossen and Bujard (Gossen and Bujard, Proc. Natl. Acad. Sci. USA, 89:5547-5551, 1992; Gossen et al., Science, 268:1766-1769, 1995). This system also allows high levels of gene expression to be regulated in response to tetracycline or tetracycline derivatives such as doxycycline. In the Tet-On™ system, gene expression is turned on in the presence of doxycycline, whereas in the Tet-Off™ system, gene expression is turned on in the absence of doxycycline. These systems are based on two regulatory elements derived from the tetracycline resistance operon of E. coli: the tetracycline operator sequence (to which the tetracycline repressor binds) and the tetracycline repressor protein. The gene of interest is cloned into a plasmid behind a promoter that has tetracycline-responsive elements present in it. A second plasmid contains a regulatory element called the tetracycline-controlled transactivator, which is composed, in the Tet-Off™ system, of the VP16 domain from the herpes simplex virus and the wild-type tetracycline repressor. Thus, in the absence of doxycycline, transcription is constitutively on. In the Tet-On™ system, the tetracycline repressor is not wild type and in the presence of doxycycline activates transcription. For gene therapy vector production, the Tet-Off™ system may be used so that the producer cells could be grown in the presence of tetracycline or doxycycline and prevent expression of a potentially toxic transgene, but when the vector is introduced to the patient, the gene expression would be constitutively on.

In some circumstances, it is desirable to regulate expression of a transgene in a gene therapy vector. For example, different viral promoters with varying strengths of activity are utilized depending on the level of expression desired. In mammalian cells, the CMV immediate early promoter is often used to provide strong transcriptional activation. The CMV promoter is reviewed in Donnelly, J. J., et al., 1997. Annu. Rev. Immunol. 15:617-48. Modified versions of the CMV promoter that are less potent have also been used when reduced levels of expression of the transgene are desired. When expression of a transgene in hematopoietic cells is desired, retroviral promoters such as the LTRs from MLV or MMTV are often used. Other viral promoters that are used depending on the desired effect include SV40, RSV LTR, HIV-1 and HIV-2 LTR, adenovirus promoters such as from the E1A, E2A, or MLP region, AAV LTR, HSV-TK, and avian sarcoma virus.

In other examples, promoters may be selected that are developmentally regulated and are active in particular differentiated cells. Thus, for example, a promoter may not be active in a pluripotent stem cell, but, for example, where the pluripotent stem cell differentiates into a more mature cell, the promoter may then be activated.

Similarly tissue specific promoters are used to effect transcription in specific tissues or cells so as to reduce potential toxicity or undesirable effects to non-targeted tissues. These promoters may result in reduced expression compared to a stronger promoter such as the CMV promoter, but may also result in more limited expression, and immunogenicity (Bojak, A., et al., 2002. Vaccine. 20:1975-79; Cazeaux, N., et al., 2002. Vaccine 20:3322-31). For example, tissue specific promoters such as the PSA associated promoter or prostate-specific glandular kallikrein, or the muscle creatine kinase gene may be used where appropriate.

Examples of tissue specific or differentiation specific promoters include, but are not limited to, the following: B29 (B cells); CD14 (monocytic cells); CD43 (leukocytes and platelets); CD45 (hematopoietic cells); CD68 (macrophages); desmin (muscle); elastase-1 (pancreatic acinar cells); endoglin (endothelial cells); fibronectin (differentiating cells, healing tissues); and Flt-1 (endothelial cells); GFAP (astrocytes).

In certain indications, it is desirable to activate transcription at specific times after administration of the gene therapy vector. This is done with such promoters as those that are hormone or cytokine regulatable. Cytokine and inflammatory protein responsive promoters that can be used include K and T kininogen (Kageyama et al., (1987) J. Biol. Chem., 262, 2345-2351), c-fos, TNF-alpha, C-reactive protein (Arcone, et al., (1988) Nucl. Acids Res., 16(8), 3195-3207), haptoglobin (Oliviero et al., (1987) EMBO J., 6, 1905-1912), serum amyloid A2, C/EBP alpha, IL-1, IL-6 (Poli and Cortese, (1989) Proc. Nat'l Acad. Sci. USA, 86, 8202-8206), Complement C3 (Wilson et al., (1990) Mol. Cell. Biol., 6181-6191), IL-8, alpha-1 acid glycoprotein (Prowse and Baumann, (1988) Mol Cell Biol, 8, 42-51), alpha-1 antitrypsin, lipoprotein lipase (Zechner et al., Mol. Cell. Biol., 2394-2401, 1988), angiotensinogen (Ron, et al., (1991) Mol. Cell. Biol., 2887-2895), fibrinogen, c-jun (inducible by phorbol esters, TNF-alpha, UV radiation, retinoic acid, and hydrogen peroxide), collagenase (induced by phorbol esters and retinoic acid), metallothionein (heavy metal and glucocorticoid inducible), Stromelysin (inducible by phorbol ester, interleukin-1 and EGF), alpha-2 macroglobulin and alpha-1 anti-chymotrypsin. Other promoters include, for example, SV40, MMTV, Human Immunodeficiency Virus (MV), Moloney virus, ALV, Epstein Barr virus, Rous Sarcoma virus, human actin, myosin, hemoglobin, and creatine.

It is envisioned that any of the above promoters alone or in combination with another can be useful depending on the action desired. Promoters, and other regulatory elements, are selected such that they are functional in the desired cells or tissue. In addition, this list of promoters should not be construed to be exhaustive or limiting; other promoters that are used in conjunction with the promoters and methods disclosed herein.

Antigen Receptors

In some embodiments of the compositions and methods of the disclosure, a modified autologous cell of the disclosure comprises an antigen receptor.

In some embodiments of the compositions and methods of the disclosure, a vector comprises a sequence encoding a chimeric antigen receptor or a portion thereof. Exemplary vectors of the disclosure include, but are not limited to, viral vectors, non-viral vectors, plasmids, nanoplasmids, minicircles, transposition systems, liposomes, polymersomes, micelles, and nanoparticles.

In some embodiments of the compositions and methods of the disclosure, a transposon comprises a sequence encoding a chimeric antigen receptor or a portion thereof. In some embodiments, the transposon is integrated onto a genomic sequence of an autologous cell by a transposase.

In some embodiments of the compositions and methods of the disclosure, a donor oligonucleotide or a donor plasmid comprises a sequence encoding a chimeric antigen receptor or a portion thereof. In some embodiments, the donor oligonucleotide or the donor plasmid are entirely or partially integrated into a chromosomal sequence of an autologous cell following a single or double-strand break and, optionally, cell-mediated repair.

Exemplary antigen receptors include non-naturally occurring transmembrane proteins that bind an antigen at a site in an extacellular domain and transduce or induce an intracellular signal through an intracellular domain.

In some embodiments, non-naturally occurring antigen receptors include, but are not limited to, recombinant, variant, chimeric, or synthetic T-cell Receptors (TCRs). In some embodiments, variant TCRs contain one or more sequence variations in either a nucleotide or amino acid sequence encoding the TCR when compared to a wild type TCR. In some embodiments, a synthetic TCR comprises at least one synthetic or modified nucleic acid or amino acid encoding the TCR. In some embodiments, a recombinant and/or chimeric TCR is encoded by a nucleic acid or amino acid sequence that either across its entire length or a portion thereof, is non-naturally occurring because the sequence is isolated or derived from one or more source sequences.

In some embodiments, non-naturally occurring antigen receptors include, but are not limited to, chimeric antigen receptors.

Chimeric Antigen Receptors

In some embodiments of the compositions and methods of the disclosure, a modified autologous cell of the disclosure comprises a chimeric antigen receptor.

In some embodiments of the compositions and methods of the disclosure, a transposon comprises a sequence encoding a chimeric antigen receptor or a portion thereof.

Chimeric antigen receptors (CARs) of the disclosure may comprise (a) an ectodomain comprising an antigen recognition region, (b) a transmembrane domain, and (c) an endodomain comprising at least one costimulatory domain. In certain embodiments, the ectodomain may further comprise a signal peptide. Alternatively, or in addition, in certain embodiments, the ectodomain may further comprise a hinge between the antigen recognition region and the transmembrane domain. In certain embodiments of the CARs of the disclosure, the signal peptide may comprise a sequence encoding a human CD2, CD3δ, CD3ε, CD3γ, CD3ζ, CD4, CD8α, CD19, CD28, 4-1BB or GM-CSFR signal peptide. In certain embodiments of the CARs of the disclosure, the signal peptide may comprise a sequence encoding a human CD8αsignal peptide. In certain embodiments, the transmembrane domain may comprise a sequence encoding a human CD2, CD3δ, CD3ε, CD3γ, CD3ζ, CD4, CD8α, CD19, CD28, 4-1BB or GM-CSFR transmembrane domain. In certain embodiments of the CARs of the disclosure, the transmembrane domain may comprise a sequence encoding a human CD8α transmembrane domain. In certain embodiments of the CARs of the disclosure, the endodomain may comprise a human CD3ζ endodomain.

In certain embodiments of the CARs of the disclosure, the at least one costimulatory domain may comprise a human 4-1BB, CD28, CD40, ICOS, MyD88, OX-40 intracellular segment, or any combination thereof. In certain embodiments of the CARs of the disclosure, the at least one costimulatory domain may comprise a CD28 and/or a 4-1BB costimulatory domain. In certain embodiments of the CARs of the disclosure, the hinge may comprise a sequence derived from a human CD8α, IgG4, and/or CD4 sequence. In certain embodiments of the CARs of the disclosure, the hinge may comprise a sequence derived from a human CD8α sequence.

The CD28 costimulatory domain may comprise an amino acid sequence comprising RVKFSRSADAPAYKQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQ EGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALP PR (SEQ ID NO: 14477) or a sequence having at least 70%, 80%, 90%, 95%, or 99% identity to the amino acid sequence comprising RVKFSRSADAPAYKQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQ EGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALP PR (SEQ ID NO: 14477). The CD28 costimulatory domain may be encoded by the nucleic acid sequence comprising cgcgtgaagtttagtcgatcagcagatgccccagcttacaaacagggacagaaccagctgtataacgagctgaatctgggccgccga gaggaatatgacgtgctggataagcggagaggacgcgaccccgaaatgggaggcaagcccaggcgcaaaaaccctcaggaagg cctgtataacgagctgcagaaggacaaaatggcagaagcctattctgagatcggcatgaagggggagcgacggagaggcaaagg gcacgatgggctgtaccagggactgagcaccgccacaaaggacacctatgatgctctgcatatgcaggcactgcctccaagg (SEQ ID NO: 14478). The 4-1BB costimulatory domain may comprise an amino acid sequence comprising KRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCEL (SEQ ID NO: 14479) or a sequence having at least 70%, 80%, 90%, 95%, or 99% identity to the amino acid sequence comprising KRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCEL (SEQ ID NO: 14479). The 4-1BB costimulatory domain may be encoded by the nucleic acid sequence comprising aagagaggcaggaagaaactgctgtatattitcaaacagcccttcatgcgccccgtgcagactacccaggaggaagacgggtgctcc tgtcgattccctgaggaagaggaaggcgggtgtgagctg (SEQ ID NO: 14480). The 4-1BB costimulatory domain may be located between the transmembrane domain and the CD28 costimulatory domain.

In certain embodiments of the CARs of the disclosure, the hinge may comprise a sequence derived from a human CD8α, IgG4, and/or CD4 sequence. In certain embodiments of the CARs of the disclosure, the hinge may comprise a sequence derived from a human CD8α sequence. The hinge may comprise a human CD8α amino acid sequence comprising TTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACD (SEQ ID NO: 14481) or a sequence having at least 70%, 80%, 90%, 95%, or 99% identity to the amino acid sequence comprising TTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACD (SEQ ID NO: 14481). The human CD8α hinge amino acid sequence may be encoded by the nucleic acid sequence comprising actaccacaccagcacctagaccaccaactccagctccaaccatcgcgagtcagcccctgagtctgagacctgaggcctgcaggcc agctgcaggaggagctgtgcacaccaggggcctggacttcgcctgcgac (SEQ ID NO: 14482).

SFv

The disclosure provides single chain variable fragment (scFv) compositions and methods for use of these compositions to recognize and bind to a specific target protein. ScFv compositions comprise a heavy chain variable region and a light chain variable region of an antibody. ScFv compositions may be incorporated into an antigen recognition region of a chimeric antigen receptor of the disclosure. ScFvs are fusion proteins of the variable regions of the heavy (VH) and light (VL) chains of immunoglobulins, and the VH and VL domains are connected with a short peptide linker. ScFvs retain the specificity of the original immunoglobulin, despite removal of the constant regions and the introduction of the linker. An exemplary linker comprises a sequence of GGGGSGTGSGGGGS (SEQ ID NO: 14483).

Centyrins

Centyrins of the disclosure specifically bind to an antigen. Chimeric antigen receptors of the disclosure comprising one or more Centyrins that specifically bind an antigen may be used to direct the specificity of a cell, (e.g. a cytotoxic immune cell) towards the specific antigen.

Centyrins of the disclosure may comprise a protein scaffold, wherein the scaffold is capable of specifically binding an antigen. Centyrins of the disclosure may comprise a protein scaffold comprising a consensus sequence of at least one fibronectin type III (FN3) domain, wherein the scaffold is capable of specifically binding an antigen. The at least one fibronectin type III (FN3) domain may be derived from a human protein. The human protein may be Tenascin-C. The consensus sequence may comprise LPAPKNLVVSEVTEDSLRLSWTAPDAAFDSFLIQYQESEKVGEAINLTVPGSERSYDL TGLKPGTEYTVSIYGVKGGHRSNPLSAEFTT (SEQ ID NO: 14488) or MLPAPKNLVVSEVTEDSLRLSWTAPDAAFDSFLIQYQESEKVGEAINLTVPGSERSY DLTGLKPGTEYTVSIYGVKGGHRSNPLSAEFTT (SEQ ID NO: 14489). The consensus sequence may comprise an amino sequence at least 74% identical to LPAPKNLVVSEVTEDSLRLSWTAPDAAFDSFLIQYQESEKVGEAINLTVPGSERSYDL TGLKPGTEYTVSIYGVKGGHRSNPLSAEFTT (SEQ ID NO: 14488) or MLPAPKNLVVSEVTEDSLRLSWTAPDAAFDSFLIQYQESEKVGEAINLTVPGSERSY DLTGLKPGTEYTVSIYGVKGGHRSNPLSAEFTT (SEQ ID NO: 14489). The consensus sequence may encoded by a nucleic acid sequence comprising atgctgcctgcaccaaagaacctggtggtgtctcatgtgacagaggatagtgccagactgtcatggactgctcccgacgcagccttcg atagttttatcatcgtgtaccgggagaacatcgaaaccggcgaggccattgtcctgacagtgccagggtccgaacgctcttatgacctg acagatctgaagcccggaactgagtactatgtgcagatcgccggcgtcaaaggaggcaatatcagcttccctctgtccgcaatcttcac caca (SEQ ID NO: 14490). The consensus sequence may be modified at one or more positions within (a) a A-B loop comprising or consisting of the amino acid residues TEDS (SEQ ID NO: 14491) at positions 13-16 of the consensus sequence; (b) a B-C loop comprising or consisting of the amino acid residues TAPDAAF (SEQ ID NO: 14492) at positions 22-28 of the consensus sequence; (c) a C-D loop comprising or consisting of the amino acid residues SEKVGE (SEQ ID NO: 14493) at positions 38-43 of the consensus sequence; (d) a D-E loop comprising or consisting of the amino acid residues GSER (SEQ ID NO: 14494) at positions 51-54 of the consensus sequence; (e) a E-F loop comprising or consisting of the amino acid residues GLKPG (SEQ ID NO: 14495) at positions 60-64 of the consensus sequence; (f) a F-G loop comprising or consisting of the amino acid residues KGGHRSN (SEQ ID NO: 14496) at positions 75-81 of the consensus sequence; or (g) any combination of (a)-(f). Centyrins of the disclosure may comprise a consensus sequence of at least 5 fibronectin type III (FN3) domains, at least 10 fibronectin type III (FN3) domains or at least 15 fibronectin type III (FN3) domains. The scaffold may bind an antigen with at least one affinity selected from a K_Dof less than or equal to 10⁻⁹M, less than or equal to 10⁻¹⁰M, less than or equal to 10⁻¹¹M, less than or equal to 10⁻¹²M, less than or equal to 10⁻¹³M, less than or equal to 10⁻¹⁴M, and less than or equal to 10⁻¹⁵M. The K_Dmay be determined by surface plasmon resonance.

The term “antibody mimetic” is intended to describe an organic compound that specifically binds a target sequence and has a structure distinct from a naturally-occurring antibody. Antibody mimetics may comprise a protein, a nucleic acid, or a small molecule. The target sequence to which an antibody mimetic of the disclosure specifically binds may be an antigen. Antibody mimetics may provide superior properties over antibodies including, but not limited to, superior solubility, tissue penetration, stability towards heat and enzymes (e.g. resistance to enzymatic degradation), and lower production costs. Exemplary antibody mimetics include, but are not limited to, an affibody, an afflilin, an affimer, an affitin, an alphabody, an anticalin, and avimer (also known as avidity multimer), a DARPin (Designed Ankyrin Repeat Protein), a Fynomer, a Kunitz domain peptide, and a monobody.

Affibody molecules of the disclosure comprise a protein scaffold comprising or consisting of one or more alpha helix without any disulfide bridges. Preferably, affibody molecules of the disclosure comprise or consist of three alpha helices. For example, an affibody molecule of the disclosure may comprise an immunoglobulin binding domain. An affibody molecule of the disclosure may comprise the Z domain of protein A.

Affilin molecules of the disclosure comprise a protein scaffold produced by modification of exposed amino acids of, for example, either gamma-B crystallin or ubiquitin. Affilin molecules functionally mimic an antibody's affinity to antigen, but do not structurally mimic an antibody. In any protein scaffold used to make an affilin, those amino acids that are accessible to solvent or possible binding partners in a properly-folded protein molecule are considered exposed amino acids. Any one or more of these exposed amino acids may be modified to specifically bind to a target sequence or antigen.

Affimer molecules of the disclosure comprise a protein scaffold comprising a highly stable protein engineered to display peptide loops that provide a high affinity binding site for a specific target sequence. Exemplary affimer molecules of the disclosure comprise a protein scaffold based upon a cystatin protein or tertiary structure thereof. Exemplary affimer molecules of the disclosure may share a common tertiary structure of comprising an alpha-helix lying on top of an anti-parallel beta-sheet.

Affitin molecules of the disclosure comprise an artificial protein scaffold, the structure of which may be derived, for example, from a DNA binding protein (e.g. the DNA binding protein Sac7d). Affitins of the disclosure selectively bind a target sequence, which may be the entirety or part of an antigen. Exemplary affitins of the disclosure are manufactured by randomizing one or more amino acid sequences on the binding surface of a DNA binding protein and subjecting the resultant protein to ribosome display and selection. Target sequences of affitins of the disclosure may be found, for example, in the genome or on the surface of a peptide, protein, virus, or bacteria. In certain embodiments of the disclosure, an affitin molecule may be used as a specific inhibitor of an enzyme. Affitin molecules of the disclosure may include heat-resistant proteins or derivatives thereof.

Alphabody molecules of the disclosure may also be referred to as Cell-Penetrating Alphabodies (CPAB). Alphabody molecules of the disclosure comprise small proteins (typically of less than 10 kDa) that bind to a variety of target sequences (including antigens). Alphabody molecules are capable of reaching and binding to intracellular target sequences. Structurally, alphabody molecules of the disclosure comprise an artificial sequence forming single chain alpha helix (similar to naturally occurring coiled-coil structures). Alphabody molecules of the disclosure may comprise a protein scaffold comprising one or more amino acids that are modified to specifically bind target proteins. Regardless of the binding specificity of the molecule, alphabody molecules of the disclosure maintain correct folding and thermostability.

Anticalin molecules of the disclosure comprise artificial proteins that bind to target sequences or sites in either proteins or small molecules. Anticalin molecules of the disclosure may comprise an artificial protein derived from a human lipocalin. Anticalin molecules of the disclosure may be used in place of, for example, monoclonal antibodies or fragments thereof. Anticalin molecules may demonstrate superior tissue penetration and thermostability than monoclonal antibodies or fragments thereof. Exemplary anticalin molecules of the disclosure may comprise about 180 amino acids, having a mass of approximately 20 kDa. Structurally, anticalin molecules of the disclosure comprise a barrel structure comprising antiparallel beta-strands pairwise connected by loops and an attached alpha helix. In preferred embodiments, anticalin molecules of the disclosure comprise a barrel structure comprising eight antiparallel beta-strands pairwise connected by loops and an attached alpha helix.

Avimer molecules of the disclosure comprise an artificial protein that specifically binds to a target sequence (which may also be an antigen). Avimers of the disclosure may recognize multiple binding sites within the same target or within distinct targets. When an avimer of the disclosure recognize more than one target, the avimer mimics function of a bi-specific antibody. The artificial protein avimer may comprise two or more peptide sequences of approximately 30-35 amino acids each. These peptides may be connected via one or more linker peptides. Amino acid sequences of one or more of the peptides of the avimer may be derived from an A domain of a membrane receptor. Avimers have a rigid structure that may optionally comprise disulfide bonds and/or calcium. Avimers of the disclosure may demonstrate greater heat stability compared to an antibody.

DARPins (Designed Ankyrin Repeat Proteins) of the disclosure comprise genetically-engineered, recombinant, or chimeric proteins having high specificity and high affinity for a target sequence. In certain embodiments, DARPins of the disclosure are derived from ankyrin proteins and, optionally, comprise at least three repeat motifs (also referred to as repetitive structural units) of the ankyrin protein. Ankyrin proteins mediate high-affinity protein-protein interactions. DARPins of the disclosure comprise a large target interaction surface.

Fynomers of the disclosure comprise small binding proteins (about 7 kDa) derived from the human Fyn SH3 domain and engineered to bind to target sequences and molecules with equal affinity and equal specificity as an antibody.

Kunitz domain peptides of the disclosure comprise a protein scaffold comprising a Kunitz domain. Kunitz domains comprise an active site for inhibiting protease activity. Structurally, Kunitz domains of the disclosure comprise a disulfide-rich alpha+beta fold. This structure is exemplified by the bovine pancreatic trypsin inhibitor. Kunitz domain peptides recognize specific protein structures and serve as competitive protease inhibitors. Kunitz domains of the disclosure may comprise Ecallantide (derived from a human lipoprotein-associated coagulation inhibitor (LACI)).

Monobodies of the disclosure are small proteins (comprising about 94 amino acids and having a mass of about 10 kDa) comparable in size to a single chain antibody. These genetically engineered proteins specifically bind target sequences including antigens. Monobodies of the disclosure may specifically target one or more distinct proteins or target sequences. In preferred embodiments, monobodies of the disclosure comprise a protein scaffold mimicking the structure of human fibronectin, and more preferably, mimicking the structure of the tenth extracellular type III domain of fibronectin. The tenth extracellular type III domain of fibronectin, as well as a monobody mimetic thereof, contains seven beta sheets forming a barrel and three exposed loops on each side corresponding to the three complementarity determining regions (CDRs) of an antibody. In contrast to the structure of the variable domain of an antibody, a monobody lacks any binding site for metal ions as well as a central disulfide bond. Multispecific monobodies may be optimized by modifying the loops BC and FG. Monobodies of the disclosure may comprise an adnectin.

VHH

In certain embodiments, the CAR comprises a single domain antibody (SdAb). In certain embodiments, the SdAb is a VHH.

The disclosure provides chimeric antigen receptors (CARs) comprising at least one VHH (a VCAR). Chimeric antigen receptors of the disclosure may comprise more than one VHH. For example, a bi-specific VCAR may comprise two VHHs that specifically bind two distinct antigens.

VHH proteins of the disclosure specifically bind to an antigen. Chimeric antigen receptors of the disclosure comprising one or more VHHs that specifically bind an antigen may be used to direct the specificity of a cell, (e.g. a cytotoxic immune cell) towards the specific antigen.

At least one VHH protein or VCAR of the disclosure can be optionally produced by a cell line, a mixed cell line, an immortalized cell or clonal population of immortalized cells, as well known in the art. See, e.g., Ausubel, et al., ed., Current Protocols in Molecular Biology, John Wiley & Sons, Inc., NY, N.Y. (1987-2001); Sambrook, et al., Molecular Cloning: A Laboratory Manual, 2nd Edition, Cold Spring Harbor. N.Y. (1989); Harlow and Lane, Antibodies, a Laboratory Manual, Cold Spring Harbor, N.Y. (1989); Colligan, et al., eds., Current Protocols in Immunology, John Wiley & Sons, Inc., NY (1994-2001); Colligan et al., Current Protocols in Protein Science, John Wiley & Sons, NY, N.Y., (1997-2001).

Amino acids from a VHH protein can be altered, added and/or deleted to reduce immunogenicity or reduce, enhance or modify binding, affinity, on-rate, off-rate, avidity, specificity, half-life, stability, solubility or any other suitable characteristic, as known in the art.

Optionally, VHH proteins can be engineered with retention of high affinity for the antigen and other favorable biological properties. To achieve this goal, the VHH proteins can be optionally prepared by a process of analysis of the parental sequences and various conceptual engineered products using three-dimensional models of the parental and engineered sequences. Three-dimensional models are commonly available and are familiar to those skilled in the art. Computer programs are available which illustrate and display probable three-dimensional conformational structures of selected candidate sequences and can measure possible immunogenicity (e.g., Immunofilter program of Xencor, Inc. of Monrovia. Calif.). Inspection of these displays permits analysis of the likely role of the residues in the functioning of the candidate sequence, i.e., the analysis of residues that influence the ability of the candidate VHH protein to bind its antigen. In this way, residues can be selected and combined from the parent and reference sequences so that the desired characteristic, such as affinity for the target antigen(s), is achieved. Alternatively, or in addition to, the above procedures, other suitable methods of engineering can be used.

Screening VHH for specific binding to similar proteins or fragments can be conveniently achieved using nucleotide (DNA or RNA display) or peptide display libraries, for example, in vitro display. This method involves the screening of large collections of peptides for individual members having the desired function or structure. The displayed nucleotide or peptide sequences can be from 3 to 5000 or more nucleotides or amino acids in length, frequently from 5-100 amino acids long, and often from about 8 to 25 amino acids long. In addition to direct chemical synthetic methods for generating peptide libraries, several recombinant DNA methods have been described. One type involves the display of a peptide sequence on the surface of a bacteriophage or cell. Each bacteriophage or cell contains the nucleotide sequence encoding the particular displayed peptide sequence. The VHH proteins of the disclosure can bind human or other mammalian proteins with a wide range of affinities (KD). In a preferred embodiment, at least one VHH of the present disclosure can optionally bind to a target protein with high affinity, for example, with a KD equal to or less than about 10⁻⁷M, such as but not limited to, 0.1-9.9 (or any range or value therein)×10⁻⁸, 10⁻⁹, 10⁻¹⁰, 10⁻¹¹, 10⁻¹², 10⁻¹³, 10⁻¹⁴, 10⁻¹⁵or any range or value therein, as determined by surface plasmon resonance or the Kinexa method, as practiced by those of skill in the art.

The affinity or avidity of a VHH or a VCAR for an antigen can be determined experimentally using any suitable method. (See, for example, Berzofsky, et al., “Antibody-Antigen Interactions,” In Fundamental Immunology, Paul, W. E., Ed., Raven Press: New York, N.Y. (1984); Kuby, Janis Immunology, W.H. Freeman and Company: New York, N.Y. (1992); and methods described herein). The measured affinity of a particular VHH-antigen or VCAR-antigen interaction can vary if measured under different conditions (e.g., salt concentration, pH). Thus, measurements of affinity and other antigen-binding parameters (e.g., KD, Kon, Koff) are preferably made with standardized solutions of VHH or VCAR and antigen, and a standardized buffer, such as the buffer described herein.

Competitive assays can be performed with the VHH or VCAR of the disclosure in order to determine what proteins, antibodies, and other antagonists compete for binding to a target protein with the VHH or VCAR of the present disclosure and/or share the epitope region. These assays as readily known to those of ordinary skill in the art evaluate competition between antagonists or ligands for a limited number of binding sites on a protein. The protein and/or antibody is immobilized or insolubilized before or after the competition and the sample bound to the target protein is separated from the unbound sample, for example, by decanting (where the protein/antibody was preinsolubilized) or by centrifuging (where the protein/antibody was precipitated after the competitive reaction). Also, the competitive binding may be determined by whether function is altered by the binding or lack of binding of the VHH or VCAR to the target protein, e.g., whether the VCAR molecule inhibits or potentiates the enzymatic activity of, for example, a label. ELISA and other functional assays may be used, as well known in the art.

In certain embodiments, the CAR comprises a single domain antibody (SdAb). In certain embodiments, the SdAb is a VH.

The disclosure provides chimeric antigen receptors (CARs) comprising a single domain antibody (VCARs). In certain embodiments, the single domain antibody comprises a VH. In certain embodiments, the VH is isolated or derived from a human sequence. In certain embodiments, VH comprises a human CDR sequence and/or a human framework sequence and a non-human or humanized sequence (e.g. a rat Fc domain). In certain embodiments, the VH is a fully humanized VH. In certain embodiments, the VH s neither a naturally occurring antibody nor a fragment of a naturally occurring antibody. In certain embodiments, the VH is not a fragment of a monoclonal antibody. In certain embodiments, the VH is a UniDab™ antibody (TeneoBio).

In certain embodiments, the VH is fully engineered using the UniRat™ (TeneoBio) system and “NGS-based Discovery” to produce the VH. Using this method, the specific VH are not naturally-occurring and are generated using fully engineered systems. The VH are not derived from naturally-occurring monoclonal antibodies (mAbs) that were either isolated directly from the host (for example, a mouse, rat or human) or directly from a single clone of cells or cell line (hybridoma). These VHs were not subsequently cloned from said cell lines. Instead, VH sequences are fully-engineered using the UniRat™ system as transgenes that comprise human variable regions (VH domains) with a rat Fc domain, and are thus human/rat chimeras without a light chain and are unlike the standard mAb format. The native rat genes are knocked out and the only antibodies expressed in the rat are from transgenes with VH domains linked to a Rat Fc (UniAbs). These are the exclusive Abs expressed in the UniRat. Next generation sequencing (NGS) and bioinformatics are used to identify the full antigen-specific repertoire of the heavy-chain antibodies generated by UniRat™ after immunization. Then, a unique gene assembly method is used to convert the antibody repertoire sequence information into large collections of fully-human heavy-chain antibodies that can be screened in vitro for a variety of functions. In certain embodiments, fully humanized VH are generated by fusing the human VH domains with human Fcs in vitro (to generate a non-naturally occurring recombinant VH antibody). In certain embodiments, the VH are fully humanized, but they are expressed in vivo as human/rat chimera (human VH, rat Fc) without a light chain. Fully humanized VHs are expressed in vivo as human/rat chimera (human VH, rat Fc) without a light chain are about 80 kDa (vs 150 kDa).

VCARs of the disclosure may comprise at least one VH of the disclosure. In certain embodiments, the VH of the disclosure may be modified to remove an Fc domain or a portion thereof. In certain embodiments, a framework sequence of the VH of the disclosure may be modified to, for example, improve expression, decrease immunogenicity or to improve function.

As used throughout the disclosure, the singular forms “a,” “and,” and “the” include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to “a method” includes a plurality of such methods and reference to “a dose” includes reference to one or more doses and equivalents thereof known to those skilled in the art, and so forth.

The term “about” or “approximately” means within an acceptable error range for the particular value as determined by one of ordinary skill in the art, which will depend in part on how the value is measured or determined, e.g., the limitations of the measurement system. For example, “about” can mean within 1 or more standard deviations. Alternatively, “about” can mean a range of up to 20%, or up to 10%, or up to 5%, or up to 1% of a given value. Alternatively, particularly with respect to biological systems or processes, the term can mean within an order of magnitude, preferably within 5-fold, and more preferably within 2-fold, of a value. Where particular values are described in the application and claims, unless otherwise stated the term “about” meaning within an acceptable error range for the particular value should be assumed.

The disclosure provides isolated or substantially purified polynucleotide or protein compositions. An “isolated” or “purified” polynucleotide or protein, or biologically active portion thereof, is substantially or essentially free from components that normally accompany or interact with the polynucleotide or protein as found in its naturally occurring environment. Thus, an isolated or purified polynucleotide or protein is substantially free of other cellular material or culture medium when produced by recombinant techniques, or substantially free of chemical precursors or other chemicals when chemically synthesized. Optimally, an “isolated” polynucleotide is free of sequences (optimally protein encoding sequences) that naturally flank the polynucleotide (i.e., sequences located at the 5′ and 3′ ends of the polynucleotide) in the genomic DNA of the organism from which the polynucleotide is derived. For example, in various embodiments, the isolated polynucleotide can contain less than about 5 kb, 4 kb, 3 kb, 2 kb, 1 kb, 0.5 kb, or 0.1 kb of nucleotide sequence that naturally flank the polynucleotide in genomic DNA of the cell from which the polynucleotide is derived. A protein that is substantially free of cellular material includes preparations of protein having less than about 30%, 20%, 10%, 5%, or 1% (by dry weight) of contaminating protein. When the protein of the disclosure or biologically active portion thereof is recombinantly produced, optimally culture medium represents less than about 30%, 20%, 10%, 5%, or 1% (by dry weight) of chemical precursors or non-protein-of-interest chemicals.

The disclosure provides fragments and variants of the disclosed DNA sequences and proteins encoded by these DNA sequences. As used throughout the disclosure, the term “fragment” refers to a portion of the DNA sequence or a portion of the amino acid sequence and hence protein encoded thereby. Fragments of a DNA sequence comprising coding sequences may encode protein fragments that retain biological activity of the native protein and hence DNA recognition or binding activity to a target DNA sequence as herein described. Alternatively, fragments of a DNA sequence that are useful as hybridization probes generally do not encode proteins that retain biological activity or do not retain promoter activity. Thus, fragments of a DNA sequence may range from at least about 20 nucleotides, about 50 nucleotides, about 100 nucleotides, and up to the full-length polynucleotide of the disclosure.

Nucleic acids or proteins of the disclosure can be constructed by a modular approach including preassembling monomer units and/or repeat units in target vectors that can subsequently be assembled into a final destination vector. Polypeptides of the disclosure may comprise repeat monomers of the disclosure and can be constructed by a modular approach by preassembling repeat units in target vectors that can subsequently be assembled into a final destination vector. The disclosure provides polypeptide produced by this method as well nucleic acid sequences encoding these polypeptides. The disclosure provides host organisms and cells comprising nucleic acid sequences encoding polypeptides produced this modular approach.

The term “antibody” is used in the broadest sense and specifically covers single monoclonal antibodies (including agonist and antagonist antibodies) and antibody compositions with polyepitopic specificity. It is also within the scope hereof to use natural or synthetic analogs, mutants, variants, alleles, homologs and orthologs (herein collectively referred to as “analogs”) of the antibodies hereof as defined herein. Thus, according to one embodiment hereof, the term “antibody hereof” in its broadest sense also covers such analogs. Generally, in such analogs, one or more amino acid residues may have been replaced, deleted and/or added, compared to the antibodies hereof as defined herein.

“Antibody fragment”, and all grammatical variants thereof, as used herein are defined as a portion of an intact antibody comprising the antigen binding site or variable region of the intact antibody, wherein the portion is free of the constant heavy chain domains (i.e. CH2, CH3, and CH4, depending on antibody isotype) of the Fc region of the intact antibody. Examples of antibody fragments include Fab, Fab′, Fab′-SH, F(ab′)₂, and Fv fragments; diabodies; any antibody fragment that is a polypeptide having a primary structure consisting of one uninterrupted sequence of contiguous amino acid residues (referred to herein as a “single-chain antibody fragment” or “single chain polypeptide”), including without limitation (1) single-chain Fv (scFv) molecules (2) single chain polypeptides containing only one light chain variable domain, or a fragment thereof that contains the three CDRs of the light chain variable domain, without an associated heavy chain moiety and (3) single chain polypeptides containing only one heavy chain variable region, or a fragment thereof containing the three CDRs of the heavy chain variable region, without an associated light chain moiety; and multispecific or multivalent structures formed from antibody fragments. In an antibody fragment comprising one or more heavy chains, the heavy chain(s) can contain any constant domain sequence (e.g. CHI in the IgG isotype) found in a non-Fc region of an intact antibody, and/or can contain any hinge region sequence found in an intact antibody, and/or can contain a leucine zipper sequence fused to or situated in the hinge region sequence or the constant domain sequence of the heavy chain(s). The term further includes single domain antibodies (“sdAB”) which generally refers to an antibody fragment having a single monomeric variable antibody domain, (for example, from camelids). Such antibody fragment types will be readily understood by a person having ordinary skill in the art.

“Binding” refers to a sequence-specific, non-covalent interaction between macromolecules (e.g., between a protein and a nucleic acid). Not all components of a binding interaction need be sequence-specific (e.g., contacts with phosphate residues in a DNA backbone), as long as the interaction as a whole is sequence-specific.

The term “comprising” is intended to mean that the compositions and methods include the recited elements, but do not exclude others. “Consisting essentially of” when used to define compositions and methods, shall mean excluding other elements of any essential significance to the combination when used for the intended purpose. Thus, a composition consisting essentially of the elements as defined herein would not exclude trace contaminants or inert carriers. “Consisting of shall mean excluding more than trace elements of other ingredients and substantial method steps. Embodiments defined by each of these transition terms are within the scope of this disclosure.

The term “epitope” refers to an antigenic determinant of a polypeptide. An epitope could comprise three amino acids in a spatial conformation, which is unique to the epitope. Generally, an epitope consists of at least 4, 5, 6, or 7 such amino acids, and more usually, consists of at least 8, 9, or 10 such amino acids. Methods of determining the spatial conformation of amino acids are known in the art, and include, for example, x-ray crystallography and two-dimensional nuclear magnetic resonance.

As used herein, “expression” refers to the process by which polynucleotides are transcribed into mRNA and/or the process by which the transcribed mRNA is subsequently being translated into peptides, polypeptides, or proteins. If the polynucleotide is derived from genomic DNA, expression may include splicing of the mRNA in a eukaryotic cell.

“Gene expression” refers to the conversion of the information, contained in a gene, into a gene product. A gene product can be the direct transcriptional product of a gene (e.g., mRNA, tRNA, rRNA, antisense RNA, ribozyme, shRNA, micro RNA, structural RNA or any other type of RNA) or a protein produced by translation of an mRNA. Gene products also include RNAs which are modified, by processes such as capping, polyadenylation, methylation, and editing, and proteins modified by, for example, methylation, acetylation, phosphorylation, ubiquitination, ADP-ribosylation, myristilation, and glycosylation.

“Modulation” or “regulation” of gene expression refers to a change in the activity of a gene. Modulation of expression can include, but is not limited to, gene activation and gene repression.

The term “operatively linked” or its equivalents (e.g., “linked operatively”) means two or more molecules are positioned with respect to each other such that they are capable of interacting to affect a function attributable to one or both molecules or a combination thereof.

Non-covalently linked components and methods of making and using non-covalently linked components, are disclosed. The various components may take a variety of different forms as described herein. For example, non-covalently linked (i.e., operatively linked) proteins may be used to allow temporary interactions that avoid one or more problems in the art. The ability of non-covalently linked components, such as proteins, to associate and dissociate enables a functional association only or primarily under circumstances where such association is needed for the desired activity. The linkage may be of duration sufficient to allow the desired effect.

A method for directing proteins to a specific locus in a genome of an organism is disclosed. The method may comprise the steps of providing a DNA localization component and providing an effector molecule, wherein the DNA localization component and the effector molecule are capable of operatively linking via a non-covalent linkage.

The term “scFv” refers to a single-chain variable fragment. scFv is a fusion protein of the variable regions of the heavy (VH) and light chains (VL) of immunoglobulins, connected with a linker peptide. The linker peptide may be from about 5 to 40 amino acids or from about 10 to 30 amino acids or about 5, 10, 15, 20, 25, 30, 35, or 40 amino acids in length. Single-chain variable fragments lack the constant Fc region found in complete antibody molecules, and, thus, the common binding sites (e.g., Protein G) used to purify antibodies. The term further includes a scFv that is an intrabody, an antibody that is stable in the cytoplasm of the cell, and which may bind to an intracellular protein.

The term “single domain antibody” means an antibody fragment having a single monomeric variable antibody domain which is able to bind selectively to a specific antigen. A single-domain antibody generally is a peptide chain of about 110 amino acids long, comprising one variable domain (VH) of a heavy-chain antibody, or of a common IgG, which generally have similar affinity to antigens as whole antibodies, but are more heat-resistant and stable towards detergents and high concentrations of urea. Examples are those derived from camelid or fish antibodies. Alternatively, single-domain antibodies can be made from common murine or human IgG with four chains.

Methods of Gene Delivery

In some embodiments of the methods of the disclosure, a composition comprises a scalable ratio of 250×10⁶primary human T cells per milliliter of buffer or other media during a delivery or an introduction step.

In some embodiments of the methods of the disclosure, a composition is delivered or introduced to a cell by electroporation or nucleofection. In some embodiments, a delivery or introduction step comprises electroporation or nucleofection.

In some embodiments of the methods of the disclosure, a composition is delivered or introduced to a cell by a method other than electroporation or nucleofection.

In some embodiments of the methods of the disclosure, a composition is delivered or introduced by one or more of topical delivery, adsorption, absorption, electroporation, spin-fection, co-culture, transfection, mechanical delivery, sonic delivery, vibrational delivery, magnetofection or by nanoparticle-mediated delivery. In some embodiments, a delivery or introduction step comprises one or more of topical delivery, adsorption, absorption, electroporation, spin-fection, co-culture, transfection, mechanical delivery, sonic delivery, vibrational delivery, magnetofection or by nanoparticle-mediated delivery.

In some embodiments of the methods of the disclosure, a composition is delivered or introduced by liposomal transfection, calcium phosphate transfection, fugene transfection, and dendrimer-mediated transfection. In some embodiments, a delivery or introduction step comprises one or more of liposomal transfection, calcium phosphate transfection, fugene transfection, and dendrimer-mediated transfection.

In some embodiments of the methods of the disclosure, a composition is delivered or introduced by mechanical transfection comprises cell squeezing, cell bombardment, or gene gun techniques. In some embodiments, a delivery or introduction step comprises one or more of mechanical transfection comprises cell squeezing, cell bombardment, or gene gun techniques.

In some embodiments of the methods of the disclosure, a composition is delivered or introduced by nanoparticle-mediated transfection comprises liposomal delivery, delivery by micelles, and delivery by polymerosomes. In some embodiments, a delivery or introduction step comprises one or more of liposomal delivery, delivery by micelles, and delivery by polymerosomes.

Construction of Nucleic Acids

The isolated nucleic acids of the disclosure can be made using (a) recombinant methods, (b) synthetic techniques, (c) purification techniques, and/or (d) combinations thereof, as well-known in the art.

The nucleic acids can conveniently comprise sequences in addition to a polynucleotide of the present disclosure. For example, a multi-cloning site comprising one or more endonuclease restriction sites can be inserted into the nucleic acid to aid in isolation of the polynucleotide. Also, translatable sequences can be inserted to aid in the isolation of the translated polynucleotide of the disclosure. For example, a hexa-histidine marker sequence provides a convenient means to purify the proteins of the disclosure. The nucleic acid of the disclosure, excluding the coding sequence, is optionally a vector, adapter, or linker for cloning and/or expression of a polynucleotide of the disclosure.

Additional sequences can be added to such cloning and/or expression sequences to optimize their function in cloning and/or expression, to aid in isolation of the polynucleotide, or to improve the introduction of the polynucleotide into a cell. Use of cloning vectors, expression vectors, adapters, and linkers is well known in the art. (See, e.g., Ausubel, supra; or Sambrook, supra).

Recombinant Methods for Constructing Nucleic Acids

The isolated nucleic acid compositions of this disclosure, such as RNA, cDNA, genomic DNA, or any combination thereof, can be obtained from biological sources using any number of cloning methodologies known to those of skill in the art. In some embodiments, oligonucleotide probes that selectively hybridize, under stringent conditions, to the polynucleotides of the present disclosure are used to identify the desired sequence in a cDNA or genomic DNA library. The isolation of RNA, and construction of cDNA and genomic libraries are well known to those of ordinary skill in the art. (See, e.g., Ausubel, supra; or Sambrook, supra).

Nucleic Add Screening and Isolation Methods

A cDNA or genomic library can be screened using a probe based upon the sequence of a polynucleotide of the disclosure. Probes can be used to hybridize with genomic DNA or cDNA sequences to isolate homologous genes in the same or different organisms. Those of skill in the art will appreciate that various degrees of stringency of hybridization can be employed in the assay; and either the hybridization or the wash medium can be stringent. As the conditions for hybridization become more stringent, there must be a greater degree of complementarity between the probe and the target for duplex formation to occur. The degree of stringency can be controlled by one or more of temperature, ionic strength, pH and the presence of a partially denaturing solvent, such as formamide. For example, the stringency of hybridization is conveniently varied by changing the polarity of the reactant solution through, for example, manipulation of the concentration of formamide within the range of 0% to 50%. The degree of complementarity (sequence identity) required for detectable binding will vary in accordance with the stringency of the hybridization medium and/or wash medium. The degree of complementarity will optimally be 100%, or 70-100%, or any range or value therein. However, it should be understood that minor sequence variations in the probes and primers can be compensated for by reducing the stringency of the hybridization and/or wash medium.

Methods of amplification of RNA or DNA are well known in the art and can be used according to the disclosure without undue experimentation, based on the teaching and guidance presented herein.

Known methods of DNA or RNA amplification include, but are not limited to, polymerase chain reaction (PCR) and related amplification processes (see, e.g., U.S. Pat. Nos. 4,683,195, 4,683,202, 4,800,159, 4,965,188, to Mullis, et al.; U.S. Pat. Nos. 4,795,699 and 4,921,794 to Tabor, et al; U.S. Pat. No. 5,142,033 to Innis; U.S. Pat. No. 5,122,464 to Wilson, et al.; U.S. Pat. No. 5,091,310 to Innis; U.S. Pat. No. 5,066,584 to Gyllensten, et al; U.S. Pat. No. 4,889,818 to Gelfand, et al; U.S. Pat. No. 4,994,370 to Silver, et al; U.S. Pat. No. 4,766,067 to Biswas; U.S. Pat. No. 4,656,134 to Ringold) and RNA mediated amplification that uses anti-sense RNA to the target sequence as a template for double-stranded DNA synthesis (U.S. Pat. No. 5,130,238 to Malek, et al, with the tradename NASBA), the entire contents of which references are incorporated herein by reference. (See, e.g., Ausubel, supra; or Sambrook, supra.)

For instance, polymerase chain reaction (PCR) technology can be used to amplify the sequences of polynucleotides of the disclosure and related genes directly from genomic DNA or cDNA libraries. PCR and other in vitro amplification methods can also be useful, for example, to clone nucleic acid sequences that code for proteins to be expressed, to make nucleic acids to use as probes for detecting the presence of the desired mRNA in samples, for nucleic acid sequencing, or for other purposes. Examples of techniques sufficient to direct persons of skill through in vitro amplification methods are found in Berger, supra, Sambrook, supra, and Ausubel, supra, as well as Mullis, et al., U.S. Pat. No. 4,683,202 (1987); and Innis, et al., PCR Protocols A Guide to Methods and Applications, Eds., Academic Press Inc., San Diego, Calif. (1990). Commercially available kits for genomic PCR amplification are known in the art. See, e.g., Advantage-GC Genomic PCR Kit (Clontech). Additionally, e.g., the T4 gene 32 protein (Boehringer Mannheim) can be used to improve yield of long PCR products.

Synthetic Methods for Constructing Nucleic Acids

The isolated nucleic acids of the disclosure can also be prepared by direct chemical synthesis by known methods (see, e.g., Ausubel, et al., supra). Chemical synthesis generally produces a single-stranded oligonucleotide, which can be converted into double-stranded DNA by hybridization with a complementary sequence, or by polymerization with a DNA polymerase using the single strand as a template. One of skill in the art will recognize that while chemical synthesis of DNA can be limited to sequences of about 100 or more bases, longer sequences can be obtained by the ligation of shorter sequences.

Recombinant Expression Cassettes

The disclosure further provides recombinant expression cassettes comprising a nucleic acid of the disclosure. A nucleic acid sequence of the disclosure, for example, a cDNA or a genomic sequence encoding a CARTyrin of the disclosure, can be used to construct a recombinant expression cassette that can be introduced into at least one desired host cell. A recombinant expression cassette will typically comprise a polynucleotide of the disclosure operably linked to transcriptional initiation regulatory sequences that will direct the transcription of the polynucleotide in the intended host cell. Both heterologous and non-heterologous (i.e., endogenous) promoters can be employed to direct expression of the nucleic acids of the disclosure.

In some embodiments, isolated nucleic acids that serve as promoter, enhancer, or other elements can be introduced in the appropriate position (upstream, downstream or in the intron) of a non-heterologous form of a polynucleotide of the disclosure so as to up or down regulate expression of a polynucleotide of the disclosure. For example, endogenous promoters can be altered in vivo or in vitro by mutation, deletion and/or substitution.

Vectors and Host Cells

The disclosure also relates to vectors that include isolated nucleic acid molecules of the disclosure, host cells that are genetically engineered with the recombinant vectors, and the production of at least one sequence by recombinant techniques, as is well known in the art. See, e.g., Sambrook, et al., supra; Ausubel, et al., supra, each entirely incorporated herein by reference.

For example, the PB-EF1a vector may be used. The vector comprises the following nucleotide sequence:

(SEQ ID NO: 17036)

tgtacatagattaaccctagaaagataatcatattgtgacgtacgttaaagataatcatgcgtaaaattgacgcatgtgtt

ttatcggtctgtatatcgaggtttatttattaatttgatagatattaagattattatatttacacttacatactaataata

aattcaacaaacaatttatttatgtttatttatttattaaaaaaaaacaaaaactcaaaatttcttctataaagtaacaaa

acttttatcgaatacctgcagcccgggggatgcagagggacagcccccccccaaagcccccagggatgtaattacgtccct

cccccgctagggggcagcagcgagccgcccggggctccgctccggtccggcgctccccccgcatccccgagccggcagcgt

gcggggacagcccgggcacggggaaggtggcacgggatcgctttcctctgaacgcttctcgctgctcagcctgcagacacc

tggggggatacggggaaaagttgactgtgcctttcgatcgaaccatggacagttagctttgcaaagatggataaagtttta

aacagagaggaatctttgcagctaatggaccttctaggtcttgaaaggagtgggaattggctccggtgcccgtcagtgggc

agagcgcacatcgcccacagtccccgagaagttggggggaggggtcggcaattgaaccggtgcctagagaaggtggcgcgg

ggtaaactgggaaagtgatgtcgtgtactggctccgcctttttcccgagggtgggggagaaccgtatataagtgcagtagt

cgccgtgaacgttctttttcgcaacgggtttgccgccagaacacaggtaagtgccgtgtgtggttcccgcgggcctggcct

ctttacgggttatggcccttgcgtgccttgaattacttccacctggctgcagtacgtgattcttgatcccgagcttcgggt

tggaagtgggtgggagagttcgaggccttgcgcttaaggagccccttcacctcgtgatgagttgagacctggcctgggcac

tggaaccgccgcgtgcgaatctggtggcaccttcgcgcctgtctcgctgctttcgataagtctctagccatttaaaatttt

tgatgacctgctgcgacgctttttttctggcaagatagtcttgtaaatgcgggccaagatctgcacactggtatttcggtt

tttggggccgcgggcgggcgacggggcccgtgcgtcccaacgcacatgttcggcgaggcggggcctgcgagcgcggccacc

gagaatcggacgggggtagtacaagctggccggcctgctctggtgcctggcctcgcgccgccgtgtatcgccccgccctgg

gcggcaaggctggcccggtcggcaccagttgcgtgagcggaaagatggccgcttcccggccctgctgcagggagctcaaaa

tggaggacgcggcgctcgggagagcgggcgggtgagtcacccacacaaaggaaaagggcctttccgtcctcagccgtcgct

tcatgtgactccacggagtaccgggcgccgtccaggcacctcgattagttctcgagcattggagtacgtcgtattagattg

gggagaggggttttatgcgatggagatccccacactgagtgggtggagactgaagttaggccagcttggcacttgatgtaa

ttctccttggaatttgccctttttgagtttggatcttggttcattctcaagcctcagacagtggttcaaagtttttttctt

ccatttcaggtgtcgtgagaattctaatacgactcactatagggtgtgctgtctcatcattttggcaaagattggccacca

agcttgtcctgcaggaggatcgacgcactagacgggcggccgctccggatccacgggtaccgatcacatatgcctttaatt

aaacactagttctatagtgtcacctaaattccattagtgagggttaatggccgtaggccgccagaattgggtccagacatg

ataagatacattgatgagtttggacaaaccacaactagaatgcagtgaaaaaaatgctttatttgtgaaatttgtgatgct

attgctttatttgtaaccattataagctgcaataaacaagttaacaacaacaattgcattcattttatgtttcaggttcag

ggggaggtgtgggaggttttttcggactctaggacctgcgcatgcgcttggcgtaatcatggtcatagctgtttcctgttt

tccccgtatccccccaggtgtctgcaggctcaaagagcagcgagaagcgttcagaggaaagcgatcccgtgccaccttccc

cgtgcccgggctgtccccgcacgctgccggctcggggatgcggggggagcgccggaccggagcggagccccgggcggctcg

ctgctgccccctagcgggggagggacgtaattacatccctgggggctttgggggggggctgtccactcaccgcggtggagc

tccagcattgttcgaattggagccccccctcgagggtatcgatgatatctataacaagaaaatatatatataataagttat

cacgtaagtagaacatgaaataacaatataattatcgtatgagttaaatcttaaaagtcacgtaaaagataatcatgcgtc

attttgactcacgcggtcgttatagttcaaaatcagtgacacttaccgcattgacaagcacgcctcacgggagctccaagc

ggcgactgagatgtcctaaatgcacagcgacggattcgcgctatttagaaagagagagcaatatttcaagaatgcatgcgt

caattttacgcagactatctttctagggttaatctagctagccttaagggcgcctattgcgttgcgctcactgcccgcttt

ccagtcgggaaacctgtcgtgccagctgcattaatgaatcggccaacgcgcggggagaggcggtttgcgtattgggcgcta

tccgcttcctccctcactgactcgctgcgctcggtcgttcggctgcggcgagcggtatcagctcactcaaaggcggtaata

cggttatccacagaatcaggggataacgcaggaaagaacataaccaaaatcccttaacgtgagtatcatccactgagcgtc

agaccccgtagaaaagatcaaaggatcttcttgagatcattttttctgcgcgtaatctgagcttgcaaacaaaaaaaccac

cgctaccagcggtggtttgtttgccggatcaagagctaccaactctttttccgaaggtaactggcttcagcagagcgcaga

taccaaatactgttcttctagtgtagccgtagttaggccaccacttcaagaactctgtagcacccctacatacctcgctag

ctaatcctgttaccagtggetgagccagtggcgataagtcgtgtcttaccgggttggactcaagacgatagttaccggtaa

ggcgcacggtcgggctgaacggggggttcgtgcacagcccagcttggagcgaacgacctacaccgaactgagatacctaca

gcgtgagctatgagaaagcgccacgcttcccgaagggagaaaggcggacaggtatccggtaagcggcagggtcggaacagg

agagcgcacgagggagatccagggggaaacgcctggtatctttatagtcctgtcgggtttcgccacctctgacttgagcgt

cgatttttgtgatgtcgtcaggggguggagcctatggaaaaacgccagcaacgcggcctttttacggttcctggccttttg

ctggccttttgctcacatgagattatcaaaaaggatcttcacctagatcatttaaattaaaaatgaagttttaaatcaatc

taaagtatatatgagtaaacttggtctgacagtcagaagaactcgtcaagaaggcgatagaaggcaatgcgctgcgaatcg

ggagcggcgataccgtaaagcacgaggaagcggtcagcccattcgccgccaagctatcagcaatatcacgggtagccaacg

ctatgtcagatagcggtccgccacacccagccggccacagtcgatgaatccagaaaagcggccatatccaccatgatattc

ggcaagcatgcatcgccatgggtcacgacgagatcctcgccgtcgggcatgctcgccttgagcctggcgaacagttcggct

ggcgcgagcccctgatgctcttcatccagatcatcctgatcgacaagaccggcttccatccgagtacgtgctcgctcgatg

cgatgtttcgcttggtggtcgaatgggcaggtagccggatcaagcgtatgcagccgccgcattgcatcagccatgatggat

actttctcggcaggagcaaggtgagatgacaggagatcctgccccggcacttcgccaatagcagccagtcccttcccgttc

agtgacaagtcgagcacagctgcaaggaacgcccgtcgtggccagccacgatagccgcgctgcctcgtcttgcagttcatt

cagggcaccggacaggtcggtcttgacaaaagaaccgggcgccctgcgctgacagccggaacacggcggcatcagagcagc

gattgtctgttgtgcccagtcatagccgaatagcctctccacccaagcggccggagaacctgcgtgcaatccatcttgttc

aatcataatattattgaagcatttatcagggttcgtctcgtcccggtctcctcccaatgcatgtcaatattggccattagc

catattattcattagttatatagcataaatcaatattggctattggccattgcatacgttgtatctatatcataata

The polynucleotides can optionally be joined to a vector containing a selectable marker for propagation in a host. Generally, a plasmid vector is introduced in a precipitate, such as a calcium phosphate precipitate, or in a complex with a charged lipid. If the vector is a virus, it can be packaged in vitro using an appropriate packaging cell line and then transduced into host cells.

The DNA insert should be operatively linked to an appropriate promoter. The expression constructs will further contain sites for transcription initiation, termination and, in the transcribed region, a ribosome binding site for translation. The coding portion of the mature transcripts expressed by the constructs will preferably include a translation initiating at the beginning and a termination codon (e.g., UAA, UGA or UAG) appropriately positioned at the end of the mRNA to be translated, with UAA and UAG preferred for mammalian or eukaryotic cell expression.

Expression vectors will preferably but optionally include at least one selectable marker. Such markers include, e.g., but are not limited to, ampicillin, zeocin (Sh bla gene), puromycin (pac gene), hygromycin B (hygB gene), G418/Geneticin (neo gene), mycophenolic acid, or glutamine synthetase (GS, U.S. Pat. Nos. 5,122,464; 5,770,359; 5,827,739), blasticidin (bsd gene), resistance genes for eukaryotic cell culture as well as ampicillin, zeocin (Sh bla gene), puromycin (pac gene), hygromycin B (hygB gene), G418/Geneticin (neo gene), kanamycin, spectinomycin, streptomycin, carbenicillin, bleomycin, erythromycin, polymyxin B, or tetracycline resistance genes for culturing in E. coli and other bacteria or prokaryotics (the above patents are entirely incorporated hereby by reference). Appropriate culture mediums and conditions for the above-described host cells are known in the art. Suitable vectors will be readily apparent to the skilled artisan. Introduction of a vector construct into a host cell can be effected by calcium phosphate transfection, DEAE-dextran mediated transfection, cationic lipid-mediated transfection, electroporation, transduction, infection or other known methods. Such methods are described in the art, such as Sambrook, supra, Chapters 1-4 and 16-18; Ausubel, supra, Chapters 1, 9, 13, 15, 16.

Expression vectors will preferably but optionally include at least one selectable cell surface marker for isolation of cells modified by the compositions and methods of the disclosure. Selectable cell surface markers of the disclosure comprise surface proteins, glycoproteins, or group of proteins that distinguish a cell or subset of cells from another defined subset of cells. Preferably the selectable cell surface marker distinguishes those cells modified by a composition or method of the disclosure from those cells that are not modified by a composition or method of the disclosure. Such cell surface markers include, e.g., but are not limited to, “cluster of designation” or “classification determinant” proteins (often abbreviated as “CD”) such as a truncated or full length form of CD19, CD271, CD34, CD22, CD20, CD33, CD52, or any combination thereof. Cell surface markers further include the suicide gene marker RQR8 (Philip B et al. Blood. 2014 Aug. 21; 124(8):1277-87).

Expression vectors will preferably but optionally include at least one selectable drug resistance marker for isolation of cells modified by the compositions and methods of the disclosure. Selectable drug resistance markers of the disclosure may comprise wild-type or mutant Neo, TYMS, FRANCF, RAD51C, GCS, MDR1, ALDH1, NKX2.2, or any combination thereof.

At least one sequence of the disclosure can be expressed in a modified form, such as a fusion protein, and can include not only secretion signals, but also additional heterologous functional regions. For instance, a region of additional amino acids, particularly charged amino acids, can be added to the N-terminus of sequence to improve stability and persistence in the host cell, during purification, or during subsequent handling and storage. Also, peptide moieties can be added to a sequence of the disclosure to facilitate purification. Such regions can be removed prior to final preparation of a sequence or at least one fragment thereof. Such methods are described in many standard laboratory manuals, such as Sambrook, supra, Chapters 17.29-17.42 and 18.1-18.74; Ausubel, supra, Chapters 16, 17 and 18.

Those of ordinary skill in the art are knowledgeable in the numerous expression systems available for expression of a nucleic acid encoding a protein of the disclosure. Alternatively, nucleic acids of the disclosure can be expressed in a host cell by turning on (by manipulation) in a host cell that contains endogenous DNA of the disclosure. Such methods are well known in the art, e.g., as described in U.S. Pat. Nos. 5,580,734, 5,641,670, 5,733,746, and 5,733,761, entirely incorporated herein by reference.

Illustrative of cell cultures useful for the production of the proteins, specified portions or variants thereof, are bacterial, yeast, and mammalian cells as known in the art. Mammalian cell systems often will be in the form of monolayers of cells although mammalian cell suspensions or bioreactors can also be used. A number of suitable host cell lines capable of expressing intact glycosylated proteins have been developed in the art, and include the COS-1 (e.g., ATCC CRL 1650), COS-7 (e.g., ATCC CRL-1651), HEK293, BHK21 (e.g., ATCC CRL-10), CHO (e.g., ATCC CRL 1610) and BSC-1 (e.g., ATCC CRL-26) cell lines, Cos-7 cells, CHO cells, hep G2 cells, P3×63Ag8.653, SP2/0-Ag14, 293 cells, HeLa cells and the like, which are readily available from, for example, American Type Culture Collection. Manassas, Va. (www.atcc.org). Preferred host cells include cells of lymphoid origin, such as myeloma and lymphoma cells. Particularly preferred host cells are P3×63Ag8.653 cells (ATCC Accession Number CRL-1580) and SP2/0-Ag14 cells (ATCC Accession Number CRL-1851). In a particularly preferred embodiment, the recombinant cell is a P3×63Ab8.653 or an SP2/0-Ag14 cell.

Expression vectors for these cells can include one or more of the following expression control sequences, such as, but not limited to, an origin of replication; a promoter (e.g., late or early SV40 promoters, the CMV promoter (U.S. Pat. Nos. 5,168,062; 5,385,839), an HSV tk promoter, a pgk (phosphoglycerate kinase) promoter, an EF-1 alpha promoter (U.S. Pat. No. 5,266,491), at least one human promoter; an enhancer, and/or processing information sites, such as ribosome binding sites, RNA splice sites, polyadenylation sites (e.g., an SV40 large T Ag poly A addition site), and transcriptional terminator sequences. See, e.g., Ausubel et al., supra; Sambrook, et al., supra. Other cells useful for production of nucleic acids or proteins of the present disclosure are known and/or available, for instance, from the American Type Culture Collection Catalogue of Cell Lines and Hybridomas (www.atcc.org) or other known or commercial sources.

When eukaryotic host cells are employed, polyadenlyation or transcription terminator sequences are typically incorporated into the vector. An example of a terminator sequence is the polyadenlyation sequence from the bovine growth hormone gene. Sequences for accurate splicing of the transcript can also be included. An example of a splicing sequence is the VP1 intron from SV40 (Sprague, et al., J. Virol. 45:773-781 (1983)). Additionally, gene sequences to control replication in the host cell can be incorporated into the vector, as known in the art.

Amino Acid Codes

The amino acids that make up compositions of the disclosure are often abbreviated. The amino acid designations can be indicated by designating the amino acid by its single letter code, its three letter code, name, or three nucleotide codon(s) as is well understood in the art (see Alberts, B., et al., Molecular Biology of The Cell, Third Ed., Garland Publishing, Inc., New York, 1994). A CARTyrin of the disclosure can include one or more amino acid substitutions, deletions or additions, from spontaneous or mutations and/or human manipulation, as specified herein. Amino acids in a composition of the disclosure that are essential for function can be identified by methods known in the art, such as site-directed mutagenesis or alanine-scanning mutagenesis (e.g., Ausubel, supra, Chapters 8, 15; Cunningham and Wells. Science 244:1081-1085 (1989)). The latter procedure introduces single alanine mutations at every residue in the molecule. The resulting mutant molecules are then tested for biological activity, such as, but not limited to, at least one neutralizing activity. Sites that are critical for CSR or CAR binding can also be identified by structural analysis, such as crystallization, nuclear magnetic resonance or photoaffinity labeling (Smith, et al., J. Mol. Biol. 224:899-904 (1992) and de Vos, et al., Science 255:306-312 (1992)).

As those of skill will appreciate, the disclosure includes at least one biologically active protein of the disclosure. Biologically active protein have a specific activity at least 20%, 30%, or 40%, and, preferably, at least 50%, 60%, or 70%, and, most preferably, at least 80%, 90%, or 95%-99% or more of the specific activity of the native (non-synthetic), endogenous or related and known protein. Methods of assaying and quantifying measures of enzymatic activity and substrate specificity are well known to those of skill in the art.

In another aspect, the disclosure relates to Centyrins and fragments, as described herein, which are modified by the covalent attachment of an organic moiety. Such modification can produce a protein fragment with improved pharmacokinetic properties (e.g., increased in vivo serum half-life). The organic moiety can be a linear or branched hydrophilic polymeric group, fatty acid group, or fatty acid ester group. In particular embodiments, the hydrophilic polymeric group can have a molecular weight of about 800 to about 120,000 Daltons and can be a polyalkane glycol (e.g., polyethylene glycol (PEG), polypropylene glycol (PPG)), carbohydrate polymer, amino acid polymer or polyvinyl pyrolidone, and the fatty acid or fatty acid ester group can comprise from about eight to about forty carbon atoms.

The modified sequence and fragments of the disclosure can comprise one or more organic moieties that are covalently bonded, directly or indirectly, to the antibody. Each organic moiety that is bonded to a sequence or fragment thereof of the disclosure can independently be a hydrophilic polymeric group, a fatty acid group or a fatty acid ester group. As used herein, the term “fatty acid” encompasses mono-carboxylic acids and di-carboxylic acids. A “hydrophilic polymeric group,” as the term is used herein, refers to an organic polymer that is more soluble in water than in octane. For example, polylysine is more soluble in water than in octane. Thus, a sequence modified by the covalent attachment of polylysine is encompassed by the disclosure. Hydrophilic polymers suitable for modifying sequences of the disclosure can be linear or branched and include, for example, polyalkane glycols (e.g., PEG, monomethoxy-polyethylene glycol (mPEG), PPG and the like), carbohydrates (e.g., dextran, cellulose, oligosaccharides, polysaccharides and the like), polymers of hydrophilic amino acids (e.g., polylysine, polyarginine, polyaspartate and the like), polyalkane oxides (e.g., polyethylene oxide, polypropylene oxide and the like) and polyvinyl pyrolidone. Preferably, the hydrophilic polymer that modifies a sequence of the disclosure has a molecular weight of about 800 to about 150,000 Daltons as a separate molecular entity. For example, PEG5000 and PEG 20,000, wherein the subscript is the average molecular weight of the polymer in Daltons, can be used. The hydrophilic polymeric group can be substituted with one to about six alkyl, fatty acid or fatty acid ester groups. Hydrophilic polymers that are substituted with a fatty acid or fatty acid ester group can be prepared by employing suitable methods. For example, a polymer comprising an amine group can be coupled to a carboxylate of the fatty acid or fatty acid ester, and an activated carboxylate (e.g., activated with N,N-carbonyl diimidazole) on a fatty acid or fatty acid ester can be coupled to a hydroxyl group on a polymer.

T Cell Isolation from a Leukapheresis Product

A leukapheresis product or blood may be collected from a subject at clinical site using a closed system and standard methods (e.g., a COBE Spectra Apheresis System). Preferably, the product is collected according to standard hospital or institutional Leukapheresis procedures in standard Leukapheresis collection bags. For example, in preferred embodiments of the methods of the disclosure, no additional anticoagulants or blood additives (heparin, etc.) are included beyond those normally used during leukapheresis.

Alternatively, white blood cells (WBC)/Peripheral Blood Mononuclear Cells (PBMC) (using Biosafe Sepax 2 (Closed/Automated)) or T cells (using CliniMACS® Prodigy (Closed/Automated)) may be isolated directly from whole blood. However, in certain subjects (e.g. those diagnosed and/or treated for cancer), the WBC/PBMC yield may be significantly lower when isolated from whole blood than when isolated by leukapheresis.

Either the leukapheresis procedure and/or the direct cell isolation procedure may be used for any subject of the disclosure.

The leukapheresis product, blood, WBC/PBMC composition and/or T-cell composition should be packed in insulated containers and should be kept at controlled room temperature (+19° C. to +25° C.) according to standard hospital of institutional blood collection procedures approved for use with the clinical protocol. The leukapheresis product, blood, WBC/PBMC composition and/or T-cell composition should not be refrigerated.

The cell concentration leukapheresis product, blood. WBC/PBMC composition and/or T-cell composition should not exceed 0.2×10⁹cells per mL during transportation. Intense mixing of the leukapheresis product, blood, WBC/PBMC composition and/or T-cell composition should be avoided.

If the leukapheresis product, blood, WBC/PBMC composition and/or T-cell composition has to be stored, e.g. overnight, it should be kept at controlled room temperature (same as above). During storage, the concentration of the leukapheresis product, blood, WBC/PBMC composition and/or T-cell composition should never exceed 0.2×10⁹cell per mL.

Preferably, cells of the leukapheresis product, blood, WBC/PBMC composition and/or T-cell composition should be stored in autologous plasma. In certain embodiments, if the cell concentration of the leukapheresis product, blood. WBC/PBMC composition and/or T-cell composition is higher than 0.2×10⁹cell per mL, the product should be diluted with autologous plasma.

Preferably, the leukapheresis product, blood, WBC/PBMC composition and/or T-cell composition should not be older than 24 hours when starting the labeling and separation procedure. The leukapheresis product, blood, WBC-PBMC composition and/or T-cell composition may be processed and/or prepared for cell labeling using a closed and/or automated system (e.g., CliniMACS Prodigy).

An automated system may perform additional buffy coat isolation, possibly by ficolation, and/or washing of the cellular product (e.g., the leukapheresis product, blood, WBC/PBMC composition and/or T cell composition).

A closed and/or automated system may be used to prepare and label cells for T-Cell isolation (from, for example, the leukapheresis product, blood, WBC/PBMC composition and/or T cell composition).

Although WBC/PBMCs may be nucleofected directly (which is easier and saves additional steps), the methods of the disclosure may include first isolating T cells prior to nucleofection. The easier strategy of directly nucleofecting PBMC requires selective expansion of modified cells that is mediated via CSR or CAR signaling, which by itself is proving to be an inferior expansion method that directly reduces the in vivo efficiency of the product by rendering T cells functionally exhausted. The product may be a heterogeneous composition of modified cells including T cells, NK cells, NKT cells, monocytes, or any combination thereof, which increases the variability in product from patient to patient and makes dosing and CRS management more difficult. Since T cells are thought to be the primary effectors in tumor suppression and killing, T cell isolation for the manufacture of an autologous product may result in significant benefits over the other more heterogeneous composition.

T cells may be isolated directly, by enrichment of labeled cells or depletion of labeled cells in a one-way labeling procedure or, indirectly, in a two-step labeling procedure. According to certain enrichment strategies of the disclosure, T cells may be collected in a Cell Collection Bag and the non-labeled cells (non-target cells) in a Negative Fraction Bag. In contrast to an enrichment strategy of the disclosure, the non-labeled cells (target cells) are collected in a Cell Collection Bag and the labeled cells (non-target cells) are collected in a Negative Fraction Bag or in the Non-Target Cell Bag, respectively. Selection reagents may include, but are not limited to, antibody-coated beads. Antibody-coated beads may either be removed prior to a modification and/or an expansion step, or, retained on the cells prior to a modification and/or an expansion step. One or more of the following non-limiting examples of cellular markers may be used to isolate T-cells: CD3, CD4, CD8, CD25, anti-biotin, CD1c, CD3/CD19, CD3/CD56, CD14, CD19, CD34, CD45RA, CD56, CD62L, CD133. CD137. CD271, CD304, IFN-gamma, TCR alpha/beta, and/or any combination thereof. Methods for the isolation of T-cells may include one or more reagents that specifically bind and/or detectably-label one or more of the following non-limiting examples of cellular markers may be used to isolate T-cells: CD3, CD4, CD8, CD25, anti-biotin, CD1c, CD3/CD19, CD3/CD56, CD14, CD19, CD34, CD45RA, CD56, CD62L, CD133, CD137, CD271, CD304, IFN-gamma, TCR alpha/beta, and/or any combination thereof. These reagents may or may not be “Good Manufacturing Practices” (“GMP”) grade. Reagents may include, but are not limited to, Thermo DynaBeads and Miltenyi CliniMACS products. Methods of isolating T-cells of the disclosure may include multiple iterations of labeling and/or isolation steps. At any point in the methods of isolating T-cells of the disclosure, unwanted cells and/or unwanted cell types may be depleted from a T cell product composition of the disclosure by positively or negatively selecting for the unwanted cells and/or unwanted cell types. A T cell product composition of the disclosure may contain additional cell types that may express CD4, CD8, and/or another T cell marker(s).

Methods of the disclosure for nucleofection of T cells may eliminate the step of T cell isolation by, for example, a process for nucleofection of T cells in a population or composition of WBC/PBMCs that, following nucleofection, includes an isolation step or a selective expansion step via TCR signaling.

Certain cell populations may be depleted by positive or negative selection before or after T cell enrichment and/or sorting. Examples of cell compositions that may be depleted from a cell product composition may include myeloid cells, CD25+ regulatory T cells (T Regs), dendritic cells, macrophages, red blood cells, mast cells, gamma-delta T cells, natural killer (NK) cells, a Natural Killer (NK)-like cell (e.g. a Cytokine Induced Killer (CIK) cell), induced natural killer (iNK) T cells, NK T cells, B cells, or any combination thereof.

T cell product compositions of the disclosure may include CD4+ and CD8+ T-Cells. CD4+ and CD8+ T-Cells may be isolated into separate collection bags during an isolation or selection procedure. CD4+ T cells and CD8+ T cells may be further treated separately, or treated after reconstitution (combination into the same composition) at a particular ratio.

The particular ratio at which CD4+ T cells and CD8+ T cells may be reconstituted may depend upon the type and efficacy of expansion technology used, cell medium, and/or growth conditions utilized for expansion of T-cell product compositions. Examples of possible CD4+: CD8+ ratios include, but are not limited to, 50%:50%, 60%:40%, 40%:60% 75%:25% and 25%:75%.

CD8+ T cells exhibit a potent capacity for tumor cell killing, while CD4+ T cells provide many of the cytokines required to support CD8+ T cell proliferative capacity and function. Because T cells isolated from normal donors are predominantly CD4+, the T-cell product compositions are artificially adjusted in vitro with respect to the CD4+:CD8+ ratio to improve upon the ratio of CD4+ T cells to CD8+ T cells that would otherwise be present in vivo. An optimized ratio may also be used for the ex vivo expansion of the autologous T− cell product composition. In view of the artificially adjusted CD4+:CD8+ ratio of the T-cell product composition, it is important to note that the product compositions of the disclosure may be significantly different and provide significantly greater advantage than any endogenously-occurring population of T-cells.

Preferred methods for T cell isolation may include a negative selection strategy for yielding untouched pan T cell, meaning that the resultant T-cell composition includes T-cells that have not been manipulated and that contain an endogenously-occurring variety/ratio of T-cells.

Reagents that may be used for positive or negative selection include, but are not limited to, magnetic cell separation beads. Magnetic cell separation beads may or may not be removed or depleted from selected populations of CD4+ T cells, CD8+ T cells, or a mixed population of both CD4+ and CD8+ T cells before performing the next step in a T-cell isolation method of the disclosure.

T cell compositions and T cell product compositions may be prepared for cryopreservation, storage in standard T Cell Culture Medium, and/or genetic modification.

T cell compositions, T cell product compositions, unstimulated T cell compositions, resting T cell compositions or any portion thereof may be cryopreserved using a standard cryopreservation method optimized for storing and recovering human cells with high recovery, viability, phenotype, and/or functional capacity. Commercially-available cryopreservation media and/or protocols may be used. Cryopreservation methods of the disclosure may include a DMSO free cryopreservant (e.g. CryoSOfree™ DMSO-free Cryopreservation Medium) reduce freezing-related toxicity.

T cell compositions, T cell product compositions, unstimulated T cell compositions, resting T cell compositions or any portion thereof may be stored in a culture medium. T cell culture media of the disclosure may be optimized for cell storage, cell genetic modification, cell phenotype and/or cell expansion. T cell culture media of the disclosure may include one or more antibiotics. Because the inclusion of an antibiotic within a cell culture media may decrease transfection efficiency and/or cell yield following genetic modification via nucleofection, the specific antibiotics (or combinations thereof) and their respective concentration(s) may be altered for optimal transfection efficiency and/or cell yield following genetic modification via nucleofection.

T cell culture media of the disclosure may include serum, and, moreover, the serum composition and concentration may be altered for optimal cell outcomes. Human AB serum is preferred over FBS/FCS for culture of T cells because, although contemplated for use in T cell culture media of the disclosure, FBS/FCS may introduce xeno-proteins. Serum may be isolated form the blood of the subject for whom the T-cell composition in culture is intended for administration, thus, a T cell culture medium of the disclosure may comprise autologous serum. Serum-free media or serum-substitute may also be used in T-cell culture media of the disclosure. In certain embodiments of the T-cell culture media and methods of the disclosure, serum-free media or serum-substitute may provide advantages over supplementing the medium with xeno-serum, including, but not limited to, healthier cells that have greater viability, nucleofect with higher efficiency, exhibit greater viability post-nucleofection, display a more desirable cell phenotype, and/or greater/faster expansion upon addition of expansion technologies.

T cell culture media may include a commercially-available cell growth media. Exemplary commercially-available cell growth media include, but are not limited to, PBS, HBSS, OptiMEM, DMEM, RPMI 1640, AIM-V, X-VIVO 15, CellGro DC Medium, CTS OpTimizer T Cell Expansion SFM, TexMACS Medium, PRIME-XV T Cell Expansion Medium, ImmunoCult-XF T Cell Expansion Medium, or any combination thereof.

T cell compositions, T cell product compositions, unstimulated T cell compositions, resting T cell compositions or any portion thereof may be prepared for genetic modification. Preparation of T cell compositions, T cell product compositions, unstimulated T cell compositions, resting T cell compositions or any portion thereof for genetic modification may include cell washing and/or resuspension in a desired nucleofection buffer. Cryopreserved T-cell compositions may be thawed and prepared for genetic modification by nucleofection. Cryopreserved cells may be thawed according to standard or known protocols. Thawing and preparation of cryopreserved cells may be optimized to yield cells that have greater viability, nucleofect with higher efficiency, exhibit greater viability post-nucleofection, display a more desirable cell phenotype, and/or greater/faster expansion upon addition of expansion technologies. For example, Grifols Albutein (25% human albumin) may be used in the thawing and/or preparation process.

Modification of an Autologous T Cell Product Composition

T cell compositions, T cell product compositions, unstimulated T cell compositions, resting T cell compositions or any portion thereof may be modified using, for example, a nucleofection strategy such as electroporation. The total number of cells to be nucleofected, the total volume of the nucleofection reaction, and the precise timing of the preparation of the sample may be optimized to yield cells that have greater viability, nucleofect with higher efficiency, exhibit greater viability post-nucleofection, display a more desirable cell phenotype, and/or greater/faster expansion upon addition of expansion technologies.

Nucleofection and/or electroporation may be accomplished using, for example. Lonza Amaxa, MaxCyte PulseAgile, Harvard Apparatus BTX, and/or Invitrogen Neon. Non-metal electrode systems, including, but not limited to, plastic polymer electrodes, may be preferred for nucleofection.

Prior to modification by nucleofection. T cell compositions, T cell product compositions, unstimulated T cell compositions, resting T cell compositions or any portion thereof may be resuspended in a nucleofection buffer. Nucleofection buffers of the disclosure include commercially-available nucleofection buffers. Nucleofection buffers of the disclosure may be optimized to yield cells that have greater viability, nucleofect with higher efficiency, exhibit greater viability post-nucleofection, display a more desirable cell phenotype, and/or greater/faster expansion upon addition of expansion technologies. Nucleofection buffers of the disclosure may include, but are not limited to, PBS, HBSS, OptiMEM, BTXpress, Amaxa Nucleofector, Human T cell nucleofection buffer and any combination thereof. Nucleofection buffers of the disclosure may comprise one or more supplemental factors to yield cells that have greater viability, nucleofect with higher efficiency, exhibit greater viability post-nucleofection, display a more desirable cell phenotype, and/or greater/faster expansion upon addition of expansion technologies. Exemplary supplemental factors include, but are not limited to, recombinant human cytokines, chemokines, interleukins and any combination thereof. Exemplary cytokines, chemokines, and interleukins include, but are not limited to, IL2, IL7, IL12, IL15, IL21, IL1, IL3, IL4, IL5, IL6, IL8, CXCL8, IL9, IL10, IL11, IL13, IL14, IL16, IL17, IL18, IL19, IL20, IL22, IL23, IL25, IL26, IL27, IL28, IL29, IL30, IL31, IL32, IL33, IL35, IL36, GM-CSF, IFN-gamma, IL-1 alpha/IL-1F1, IL-1 beta/IL-1F2, IL-12 p70, IL-12/IL-35 p35, IL-13, IL-17/IL-17A, IL-17A/F Heterodimer, IL-17F, IL-18/IL-1F4, IL-23, IL-24, IL-32, IL-32 beta, IL-32 gamma, IL-33, LAP (TGF-beta 1), Lymphotoxin-alpha/TNF-beta, TGF-beta, TNF-alpha, TRANCE/TNFSF11/RANK L and any combination thereof. Exemplary supplemental factors include, but are not limited to, salts, minerals, metabolites or any combination thereof. Exemplary salts, minerals, and metabolites include, but are not limited to, HEPES, Nicotinamide, Heparin, Sodium Pyruvate, L-Glutamine, MEM Non-Essential Amino Acid Solution, Ascorbic Acid. Nucleosides, FBS/FCS, Human serum, serum-substitute, anti-biotics, pH adjusters, Earle's Salts, 2-Mercaptoethanol, Human transferrin, Recombinant human insulin, Human serum albumin, Nucleofector PLUS Supplement, KCL, MgCl2, Na2HPO4, NAH2PO4, Sodium lactobionate, Manitol, Sodium succinate, Sodium Chloride, CINa, Glucose, Ca(NO3)2, Tris/HCl, K2HPO4, KH2PO4, Polyethylenimine, Poly-ethylene-glycol, Poloxamer 188, Poloxamer 181, Poloxamer 407, Poly-vinylpyrrolidone, Pop313, Crown-5, and any combination thereof. Exemplary supplemental factors include, but are not limited to, media such as PBS, HBSS. OptiMEM, DMEM, RPMI 1640, AIM-V, X-VIVO 15. CellGro DC Medium. CTS OpTimizer T Cell Expansion SFM, TexMACS Medium. PRIME-XV T Cell Expansion Medium, ImmunoCult-XF T Cell Expansion Medium and any combination thereof. Exemplary supplemental factors include, but are not limited to, inhibitors of cellular DNA sensing, metabolism, differentiation, signal transduction, the apoptotic pathway and combinations thereof. Exemplary inhibitors include, but are not limited to, inhibitors of TLR9, MyD88, IRAK, TRAF6, TRAF3, IRF-7, NF-KB, Type 1 Interferons, pro-inflammatory cytokines, cGAS, STING, Sec5, TBK1, IRF-3, RNA pol III, RIG-1, IPS-1, FADD, RIP1, TRAF3, AIM2, ASC, Caspase1, Pro-IL1B, PI3K. Akt, Wnt3A, inhibitors of glycogen synthase kinase-3β (GSK-3β) (e.g. TWS119), Bafilomycin, Chloroquine, Quinacrine, AC-YVAD-CMK, Z-VAD-FMK, Z-IETD-FMK and any combination thereof. Exemplary supplemental factors include, but are not limited to, reagents that modify or stabilize one or more nucleic acids in a way to enhance cellular delivery, enhance nuclear delivery or transport, enhance the facilitated transport of nucleic acid into the nucleus, enhance degradation of epi-chromosomal nucleic acid, and/or decrease DNA-mediated toxicity. Exemplary reagents that modify or stabilize one or more nucleic acids include, but are not limited to, pH modifiers, DNA-binding proteins, lipids, phospholipids, CaPO4, net neutral charge DNA binding peptides with or without NLS sequences, TREX1 enzyme, and any combination thereof.

Transposition reagents, including a transposon and a transposase, may be added to a nucleofection reaction of the disclosure prior to, simultaneously with, or after an addition of cells to a nucleofection buffer (optionally, contained within a nucleofection reaction vial or cuvette). Transposons of the disclosure may comprise plasmid DNA, linearized plasmid DNA, a PCR product, nanoplasmid, DOGGYBONET™ DNA, an mRNA template, a single or double-stranded DNA, a protein-nucleic acid combination or any combination thereof. Transposons of the disclosure may comprised one or more sequences that encode one or more TTAA site(s), one or more inverted terminal repeat(s) (ITRs), one or more long terminal repeat(s) (LTRs), one or more insulator(s), one or more promotor(s), one or more full-length or truncated gene(s), one or more polyA signal(s), one or more self-cleaving 2A peptide cleavage site(s), one or more internal ribosome entry site(s) (IRES), one or more enhancer(s), one or more regulator(s), one or more replication origin(s), and any combination thereof.

Transposons of the disclosure may comprise one or more sequences that encode one or more full-length or truncated gene(s). Full-length and/or truncated gene(s) introduced by transposons of the disclosure may encode one or more of a signal peptide, a hinge, a transmembrane domain, a costimulatory domain, a chimeric antigen receptor (CAR), a chimeric T-cell receptor (CAR-T, a CARTyrin or a VCAR), a receptor, a ligand, a cytokine, a drug resistance gene, a tumor antigen, an allo or auto antigen, an enzyme, a protein, a peptide, a poly-peptide, a fluorescent protein, a mutein or any combination thereof.

Transposons of the disclosure may be prepared in water, TAE, TBE, PBS, HBSS, media, a supplemental factor of the disclosure or any combination thereof.

Transposons of the disclosure may be designed to optimize clinical safety and/or improve manufacturability. As a non-limiting example, transposons of the disclosure may be designed to optimize clinical safety and/or improve manufacturability by eliminating unnecessary sequences or regions and/or including a non-antibiotic selection marker. Transposons of the disclosure may or may not be GMP grade.

Transposase enzymes of the disclosure may be encoded by one or more sequences of plasmid DNA, mRNA, protein, protein-nucleic acid combination or any combination thereof.

Transposase enzymes of the disclosure may be prepared in water, TAE, TBE, PBS, HBSS, media, a supplemental factor of the disclosure or any combination thereof. Transposase enzymes of the disclosure or the sequences/constructs encoding or delivering them may or may not be GMP grade.

Transposons and transposase enzymes of the disclosure may be delivered to a cell by any means.

Although compositions and methods of the disclosure include delivery of a transposon and/or transposase of the disclosure to a cell by plasmid DNA (pDNA), the use of a plasmid for delivery may allow the transposon and/or transposase to be integrated into the chromosomal DNA of the cell, which may lead to continued transposase expression. Accordingly, transposon and/or transposase enzymes of the disclosure may be delivered to a cell as either mRNA or protein to remove any possibility for chromosomal integration.

Transposons and transposases of the disclosure may be pre-incubated alone or in combination with one another prior to the introduction of the transposon and/or transposase into a nucleofection reaction. The absolute amounts of each of the transposon and the transposase, as well as the relative amounts, e.g., a ratio of transposon to transposase may be optimized.

Following preparation of nucleofection reaction, optionally, in a vial or cuvette, the reaction may be loaded into a nucleofector apparatus and activated for delivery of an electric pulse according to the manufacturer's protocol. Electric pulse conditions used for delivery of a transposon and/or a transposase of the disclosure (or a sequence encoding a transposon and/or a transposase of the disclosure) to a cell may be optimized for yielding cells with enhanced viability, higher nucleofection efficiency, greater viability post-nucleofection, desirable cell phenotype, and/or greater/faster expansion upon addition of expansion technologies. When using Amaxa nucleofector technology, each of the various nucleofection programs for the Amaxa 2B or 4D nucleofector are contemplated.

Following a nucleofection reaction of the disclosure, cells may be gently added to a cell medium. For example, when T cells undergo the nucleofection reaction, the T cells may be added to a T cell medium. Post-nucleofection cell media of the disclosure may comprise any one or more commercially-available media. Post-nucleofection cell media of the disclosure (including post-nucleofection T cell media of the disclosure) may be optimized to yield cells with greater viability, higher nucleofection efficiency, exhibit greater viability post-nucleofection, display a more desirable cell phenotype, and/or greater/faster expansion upon addition of expansion technologies. Post-nucleofection cell media of the disclosure (including post-nucleofection T cell media of the disclosure) may comprise PBS, HBSS, OptiMEM, DMEM, RPMI 1640, AIM-V, X-VIVO 15, CellGro DC Medium, CTS OpTimizer T Cell Expansion SFM, TexMACS Medium, PRIME-XV T Cell Expansion Medium, ImmunoCult-XF T Cell Expansion Medium and any combination thereof. Post-nucleofection cell media of the disclosure (including post-nucleofection T cell media of the disclosure) may comprise one or more supplemental factors of the disclosure to enhance viability, nucleofection efficiency, viability post-nucleofection, cell phenotype, and/or greater/faster expansion upon addition of expansion technologies. Exemplary supplemental factors include, but are not limited to, recombinant human cytokines, chemokines, interleukins and any combination thereof. Exemplary cytokines, chemokines, and interleukins include, but are not limited to, IL2, IL7, IL12, IL15, IL21, IL1, IL3, IL4, IL5, IL6, IL8, CXCL8, IL9, IL10, IL11, IL13, IL14, IL16, IL17, IL18, IL19, IL20, IL22, IL23, IL25, IL26, IL27, IL28, IL29, IL30, IL31, IL32, IL33, IL35, IL36, GM-CSF, IFN-gamma, IL-1 alpha/IL-1F1, IL-1 beta/IL-1F2, IL-12 p70, IL-12/IL-35 p35, IL-13, IL-17/IL-17A, IL-17A/F Heterodimer, IL-17F, IL-18/IL-1F4, IL-23, IL-24, IL-32, IL-32 beta, IL-32 gamma, IL-33, LAP (TGF-beta 1), Lymphotoxin-alpha/TNF-beta, TGF-beta, TNF-alpha, TRANCE/TNFSF11/RANK L and any combination thereof. Exemplary supplemental factors include, but are not limited to, salts, minerals, metabolites or any combination thereof. Exemplary salts, minerals, and metabolites include, but are not limited to, HEPES, Nicotinamide, Heparin, Sodium Pyruvate, L-Glutamine, MEM Non-Essential Amino Acid Solution, Ascorbic Acid, Nucleosides, FBS/FCS, Human serum, serum-substitute, anti-biotics, pH adjusters, Earle's Salts, 2-Mercaptoethanol, Human transferrin, Recombinant human insulin, Human serum albumin, Nucleofector PLUS Supplement, KCL, MgCl2, Na2HPO4, NAH2PO4, Sodium lactobionate, Manitol, Sodium succinate, Sodium Chloride, CINa, Glucose. Ca(NO3)2, Tris/HCl, K2HPO4, KH2PO4, Polyethylenimine, Poly-ethylene-glycol, Poloxamer 188, Poloxamer 181, Poloxamer 407, Poly-vinylpyrrolidone, Pop313, Crown-5, and any combination thereof. Exemplary supplemental factors include, but are not limited to, media such as PBS, HBSS, OptiMEM, DMEM, RPMI 1640, AIM-V, X-VIVO 15, CellGro DC Medium, CTS OpTimizer T Cell Expansion SFM, TexMACS Medium, PRIME-XV T Cell Expansion Medium, ImmunoCult-XF T Cell Expansion Medium and any combination thereof. Exemplary supplemental factors include, but are not limited to, inhibitors of cellular DNA sensing, metabolism, differentiation, signal transduction, the apoptotic pathway and combinations thereof. Exemplary inhibitors include, but are not limited to, inhibitors of TLR9, MyD88, IRAK, TRAF6, TRAF3, IRF-7, NF-KB, Type 1 Interferons, pro-inflammatory cytokines, cGAS, STING, Sec5, TBK1, IRF-3, RNA pol 111, RIG-1. IPS-1, FADD, RIP1, TRAF3, AIM2, ASC, Caspase1, Pro-IL1B, PI3K. Akt, Wnt3A, inhibitors of glycogen synthase kinase-3β (GSK-3β) (e.g. TWS119), Bafilomycin, Chloroquine, Quinacrine, AC-YVAD-CMK, Z-VAD-FMK, Z-IETD-FMK and any combination thereof. Exemplary supplemental factors include, but are not limited to, reagents that modify or stabilize one or more nucleic acids in a way to enhance cellular delivery, enhance nuclear delivery or transport, enhance the facilitated transport of nucleic acid into the nucleus, enhance degradation of epi-chromosomal nucleic acid, and/or decrease DNA-mediated toxicity. Exemplary reagents that modify or stabilize one or more nucleic acids include, but are not limited to, pH modifiers, DNA-binding proteins, lipids, phospholipids, CaPO4, net neutral charge DNA binding peptides with or without NLS sequences, TREX1 enzyme, and any combination thereof.

Post-nucleofection cell media of the disclosure (including post-nucleofection T cell media of the disclosure) may be used at room temperature or pre-warmed to, for example to between 32° C. to 37° C., inclusive of the endpoints. Post-nucleofection cell media of the disclosure (including post-nucleofection T cell media of the disclosure) may be pre-warmed to any temperature that maintains or enhances cell viability and/or expression of a transposon or portion thereof of the disclosure.

Post-nucleofection cell media of the disclosure (including post-nucleofection T cell media of the disclosure) may be contained in tissue culture flasks or dishes, G-Rex flasks, Bioreactor or cell culture bags, or any other standard receptacle. Post-nucleofection cell cultures of the disclosure (including post-nucleofection T cell cultures of the disclosure) may be may be kept still, or, alternatively, they may be perturbed (e.g. rocked, swirled, or shaken).

Post-nucleofection cell cultures may comprise modified cells. Post-nucleofection T cell cultures may comprise modified T cells. Modified cells of the disclosure may be either rested for a defined period of time or stimulated for expansion by, for example, the addition of a T Cell Expander technology. In certain embodiments, modified cells of the disclosure may be either rested for a defined period of time or immediately stimulated for expansion by, for example, the addition of a T Cell Expander technology. Modified cells of the disclosure may be rested to allow them sufficient time to acclimate, time for transposition to occur, and/or time for positive or negative selection, resulting in cells with enhanced viability, higher nucleofection efficiency, greater viability post-nucleofection, desirable cell phenotype, and/or greater/faster expansion upon addition of expansion technologies. Modified cells of the disclosure may be rested, for example, for 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or more hours. In certain embodiments, genetically modified cells of the disclosure may be rested, for example, for an overnight. In certain aspects, an overnight is about 12 hours. Modified cells of the disclosure may be rested, for example, for 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or more days.

Modified cells of the disclosure may be selected following a nucleofection reaction and prior to addition of an expander technology. For optimal selection of modified cells, the cells may be allowed to rest in a post-nucleofection cell medium for at least 2-14 days to facilitate identification of modified cells (e.g., differentiation of modified from non-modified cells).

As early as 24-hours post-nucleofection, expression of a Centyrin or CARTyrin and selection marker of the disclosure may be detectable in modified T cells upon successful nucleofection of a transposon of the disclosure. Due to epi-chromosomal expression of the transposon, expression of a selection marker alone may not differentiate modified T cells (those cells in which the transposon has been successfully integrated) from unmodified T cells (those cells in which the transposon was not successfully integrated). When epi-chromosomal expression of the transposon obscures the detection of modified cells by the selection marker, the nucleofected cells (both modified and unmodified cells) may be rested for a period of time (e.g. 2-14 days) to allow the cells to cease expression or lose all epi-chromosomal transposon expression. Following this extended resting period, only modified T cells should remain positive for expression of selection marker. The length of this extended resting period may be optimized for each nucleofection reaction and selection process. When epi-chromosomal expression of the transposon obscures the detection of modified cells by the selection marker, selection may be performed without this extended resting period, however, an additional selection step may be included at a later time point (e.g. either during or after the expansion stage).

Selection of modified cells of the disclosure may be performed by any means. In certain embodiments of the methods of the disclosure, selection of modified cells of the disclosure may be performed by isolating cells expressing a specific selection marker. Selection markers of the disclosure may be encoded by one or more sequences in the transposon. Selection markers of the disclosure may be expressed by the modified cell as a result of successful transposition (i.e., not encoded by one or more sequences in the transposon). In certain embodiments, modified cells of the disclosure contain a selection marker that confers resistance to a deleterious compound of the post-nucleofection cell medium. The deleterious compound may comprise, for example, an antibiotic or a drug that, absent the resistance conferred by the selection marker to the modified cells, would result in cell death. Exemplary selection markers include, but are not limited to, wild type (WT) or mutant forms of one or more of the following genes: neo, DHFR, TYMS, ALDH, MDR1, MGMT, FANCF, RAD51C, GCS, and NKX2.2. Exemplary selection markers include, but are not limited to, a surface-expressed selection marker or surface-expressed tag may be targeted by Ab-coated magnetic bead technology or column selection, respectively. A cleavable tag such as those used in protein purification may be added to a selection marker of the disclosure for efficient column selection, washing, and elution. In certain embodiments, selection markers of the disclosure are not expressed by the modified cells (including modified T cells) endogenously and, therefore, may be useful in the physical isolation of modified cells (by, for example, cell sorting techniques). Exemplary selection markers of the disclosure are not expressed by the modified cells (including modified T cells) endogenously include, but are not limited to, full-length, mutated, or truncated forms of CD271, CD19 CD52. CD34. RQR8, CD22, CD20, CD33 and any combination thereof.

In some embodiments of the modified cells of the disclosure, the selection marker comprises a protein that is active in dividing cells and not active in non-dividing cells. In some embodiments, the selection marker comprises a metabolic marker. In some embodiments, the selection marker comprises a dihydrofolate reductase (DHFR) mutein enzyme. In some embodiments, the DHFR mutein enzyme comprises or consists of the amino acid sequence of:

(SEQ ID NO: 17012)

1
MVGSLNCIVA VSQNMGIGKN GDFPWPPLRN ESRYFQRMTI

TSSVEGKQNL

61
VIMGKKTWFS IPEKNRPLKG RINLVLSREL KEPPOGAHFL

SRSLDDALKL

121
TEQPELANKV DMVWIVGGSS VYKEAMNHPG HLKLFVTRIM

QDFESDTFFP

181
EIDLEKYKLL PEYPGVLSDV QEEKGIKYKF EVYEKND.

In some embodiments, the amino acid sequence of the DHFR mutein enzyme further comprises a mutation at one or more of positions 80, 113, or 153. In some embodiments, the amino acid sequence of the DHFR mutein enzyme comprises one or more of a substitution of a Phenylalanine (F) or a Leucine (L) at position 80, a substitution of a Leucine (L) or a Valine (V) at position 113, and a substitution of a Valine (V) or an Aspartic Acid (D) at position 153.

Modified cells of the disclosure may be selective expanded following a nucleofection reaction. In certain embodiments, modified T cells comprising a CARTyrin may be selectively expanded by CARTyrin stimulation. Modified T cells comprising a CARTyrin may be stimulated by contact with a target-covered reagent (e.g. a tumor line or a normal cell line expressing a target or expander beads covered in a target). Alternatively, modified T cells comprising a CARTyrin may be stimulated by contact with an irradiated tumor cell, an irradiated allogeneic normal cell, an irradiated autologous PBMC. To minimize contamination of cell product compositions of the disclosure with a target-expressing cell used for stimulation, for example, when the cell product composition may be administered directly to a subject, the stimulation may be performed using expander beads coated with CARTyrin target protein. Selective expansion of modified T cells comprising a CARTyrin by CARTyrin stimulation may be optimized to avoid functionally-exhausting the modified T-cells.

Selected modified cells of the disclosure may be cryopreserved, rested for a defined period of time, or stimulated for expansion by the addition of a Cell Expander technology. Selected modified cells of the disclosure may be cryopreserved, rested for a defined period of time, or immediately stimulated for expansion by the addition of a Cell Expander technology. When the selected modified cells are T cells, the T cells may be stimulated for expansion by the addition of a T-Cell Expander technology. Selected modified cells of the disclosure may be rested, for example, for 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or more hours. In certain embodiments, selected modified cells of the disclosure may be rested, for example, for an overnight. In certain aspects, an overnight is about 12 hours. Selected modified cells of the disclosure may be rested, for example, for 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or more days. Selected modified cells of the disclosure may be rested for any period of time resulting in cells with enhanced viability, higher nucleofection efficiency, greater viability post-nucleofection, desirable cell phenotype, and/or greater/faster expansion upon addition of expansion technologies.

Selected modified cells (including selected modified T cells of the disclosure) may be cryopreserved using any standard cryopreservation method, which may be optimized for storing and/or recovering human cells with high recovery, viability, phenotype, and/or functional capacity. Cryopreservation methods of the disclosure may include commercially-available cryopreservation media and/or protocols.

A transposition efficiency of selected modified cells (including selected modified T cells of the disclosure) may be assessed by any means. For example, prior to the application of an expander technology, expression of the transposon by selected modified cells (including selected modified T cells of the disclosure) may be measured by fluorescence-activated cell sorting (FACS). Determination of a transposition efficiency of selected modified cells (including selected modified T cells of the disclosure) may include determining a percentage of selected cells expressing the transposon (e.g. a CARTyrin). Alternatively, or in addition, a purity of T cells, a Mean Fluorescence Intensity (MFI) of the transposon expression (e.g. CARTyrin expression), an ability of a CARTyrin (delivered in the transposon) to mediate degranulation and/or killing of a target cell expressing the CARTyrin ligand, and/or a phenotype of selected modified cells (including selected modified T cells of the disclosure) may be assessed by any means.

Cell product compositions of the disclosure may be released for administration to a subject upon meeting certain release criteria. Exemplary release criteria may include, but are not limited to, a particular percentage of modified, selected and/or expanded T cells expressing detectable levels of a CARTyrin on the cell surface.

Modification of an Autologous T Cell Product Composition

Modified cells (including modified T cells) of the disclosure may be expanded using an expander technology. Expander technologies of the disclosure may comprise a commercially-available expander technology. Exemplary expander technologies of the disclosure include stimulation a modified T cell of the disclosure via the TCR. While all means for stimulation of a modified T cell of the disclosure are contemplated, stimulation a modified T cell of the disclosure via the TCR is a preferred method, yielding a product with a superior level of killing capacity.

To stimulate a modified T cell of the disclosure via the TCR, Thermo Expander DynaBeads may be used at a 3:1 bead to T cell ratio. If the expander beads are not biodegradable, the beads may be removed from the expander composition. For example, the beads may be removed from the expander composition after about 5 days. To stimulate a modified T cell of the disclosure via the TCR, a Miltenyi T Cell Activation/Expansion Reagent may be used. To stimulate a modified T cell of the disclosure via the TCR. StemCell Technologies' ImmunoCult Human CD3/CD28 or CD3/CD28/CD2 T Cell Activator Reagent may be used. This technology may be preferred since the soluble tetrameric antibody complexes would degrade after a period and would not require removal from the process.

Artificial antigen presenting cells (APCs) may be engineered to co-express the target antigen and may be used to stimulate a cell or T-cell of the disclosure through a TCR and/or CARTyrin of the disclosure. Artificial APCs may comprise or may be derived from a tumor cell line (including, for example, the immortalized myelogenous leukemia line K562) and may be engineered to co-express multiple costimulatory molecules or technologies (such as CD28, 4-1BBL, CD64, mbIL-21, mbIL-15, CAR target molecule, etc.). When artificial APCs of the disclosure are combined with costimulatory molecules, conditions may be optimized to prevent the development or emergence of an undesirable phenotype and functional capacity, namely terminally-differentiated effector T cells.

Irradiated PBMCs (auto or allo) may express some target antigens, such as CD19, and may be used to stimulate a cell or T-cell of the disclosure through a TCR and/or CARTyrin of the disclosure. Alternatively, or in addition, irradiated tumor cells may express some target antigens and may be used to stimulate a cell or T-cell of the disclosure through a TCR and/or CARTyrin of the disclosure.

Plate-bound and/or soluble anti-CD3, anti-CD2 and/or anti-CD28 stimulate may be used to stimulate a cell or T-cell of the disclosure through a TCR and/or CARTyrin of the disclosure.

Antigen-coated beads may display target protein and may be used to stimulate a cell or T-cell of the disclosure through a TCR and/or CAR of the disclosure. Alternatively, or in addition, expander beads coated with a CARTyrin target protein may be used to stimulate a cell or T-cell of the disclosure through a TCR and/or CARTyrin of the disclosure.

Expansion methods drawn to stimulation of a cell or T-cell of the disclosure through the TCR or CARTyrin and via surface-expressed CD2, CD3, CD28, 4-1BB, and/or other markers on modified T cells.

An expansion technology may be applied to a cell of the disclosure immediately post-nucleofection until approximately 24 hours post-nucleofection. While various cell media may be used during an expansion procedure, a desirable T Cell Expansion Media of the disclosure may yield cells with, for example, greater viability, cell phenotype, total expansion, or greater capacity for in vivo persistence, engraftment, and/or CAR-mediated killing. Cell media of the disclosure may be optimized to improve/enhance expansion, phenotype, and function of modified cells of the disclosure. A preferred phenotype of expanded T cells may include a mixture of T stem cell memory. T central, and T effector memory cells. Expander Dynabeads may yield mainly central memory T cells which may lead to superior performance in the clinic.

Exemplary T cell expansion media of the disclosure may include, in part or in total, PBS, HBSS, OptiMEM, DMEM, RPMI 1640, AIM-V, X-VIVO 15, CellGro DC Medium. CTS OpTimizer T Cell Expansion SFM, TexMACS Medium. PRIME-XV T Cell Expansion Medium, ImmunoCult-XF T Cell Expansion Medium, or any combination thereof. T cell expansion media of the disclosure may further include one or more supplemental factors. Supplemental factors that may be included in a T cell expansion media of the disclosure enhance viability, cell phenotype, total expansion, or increase capacity for in vivo persistence, engraftment, and/or CARTyrin-mediated killing. Supplemental factors that may be included in a T cell expansion media of the disclosure include, but are not limited to, recombinant human cytokines, chemokines, and/or interleukins such as IL2, IL7, IL12, IL15, IL21, IL1, IL3, IL4, IL5, IL6, IL8, CXCL8, IL9, IL10, IL11, IL13, IL14, IL16, IL17, IL18, IL19, IL20, IL22, IL23, IL25, IL26, IL27, IL28, IL29, IL30, IL31, IL32, IL33, IL35, IL36, GM-CSF, IFN-gamma, IL-1 alpha/IL-1F1, IL-1 beta/IL-1F2, IL-12 p70, IL-12/IL-35 p35, IL-13, IL-17/IL-17A, IL-17A/F Heterodimer, IL-17F, IL-18/IL-1F4, IL-23, IL-24, IL-32, IL-32 beta, IL-32 gamma, IL-33, LAP (TGF-beta 1), Lymphotoxin-alpha/TNF-beta, TGF-beta, TNF-alpha. TRANCE/TNFSF11/RANK L. or any combination thereof. Supplemental factors that may be included in a T cell expansion media of the disclosure include, but are not limited to, salts, minerals, and/or metabolites such as HEPES, Nicotinamide, Heparin. Sodium Pyruvate, L-Glutamine, MEM Non-Essential Amino Acid Solution, Ascorbic Acid, Nucleosides, FBS/FCS, Human serum, serum-substitute, anti-biotics, pH adjusters, Earle's Salts, 2-Mercaptoethanol, Human transferrin, Recombinant human insulin, Human serum albumin, Nucleofector PLUS Supplement, KCL, MgCl2, Na2HPO4, NAH2PO4. Sodium lactobionate, Manitol, Sodium succinate, Sodium Chloride, CINa, Glucose, Ca(NO3)2. Tris/HCl, K2HPO4, KH2PO4, Polyethylenimine, Poly-ethylene-glycol, Poloxamer 188, Poloxamer 181, Poloxamer 407, Poly-vinylpyrrolidone. Pop313. Crown-5 or any combination thereof. Supplemental factors that may be included in a T cell expansion media of the disclosure include, but are not limited to, inhibitors of cellular DNA sensing, metabolism, differentiation, signal transduction, and/or the apoptotic pathway such as inhibitors of TLR9, MyD88, IRAK. TRAF6, TRAF3, IRF-7, NF-KB, Type 1 Interferons, pro-inflammatory cytokines, cGAS, STING, Sec5, TBK1, IRF-3, RNA pol III, RIG-1, IPS-1, FADD, RIP1, TRAF3, AIM2, ASC, Caspase1, Pro-IL1B, PI3K, Akt, Wnt3A, inhibitors of glycogen synthase kinase-3β (GSK-3β) (e.g. TWS119), Bafilomycin, Chloroquine, Quinacrine, AC-YVAD-CMK, Z-VAD-FMK, Z-IETD-FMK, or any combination thereof.

Supplemental factors that may be included in a T cell expansion media of the disclosure include, but are not limited to, reagents that modify or stabilize nucleic acids in a way to enhance cellular delivery, enhance nuclear delivery or transport, enhance the facilitated transport of nucleic acid into the nucleus, enhance degradation of epi-chromosomal nucleic acid, and/or decrease DNA-mediated toxicity, such as pH modifiers, DNA-binding proteins, lipids, phospholipids, CaPO4, net neutral charge DNA binding peptides with or without NLS sequences, TREX1 enzyme, or any combination thereof.

Modified cells of the disclosure may be selected during the expansion process by the use of selectable drugs or compounds. For example, in certain embodiments, when a transposon of the disclosure may encode a selection marker that confers to modified cells resistance to a drug added to the culture medium, selection may occur during the expansion process and may require approximately 1-14 days of culture for selection to occur. Examples of drug resistance genes that may be used as selection markers encoded by a transposon of the disclosure, include, but are not limited to, wild type (WT) or mutant forms of the genes neo, DHFR, TYMS, ALDH, MDR1, MGMT. FANCF, RAD51C. GCS, NKX2.2, or any combination thereof. Examples of corresponding drugs or compounds that may be added to the culture medium to which a selection marker may confer resistance include, but are not limited to, G418, Puromycin, Ampicillin, Kanamycin, Methotrexate, Mephalan, Temozolomide, Vincristine, Etoposide, Doxorubicin, Bendamustine, Fludarabine, Aredia (Pamidronate Disodium), Becenum (Carmustine), BiCNU (Carmustine), Bortezomib, Carfilzomib, Carmubris (Carmustine), Carmustine, Clafen (Cyclophosphamide), Cyclophosphamide, Cytoxan (Cyclophosphamide). Daratumumab, Darzalex (Daratumumab), Doxil (Doxorubicin Hydrochloride Liposome), Doxorubicin Hydrochloride Liposome, Dox-SL (Doxorubicin Hydrochloride Liposome), Elotuzumab, Empliciti (Elotuzumab), Evacet (Doxorubicin Hydrochloride Liposome), Farydak (Panobinostat), Ixazomib Citrate, Kyprolis (Carfilzomib), Lenalidomide, LipoDox (Doxorubicin Hydrochloride Liposome), Mozobil (Plerixafor), Neosar (Cyclophosphamide), Ninlaro (Ixazomib Citrate), Pamidronate Disodium. Panobinostat, Plerixafor, Pomalidomide, Pomalyst (Pomalidomide), Revlimid (Lenalidomide), Synovir (Thalidomide), Thalidomide, Thalomid (Thalidomide), Velcade (Bortezomib), Zoledronic Acid, Zometa (Zoledronic Acid), or any combination thereof.

A T-Cell Expansion process of the disclosure may occur in a cell culture bag in a WAVE Bioreactor, a G-Rex flask, or in any other suitable container and/or reactor.

A cell or T-cell culture of the disclosure may be kept steady, rocked, swirled, or shaken.

A cell or T-cell expansion process of the disclosure may optimize certain conditions, including, but not limited to culture duration, cell concentration, schedule for T cell medium addition/removal, cell size, total cell number, cell phenotype, purity of cell population, percentage of modified cells in growing cell population, use and composition of supplements, the addition/removal of expander technologies, or any combination thereof.

A cell or T-cell expansion process of the disclosure may continue until a predefined endpoint prior to formulation of the resultant expanded cell population. For example, a cell or T-cell expansion process of the disclosure may continue for a predetermined amount of time: at least, 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24 hours; at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30 days; at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 weeks; at least 1, 2, 3, 4, 5, 6, months, or at least 1 year. A cell or T-cell expansion process of the disclosure may continue until the resultant culture reaches a predetermined overall cell density: 1, 10, 100, 1000, 104, 105, 106, 107, 108, 109, 1010 cells per volume (p0, ml, L) or any density in between. A cell or T-cell expansion process of the disclosure may continue until the modified cells of a resultant culture demonstrate a predetermined level of expression of a transposon of the disclosure: 1%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100% or any percentage in between of a threshold level of expression (a minimum, maximum or mean level of expression indicating the resultant modified cells are clinically-efficacious). A cell or T-cell expansion process of the disclosure may continue until the proportion of modified cells of a resultant culture to the proportion of unmodified cells reaches a predetermined threshold: at least 1:10, 1:9, 1:8, 1:7, 1:6, 1:5, 1:4, 1:3, 1:2, 1:1, 2:1, 2:1, 4:1, 5:1, 6:1, 7:1, 8:1, 9:1 10:1 or any ratio in between.

Analysis of Modified Autologous T Cells for Release

A percentage of modified cells may be assessed during or after an expansion process of the disclosure. Cellular expression of a transposon by a modified cell of the disclosure may be measured by fluorescence-activated cell sorting (FACS). For example, FACS may be used to determine a percentage of cells or T cells expressing a CARTyrin of the disclosure. Alternatively, or in addition, a purity of modified cells or T cells, the Mean Fluorescence Intensity (MFI) of a CARTyrin expressed by a modified cell or T cell of the disclosure, an ability of the CARTyrin to mediate degranulation and/or killing of a target cell expressing the CARTyrin ligand, and/or a phenotype of CARTyrin+ T cells may be assessed.

Compositions of the disclosure intended for administration to a subject may be required to meet one or more “release criteria” that indicate that the composition is safe and efficacious for formulation as a pharmaceutical product and/or administration to a subject. Release criteria may include a requirement that a composition of the disclosure (e.g. a T-cell product of the disclosure) comprises a particular percentage of T cells expressing detectable levels of a CARTyrin of the disclosure on their cell surface.

The expansion process should be continued until a specific criterion has been met (e.g. achieving a certain total number of cells, achieving a particular population of memory cells, achieving a population of a specific size).

Certain criterion signal a point at which the expansion process should end. For example, cells should be formulated, reactivated, or cryopreserved once they reach a cell size of 300fL (otherwise, cells reaching a size above this threshold may start to die). Cryopreservation immediately once a population of cells reaches an average cell size of less than 300 fL may yield better cell recovery upon thawing and culture because the cells haven't yet reached a fully quiescent state prior to cryopreservation (a fully quiescent size is approximately 180 fL). Prior to expansion, T cells of the disclosure may have a cell size of about 180 fL, but may more than quadruple their cell size to approximately 900 fL at 3 days post-expansion. Over the next 6-12 days, the population of T-cells will slowly decrease cell size to full quiescence at 180 fL.

A process for preparing a cell population for formulation may include, but is not limited to the steps of, concentrating the cells of the cell population, washing the cells, and/or further selection of the cells via drug resistance or magnetic bead sorting against a particular surface-expressed marker. A process for preparing a cell population for formulation may further include a sorting step to ensure the safety and purity of the final product. For example, if a tumor cell from a patient has been used to stimulate a modified T-cell of the disclosure or that have been modified in order to stimulate a modified T-cell of the disclosure that is being prepared for formulation, it is critical that no tumor cells from the patient are included in the final product.

Cell Product Infusion and/or Cryopreservation for Infusion

A pharmaceutical formulation of the disclosure may be distributed into bags for infusion, cryopreservation, and/or storage.

A pharmaceutical formulation of the disclosure may be cryopreserved using a standard protocol and, optionally, an infusible cryopreservation medium. For example, a DMSO free cryopreservant (e.g. CryoSOfree™ DMSO-free Cryopreservation Medium) may be used to reduce freezing-related toxicity. A cryopreserved pharmaceutical formulation of the disclosure may be stored for infusion to a patient at a later date. An effective treatment may require multiple administrations of a pharmaceutical formulation of the disclosure and, therefore, pharmaceutical formulations may be packaged in pre-aliquoted “doses” that may be stored frozen but separated for thawing of individual doses.

A pharmaceutical formulation of the disclosure may be stored at room temperature. An effective treatment may require multiple administrations of a pharmaceutical formulation of the disclosure and, therefore, pharmaceutical formulations may be packaged in pre-aliquoted “doses” that may be stored together but separated for administration of individual doses.

A pharmaceutical formulation of the disclosure may be archived for subsequent re-expansion and/or selection for generation of additional doses to the same patient in the case of an allogenic therapy who may need an administration at a future date following, for example, a remission and relapse of a condition.

Formulations

As noted above, the disclosure provides for stable formulations, which preferably comprise a phosphate buffer with saline or a chosen salt, as well as preserved solutions and formulations containing a preservative as well as multi-use preserved formulations suitable for pharmaceutical or veterinary use, comprising at least one modified cell in a pharmaceutically acceptable formulation. Preserved formulations contain at least one known preservative or optionally selected from the group consisting of at least one phenol, m-cresol, p-cresol, o-cresol, chlorocresol, benzyl alcohol, phenylmercuric nitrite, phenoxyethanol, formaldehyde, chlorobutanol, magnesium chloride (e.g., hexahydrate), alkylparaben (methyl, ethyl, propyl, butyl and the like), benzalkonium chloride, benzethonium chloride, sodium dehydroacetate and thimerosal, polymers, or mixtures thereof in an aqueous diluent. Any suitable concentration or mixture can be used as known in the art, such as about 0.0015%, or any range, value, or fraction therein. Non-limiting examples include, no preservative, about 0.1-2% m-cresol (e.g., 0.2, 0.3, 0.4, 0.5, 0.9, 1.0%), about 0.1-3% benzyl alcohol (e.g., 0.5, 0.9, 1.1, 1.5, 1.9, 2.0, 2.5%), about 0.001-0.5% thimerosal (e.g., 0.005, 0.01), about 0.001-2.0% phenol (e.g., 0.05, 0.25, 0.28, 0.5, 0.9, 1.0%), 0.0005-1.0% alkylparaben(s) (e.g., 0.00075, 0.0009, 0.001, 0.002, 0.005, 0.0075, 0.009, 0.01, 0.02, 0.05, 0.075, 0.09, 0.1, 0.2, 0.3, 0.5, 0.75, 0.9, 1.0%), and the like.

As noted above, the disclosure provides an article of manufacture, comprising packaging material and at least one vial comprising a solution of at least one modified cell with the prescribed buffers and/or preservatives, optionally in an aqueous diluent, wherein said packaging material comprises a label that indicates that such solution can be held over a period of 1, 2, 3, 4, 5, 6, 9, 12, 18, 20, 24, 30, 36, 40, 48, 54, 60, 66, 72 hours or greater.

The present claimed articles of manufacture are useful for administration over a period ranging from immediate to twenty-four hours or greater. Accordingly, the presently claimed articles of manufacture offer significant advantages to the patient. Formulations of the disclosure can optionally be safely stored at temperatures of from about 2° C. to about 40° C. and retain the biological activity of the protein for extended periods of time, thus allowing a package label indicating that the solution can be held and/or used over a period of 6, 12, 18, 24, 36, 48, 72, or 96 hours or greater.

The products presently claimed include packaging material. The packaging material provides, in addition to the information required by the regulatory agencies, the conditions under which the product can be used.

Therapeutic Applications

The present disclosure also provides a method for modulating or treating a disease, in a cell, tissue, organ, animal, or patient, as known in the art or as described herein, using at least one composition of the disclosure. e.g., administering or contacting the cell, tissue, organ, animal, or patient with a therapeutic effective amount of a composition of the disclosure. The present disclosure also provides a method for modulating or treating a disease, in a cell, tissue, organ, animal, or patient including, but not limited to, a malignant disease.

The present disclosure also provides a method for modulating or treating at least one malignant disease in a cell, tissue, organ, animal or patient, including, but not limited to, at least one of: leukemia, acute leukemia, acute lymphoblastic leukemia (ALL), acute lymphocytic leukemia. B-cell, T-cell or FAB ALL, acute myeloid leukemia (AML), acute myelogenous leukemia, chronic myelocytic leukemia (CML), chronic lymphocytic leukemia (CLL), hairy cell leukemia, myelodyplastic syndrome (MDS), a lymphoma, Hodgkin's disease, a malignant lymphoma, non-Hodgkin's lymphoma, Burkitt's lymphoma, multiple myeloma. Kaposi's sarcoma, colorectal carcinoma, pancreatic carcinoma, nasopharyngeal carcinoma, malignant histiocytosis, paraneoplastic syndrome/hypercalcemia of malignancy, solid tumors, bladder cancer, breast cancer, colorectal cancer, endometrial cancer, head cancer, neck cancer, hereditary nonpolyposis cancer, Hodgkin's lymphoma, liver cancer, lung cancer, non-small cell lung cancer, ovarian cancer, pancreatic cancer, prostate cancer, renal cell carcinoma, testicular cancer, adenocarcinomas, sarcomas, malignant melanoma, hemangioma, metastatic disease, cancer related bone resorption, cancer related bone pain, and the like.

Any method of the present disclosure can comprise administering an effective amount of a composition or pharmaceutical composition to a cell, tissue, organ, animal or patient in need of such modulation, treatment or therapy. Such a method can optionally further comprise co-administration or combination therapy for treating such diseases or disorders, wherein the administering of said at least one composition, further comprises administering, before concurrently, and/or after, at least one selected from at least one of a second therapeutic agent. Suitable dosages are well known in the art. See, e.g., Wells et al., eds., Pharmacotherapy Handbook, 2nd Edition, Appleton and Lange, Stamford, Conn. (2000); PDR Pharmacopoeia, Tarascon Pocket Pharmacopoeia 2000, Deluxe Edition, Tarascon Publishing, Loma Linda, Calif. (2000); Nursing 2001 Handbook of Drugs, 21st edition, Springhouse Corp., Springhouse, Pa., 2001; Health Professional's Drug Guide 2001, ed., Shannon, Wilson, Stang, Prentice-Hall, Inc, Upper Saddle River, N.J. each of which references are entirely incorporated herein by reference.

Infusion of Modified Cells as Adoptive Cell Therapy

The disclosure provides modified cells that express one or more CSRs and/or CARs of the disclosure that have been selected and/or expanded for administration to a subject in need thereof. Modified cells of the disclosure may be formulated for storage at any temperature including room temperature and body temperature. Modified cells of the disclosure may be formulated for cryopreservation and subsequent thawing. Modified cells of the disclosure may be formulated in a pharmaceutically acceptable carrier for direct administration to a subject from sterile packaging. Modified cells of the disclosure may be formulated in a pharmaceutically acceptable carrier with an indicator of cell viability and/or protein expression level to ensure a minimal level of cell function and protein expression. Modified cells of the disclosure may be formulated in a pharmaceutically acceptable carrier at a prescribed density with one or more reagents to inhibit further expansion and/or prevent cell death.

Armored T-Cells “Knock-Down” Strategy

T-cells of the disclosure may be modified to enhance their therapeutic potential. Alternatively, or in addition, T-cells of the disclosure may be modified to render them less sensitive to immunologic and/or metabolic checkpoints. Modifications of this type “armor” the T cells of the disclosure, which, following the modification, may be referred to here as “armored” T cells. Armored T cells of the disclosure may be produced by, for example, blocking and/or diluting specific endogenous checkpoint signals delivered to the T-cells (i.e. checkpoint inhibition) within the tumor immunosuppressive microenvironment, for example.

In some embodiments, an armored T-cell of the disclosure is derived from a T cell, a NK cell, a hematopoietic progenitor cell, a peripheral blood (PB) derived T cell (including a T cell isolated or derived from G-CSF-mobilized peripheral blood), or an umbilical cord blood (UCB) derived T cell. In some embodiments, an armored T-cell of the disclosure comprises one or more of a chimeric ligand receptor (CLR comprising a protein scaffold, an antibody, an ScFv, or an antibody mimetic)/chimeric antigen receptor (CAR comprising a protein scaffold, an antibody, an ScFv, or an antibody mimetic), a CARTyrin (a CAR comprising a Centyrin), and/or a VCAR (a CAR comprising a camelid VHH or a single domain VH) of the disclosure. In some embodiments, an armored T-cell of the disclosure comprises an inducible proapoptotic polypeptide comprising (a) a ligand binding region, (b) a linker, and (c) a truncated caspase 9 polypeptide, wherein the inducible proapoptotic polypeptide does not comprise a non-human sequence. In some embodiments, the non-human sequence is a restriction site. In some embodiments, the ligand binding region inducible caspase polypeptide comprises a FK506 binding protein 12 (FKBP12) polypeptide. In some embodiments, the amino acid sequence of the FK506 binding protein 12 (FKBP12) polypeptide comprises a modification at position 36 of the sequence. In some embodiments, the modification is a substitution of valine (V) for phenylalanine (F) at position 36 (F36V). In some embodiments, an armored T-cell of the disclosure comprises an exogenous sequence. In some embodiments, the exogenous sequence comprises a sequence encoding a therapeutic protein. Exemplary therapeutic proteins may be nuclear, cytoplasmic, intracellular, transmembrane, cell-surface bound, or secreted proteins. Exemplary therapeutic proteins expressed by the armored T cell may modify an activity of the armored T cell or may modify an activity of a second cell. In some embodiments, an armored T-cell of the disclosure comprises a selection gene or a selection marker. In some embodiments, an armored T-cell of the disclosure comprises a synthetic gene expression cassette (also referred to herein as an inducible transgene construct).

In some embodiments, a T-cell of the disclosure is modified to silence or reduce expression one or more gene(s) encoding receptor(s) of inhibitory checkpoint signals to produce an armored T-cell of the disclosure. Examples of inhibitory checkpoint signals include, but are not limited to, a PD-L1 ligand binding to a PD-1 receptor on a CAR-T cell of the disclosure or a TGFβ cytokine binding to a TGFβRII receptor on a CAR-T cell. Receptors of inhibitory checkpoint signals are expressed on the cell surface or within the cytoplasm of a T-cell. Silencing or reducing expressing of the gene encoding the receptor of the inhibitory checkpoint signal results a loss of protein expression of the inhibitory checkpoint receptors on the surface or within the cytoplasm of an armored T-cell of the disclosure. Thus, armored T cells of the disclosure having silenced or reduced expression of one or more genes encoding an inhibitory checkpoint receptor is resistant, non-receptive or insensitive to checkpoint signals. The armored T cell's resistance or decreased sensitivity to inhibitory checkpoint signals enhances the armored T cell's therapeutic potential in the presence of these inhibitory checkpoint signals. Inhibitory checkpoint signals include but are not limited to the examples listed in Table 1. Exemplary inhibitory checkpoint signals that may be silenced in an armored T cell of the disclosure include, but are not limited to, PD-1 and TGFβRII.

TABLE 1

Exemplary Inhibitory Checkpoint Signals (and proteins that

induce immunosuppression). A CSR of the disclosure may comprise

an endodomain of any one of the proteins of this table.

Full Name
Abbreviation
SEQ ID NO:

Programmed cell death protein 1
PD1
14643-14644

transforming growth factor β Receptor 1
TGFβR1
14645

transforming growth factor β Receptor 2
TGFβR2
14646

T-cell immunoglobulin and mucin-domain
TIM3
14647

containing-3

Lymphocyte-activation gene 3
LAG3
14648

Cytotoxic T-lymphocyte protein 4
CTLA4
14649

B- and T-lymphocyte attenuator
BTLA
14650

Killer cell immunoglobulin-like receptor
KIR
14651

Alpha-2A adrenergic receptor
A2aR
14652

V-type immunoglobulin domain-containing
VISTA
14653

suppressor of T-cell activation

T-cell immunoreceptor with Ig and ITIM
TIGIT
14654

domains

Programmed cell death 1 ligand 1
B7H1 or PD-L1
14655

Programmed cell death 1 ligand 2
B7DC or PD-L2
14656

T-lymphocyte activation antigen CD80
B7-1 or CD80
14657

T-lymphocyte activation antigen CD86
B7-2 or CD86
14658

CD160 antigen
CD160
14659

Leukocyte-associated immunoglobulin-like
LAIR1
14660

receptor 1

T-cell immunoglobulin and mucin domain-
TIM4 or TIMD4
14661

containing protein 4

Natural killer cell receptor 2B4
2B4 or CD244
14662

Major Histocompatibility Complex type I
MHC I
14663

Major Histocompatibility Complex type II
MHC II

Putative 2-methylcitrate dehydratase receptor
PDH1R

T-cell immunoglobulin and mucin domain 1
TIM1R

receptor

T-cell immunoglobulin and mucin domain 4
TIM4R

receptor

B7-H3 receptor
B7H3R or CD176

Receptor

B7-H4 receptor
B7H4R

Immunoglobulin-like transcript (ILT) 3 receptor
ILT3R

phosphoinositide 3-kinase, subunit alpha
PI3K alpha
14664

phosphoinositide 3-kinase, subunit gamma
PI3K gamma
14665

Tyrosine-protein phosphatase non-receptor type
SHP2 or PTPN11
14666

11

Protein phosphatase 2, subunit gamma
PP2A gamma
14667

Protein phosphatase 2, subunit beta
PP2A beta
14668

Protein phosphatase 2, subunit delta
PP2A delta
14669

Protein phosphatase 2, subunit epsilon
PP2A epsilon
14670

Protein phosphatase 2, subunit alpha
PP2A alpha
14671

T-cell Receptor, subunit alpha
TCR alpha
14672

T-cell Receptor, subunit beta
TCR beta
14673

T-cell Receptor, subunit zeta
TCR zeta
14674

T-cell Receptor, subunit CD3 epsilon
TCR CD3 epsilon
14675

T-cell Receptor, subunit CD3 gamma
TCR CD3 gamma
14676

T-cell Receptor, subunit CD3 delta
TCR CD3 delta
14677

Cluster of Differentiation 28
CD28
14678

Galectins
Galectins

Galectin 9
Galectin 9
14679

High Mobility Group Box 1
HMGB1
14680

Arginase 1
ARG1
14681

Prostaglandin-Endoperoxide Synthase 1
PTGS1
14682

Prostaglandin-Endoperoxide Synthase 2
PTGS2
14683

Mucin 1, Cell Surface Associated
MUC1
14684

Mucin 2, Oligomeric Mucus/Gel-Forming
MUC2
14685

Mucin 3A, Cell Surface Associated
MUC3A
14686

Mucin 3B, Cell Surface Associated
MUC3B
14687

Mucin 4, Cell Surface Associated
MUC4
14688

Mucin 5AC, Oligomeric Mucus/Gel-Forming
MUC5AC
14689

Mucin 5B, Oligomeric Mucus/Gel-Forming
MUC5B
14690

Mucin 6, Oligomeric Mucus/Gel-Forming
MUC6
14691

Mucin 7, Secreted
MUC7
14692

Mucin 8
MUC8

Mucin 12, Cell Surface Associated
MUC12
14693

Mucin 13, Cell Surface Associated
MUC13
14694

Mucin 15, Cell Surface Associated
MUC15
14695

Mucin 16, Cell Surface Associated
MUC16
14696

Mucin 17, Cell Surface Associated
MUC17
14697

Mucin 19, Oligomeric
MUC19
14698

Mucin 20, Cell Surface Associated
MUC20
14699

Mucin 21, Cell Surface Associated
MUC21
14700

Mucin 22
MUC22
14701

Indoleamine 2,3-Dioxygenase 1
IDO1
14702

Indoleamine 2,3-Dioxygenase 2
IDO2
14703

Inducible T Cell Costimulator Ligand
ICOSLG
14704

ROS Proto-Oncogene 1, Receptor Tyrosine
ROS1
14705

Kinase

Tumor Necrosis Factor Receptor Superfamily
4-1BB, CD137, ILA or
14706

Member 9
TNFRSF9

4-1BB Ligand
4-1BB-L
14707

Glucocorticoid-induced TNFR family related
GITR
14708

gene

Glucocorticoid-induced TNFR family related
GITRL
14709

gene ligand

In some embodiments, a T-cell of the disclosure is modified to silence or reduce expression of one or more gene(s) encoding intracellular proteins involved in checkpoint signaling to produce an armored T-cell of the disclosure. The activity of a T-cell of the disclosure may be enhanced by targeting any intracellular signaling protein involved in a checkpoint signaling pathway, thereby achieving checkpoint inhibition or interference to one or more checkpoint pathways. Intracellular signaling proteins involved in checkpoint signaling include, but are not limited to, exemplary intracellular signaling proteins listed in Table 2.

TABLE 2

Exemplary Intracellular Signaling Proteins.

Full Name
Abbreviation
SEQ ID NO:

phosphoinositide 3-kinase, subunit alpha
PI3K alpha
14710

phosphoinositide 3-kinase, subunit gamma
PI3K gamma
14711

Tyrosine-protein phosphatase non-receptor type
SHP2 or PTPN11
14712

11

Protein phosphatase 2, subunit gamma
PP2A gamma
14713

Protein phosphatase 2, subunit beta
PP2A beta
14714

Protein phosphatase 2, subunit delta
PP2A delta
14715

Protein phosphatase 2, subunit epsilon
PP2A epsilon
14716

Protein phosphatase 2, subunit alpha
PP2A alpha
14717

RAC-alpha serine/threonine-protein kinase
AKT or PKB
14718

Tyrosine-protein kinase ZAP-70
ZAP70
14719

Amino acid sequence (KIEELE)-containing
KIEELE-domain

domain protein
containing proteins

BCL2 associated athanogene 6
Bat3, Bag6 or Scythe
14720

B-cell lymphoma-extra large
Bcl-xL
14721

Bcl-2-related protein A1
Bfl-1 or BCL2A1
14722

In some embodiments, a T-cell of the disclosure is modified to silence or reduce expression of one or more gene(s) encoding a transcription factor that hinders the efficacy of a therapy to produce an armored T-cell of the disclosure. The activity of armored T-cells may be enhanced or modulated by silencing or reducing expression (or repressing a function) of a transaction factor that hinders the efficacy of a therapy. Exemplary transcription factors that may be modified to silence or reduce expression or to repress a function thereof include, but are not limited tom the exemplary transcription factors listed in Table 3. For example expression of a FOXP3 gene may be silenced or reduced in an armored T cell of the disclosure to prevent or reduce the formation of T regulatory CAR-T-cells (CAR-Treg cells), the expression or activity of which may reduce efficacy of a therapy

TABLE 3

Exemplary Transcription Factors.

Full Name
Abbreviation
SEQ ID NO:

activity-dependent neuroprotector homeobox
ADNP
14723

ADNP homeobox 2
ADNP2
14724

AE binding protein 1
AEBP1
14725

AE binding protein 2
AEBP2
14726

AF4/FMR2 family member 1
AFF1
14727

AF4/FMR2 family member 2
AFF2
14728

AF4/FMR2 family member 3
AFF3
14729

AF4/FMR2 family member 4
AFF4
14730

AT-hook containing transcription factor 1
AHCTF1
14731

aryl hydrocarbon receptor
AHR
14732

aryl-hydrocarbon receptor repressor
AHRR
14733

autoimmune regulator
AIRE
14734

AT-hook transcription factor
AKNA
14735

ALX homeobox 1
ALX1
14736

ALX homeobox 3
ALX3
14737

ALX homeobox 4
ALX4
14738

ankyrin repeat and zinc finger domain containing 1
ANKZF1
14739

adaptor related protein complex 5 zeta 1 subunit
AP5Z1
14740

androgen receptor
AR
14741

arginine-fifty homeobox
ARGFX
14742

Rho GTPase activating protein 35
ARHGAP35
14743

AT-rich interaction domain 1A
ARID1A
14744

AT-rich interaction domain 1B
ARID1B
14745

AT-rich interaction domain 2
ARID2
14746

AT-rich interaction domain 3A
ARID3A
14747

AT-rich interaction domain 3B
ARID3B
14748

AT-rich interaction domain 3C
ARID3C
14749

AT-rich interaction domain 4A
ARID4A
14750

AT-rich interaction domain 4B
ARID4B
14751

AT-rich interaction domain 5A
ARID5A
14752

AT-rich interaction domain 5B
ARID5B
14753

aryl hydrocarbon receptor nuclear translocator
ARNT
14754

aryl hydrocarbon receptor nuclear translocator 2
ARNT2
14755

aryl hydrocarbon receptor nuclear translocator like
ARNTL
14756

aryl hydrocarbon receptor nuclear translocator like 2
ARNTL2
14757

aristaless related homeobox
ARX
14758

achaete-scute family bHLH transcription factor 1
ASCL1
14759

achaete-scute family bHLH transcription factor 2
ASCL2
14760

achaete-scute family bHLH transcription factor 3
ASCL3
14761

achaete-scute family bHLH transcription factor 4
ASCL4
14762

achaete-scute family bHLH transcription factor 5
ASCL5
14763

ash1 (absent, small, or homeotic)-like (Drosophila)
ASH1L
14764

ash2 (absent, small, or homeotic)-like (Drosophila)
ASH2L
14765

activating transcription factor 1
ATF1
14766

activating transcription factor 2
ATF2
14767

activating transcription factor 3
ATF3
14768

activating transcription factor 4
ATF4
14769

activating transcription factor 5
ATF5
14770

activating transcription factor 6
ATF6
14771

activating transcription factor 6 beta
ATF6B
14772

activating transcription factor 7
ATF7
14773

atonal bHLH transcription factor 1
ATOH1
14774

atonal bHLH transcription factor 7
ATOH7
14775

atonal bHLH transcription factor 8
ATOH8
14776

alpha thalassemia/mental retardation syndrome X-
ATRX
14777

linked

ataxin 7
ATXN7
14778

BTB and CNC homology 1, basic leucine zipper
BACH1
14779-14780

transcription factor 1

BTB domain and CNC homolog 2
BACH2
14781

BarH like homeobox 1
BARHL1
14782

BarH like homeobox 2
BARHL2
14783

BARX homeobox 1
BARX1
14784

BARX homeobox 2
BARX2
14785

Basic Leucine Zipper ATF-Like Transcription Factor,
Batf
14786

basic leucine zipper transcription factor, ATF-like
BATF
14786

basic leucine zipper transcription factor, ATF-like 2
BATF2
14787

basic leucine zipper transcription factor, ATF-like 3
BATF3
14788

bobby sox homolog (Drosophila)
BBX
14789

B-cell CLL/lymphoma 11A
BCL11A
14790

B-cell CLL/lymphoma 11B
BCL11B
14791

B-cell CLL/lymphoma 3
BCL3
14792

B-cell CLL/lymphoma 6
BCL6
14793

B-cell CLL/lymphoma 6, member B
BCL6B
14794

BCL2 associated transcription factor 1
BCLAF1
14795

basic helix-loop-helix family member a15
BHLHA15
14796

basic helix-loop-helix family member a9
BHLHA9
14797

basic helix-loop-helix domain containing, class B, 9
BHLHB9
14798

basic helix-loop-helix family member e22
BHLHE22
14799

basic helix-loop-helix family member e23
BHLHE23
14800

basic helix-loop-helix family member e40
BHLHE40
14801

basic helix-loop-helix family member e41
BHLHE41
14802

Beta-Interferon Gene Positive-Regulatory Domain I
Blimp-1
14803

Binding Factor

bone morphogenetic protein 2
BMP2
14804

basonuclin 1
BNC1
14805

basonuclin 2
BNC2
14806

bolA family member 1
BOLA1
14807

bolA family member 2
BOLA2
14808

bolA family member 3
BOLA3
14809

bromodomain PHD finger transcription factor
BPTF
14810

breast cancer 1
BRCA1
14811

brain specific homeobox
BSX
14812

chromosome 20 open reading frame 194
C20orf194
14813

calmodulin binding transcription activator 1
CAMTA1
14814

calmodulin binding transcription activator 2
CAMTA2
14815

calcium regulated heat stable protein 1
CARHSP1
14816

castor zinc finger 1
CASZ1
14817

core-binding factor, beta subunit
CBFB
14818

coiled-coil domain containing 79
CCDC79
14819

cell division cycle 5 like
CDC5L
14820

caudal type homeobox 1
CDX1
14821

caudal type homeobox 2
CDX2
14822

caudal type homeobox 4
CDX4
14823

CCAAT/enhancer binding protein alpha
CEBPA
14824

CCAAT/enhancer binding protein beta
CEBPB
14825

CCAAT/enhancer binding protein delta
CEBPD
14826

CCAAT/enhancer binding protein epsilon
CEBPE
14827

CCAAT/enhancer binding protein gamma
CEBPG
14828

CCAAT/enhancer binding protein zeta
CEBPZ
14829

centromere protein T
CENPT
14830

ceramide synthase 3
CERS3
14831

ceramide synthase 6
CERS6
14832

chromosome alignment maintaining phosphoprotein 1
CHAMP1
14833

capicua transcriptional repressor
CIC
14834

CDKN1A interacting zinc finger protein 1
CIZ1
14835

clock circadian regulator
CLOCK
14836

CCR4-NOT transcription complex subunit 4
CNOT4
14837

CPX chromosome region, candidate 1
CPXCR1
14838

cramped chromatin regulator homolog 1
CRAMP1
14839

cAMP responsive element binding protein 1
CREB1
14840

cAMP responsive element binding protein 3
CREB3
14841

cAMP responsive element binding protein 3-like 1
CREB3L1
14842

cAMP responsive element binding protein 3-like 2
CREB3L2
14843

cAMP responsive element binding protein 3-like 3
CREB3L3
14844

cAMP responsive element binding protein 3-like 4
CREB3L4
14845

cAMP responsive element binding protein 5
CREB5
14846

CREB binding protein
CREBBP
14847

cAMP responsive element binding protein-like 2
CREBL2
14848

CREB3 regulatory factor
CREBRF
14849

CREB/ATF bZIP transcription factor
CREBZF
14850

cAMP responsive element modulator
CREM
14851

cone-rod homeobox
CRX
14852

cysteine-serine-rich nuclear protein 1
CSRNP1
14853

cysteine-serine-rich nuclear protein 2
CSRNP2
14854

cysteine-serine-rich nuclear protein 3
CSRNP3
14855

CCCTC-binding factor (zinc finger protein)
CTCF
14856

CCCTC-binding factor like
CTCFL
14857

cut-like homeobox 1
CUX1
14858-14859

cut-like homeobox 2
CUX2
14860

CXXC finger protein 1
CXXC1
14861

dachshund family transcription factor 1
DACH1
14862

dachshund family transcription factor 2
DACH2
14863

D site of albumin promoter (albumin D-box) binding
DBP
14864

protein

developing brain homeobox 1
DBX1
14865

developing brain homeobox 2
DBX2
14866

damage specific DNA binding protein 2
DDB2
14867

DNA damage inducible transcript 3
DDIT3
14868

DEAF1, transcription factor
DEAF1
14869

distal-less homeobox 1
DLX1
14870

distal-less homeobox 2
DLX2
14871

distal-less homeobox 3
DLX3
14872

distal-less homeobox 4
DLX4
14873

distal-less homeobox 5
DLX5
14874

distal-less homeobox 6
DLX6
14875

DNA methyltransferase 1 associated protein 1
DMAP1
14876

diencephalon/mesencephalon homeobox 1
DMBX1
14877

doublesex and mab-3 related transcription factor 1
DMRT1
14878

doublesex and mab-3 related transcription factor 2
DMRT2
14879

doublesex and mab-3 related transcription factor 3
DMRT3
14880

DMRT like family Al
DMRTA1
14881

DMRT like family A2
DMRTA2
14882

DMRT like family B with proline rich C-terminal 1
DMRTB1
14883

DMRT like family C1
DMRTC1
14884

DMRT like family C1B
DMRTC1B
14884

DMRT like family C2
DMRTC2
14885

cyclin D binding myb like transcription factor 1
DMTF1
14886

DnaJ heat shock protein family (Hsp40) member C1
DNAJC1
14887

DnaJ heat shock protein family (Hsp40) member C2
DNAJC2
14888

DnaJ heat shock protein family (Hsp40) member C21
DNAJC21
14889

DNA (cytosine-5-)-methyltransferase 1
DNMT1
14890

DNA (cytosine-5-)-methyltransferase 3 alpha
DNMT3A
14891

DNA (cytosine-5-)-methyltransferase 3 beta
DNMT3B
14892

DNA (cytosine-5-)-methyltransferase 3-like
DNMT3L
14893

double PHD fingers 1
DPF1
14894

double PHD fingers 2
DPF2
14895

double PHD fingers 3
DPF3
14896

divergent-paired related homeobox
DPRX
14897

down-regulator of transcription 1
DR1
14898

DR1 associated protein 1
DRAP1
14899

dorsal root ganglia homeobox
DRGX
14900

double homeobox 4
DUX4
14901

double homeobox 4 like 9
DUX4L9
14902

double homeobox A
DUXA
14903

E2F transcription factor 1
E2F1
14904

E2F transcription factor 2
E2F2
14905

E2F transcription factor 3
E2F3
14906

E2F transcription factor 4
E2F4
14907

E2F transcription factor 5
E2F5
14908

E2F transcription factor 6
E2F6
14909

E2F transcription factor 7
E2F7
14910

E2F transcription factor 8
E2F8
14911

E4F transcription factor 1
E4F1
14912

early B-cell factor 1
EBF1
14913

early B-cell factor 2
EBF2
14914

early B-cell factor 3
EBF3
14915

early B-cell factor 4
EBF4
14916

early growth response 1
EGR1
14917

early growth response 2
EGR2
14918

early growth response 3
EGR3
14919

early growth response 4
EGR4
14920

ets homologous factor
EHF
14921

E74-like factor 1 (ets domain transcription factor)
ELF1
14922

E74-like factor 2 (ets domain transcription factor)
ELF2
14923

E74-like factor 3 (ets domain transcription factor,
ELF3
14924

epithelial-specific)

E74-like factor 4 (ets domain transcription factor)
ELF4
14925

E74-like factor 5 (ets domain transcription factor)
ELF5
14926

ELK1, member of ETS oncogene family
ELK1
14927

ELK3, ETS-domain protein (SRF accessory protein 2)
ELK3
14928

ELK4, ETS-domain protein (SRF accessory protein 1)
ELKA
14929

ELM2 and Myb/SANT-like domain containing 1
ELMSAN1
14930

empty spiracles homeobox 1
EMX1
14931

empty spiracles homeobox 2
EMX2
14932

engrailed homeobox 1
EN1
14933

engrailed homeobox 2
EN2
14934

enolase 1, (alpha)
ENO1
14935

eomesodermin
EOMES
14936

endothelial PAS domain protein 1
EPAS1
14937

Ets2 repressor factor
ERF
14938

v-ets avian erythroblastosis virus E26 oncogene
ERG
14939-14940

homolog

estrogen receptor 1
ESR1
14941

estrogen receptor 2 (ER beta)
ESR2
14942

estrogen related receptor alpha
ESRRA
14943

estrogen related receptor beta
ESRRB
14944

estrogen related receptor gamma
ESRRG
14945

ESX homeobox 1
ESX1
14946

v-ets avian erythroblastosis virus E26 oncogene
ETS1
14947

homolog 1

v-ets avian erythroblastosis virus E26 oncogene
ETS2
14948

homolog 2

ets variant 1
ETV1
14949

ets variant 2
ETV2
14950

ets variant 3
ETV3
14951

ets variant 3-like
ETV3L
14952

ets variant 4
ETV4
14953

ets variant 5
ETV5
14954

ets variant 6
ETV6
14955

ets variant 7
ETV7
14956

even-skipped homeobox 1
EVX1
14957

even-skipped homeobox 2
EVX2
14958

enhancer of zeste 1 poly comb repressive complex 2
EZH1
14959

subunit

enhancer of zeste 2 poly comb repressive complex 2
EZH2
14960

subunit

family with sequence similarity 170 member A
FAM170A
14961

Fer3-like bHLH transcription factor
FERD3L
14962

FEV (ETS oncogene family)
FEV
14963

FEZ family zinc finger 1
FEZF1
14964

FEZ family zinc finger 2
FEZF2
14965

folliculogenesis specific bHLH transcription factor
FIGLA
14966

FLT3-interacting zinc finger 1
FIZ1
14967

Fli-1 proto-oncogene, ETS transcription factor
FLI1
14968

FBJ murine osteosarcoma viral oncogene homolog
FOS
14969

FBJ murine osteosarcoma viral oncogene homolog B
FOSB
14970

FOS like antigen 1
FOSL1
14971

FOS like antigen 2
FOSL2
14972

forkhead box A1
FOXA1
14973

forkhead box A2
FOXA2
14974

forkhead box A3
FOXA3
14975

forkhead box B1
FOXB1
14976

forkhead box B2
FOXB2
14977

forkhead box C1
FOXC1
14978

forkhead box C2
FOXC2
14979

forkhead box D1
FOXD1
14980

forkhead box D2
FOXD2
14981

forkhead box D3
FOXD3
14982

forkhead box D4
FOXD4
14983

forkhead box D4-like 1
FOXD4L1
14984

forkhead box D4-like 3
FOXD4L3
14985

forkhead box D4-like 4
FOXD4L4
14986

forkhead box D4-like 5
FOXD4L5
14987

forkhead box D4-like 6
FOXD4L6
14988

forkhead box E1
FOXE1
14989

forkhead box E3
FOXE3
14990

forkhead box F1
FOXF1
14991

forkhead box F2
FOXF2
14992

forkhead box G1
FOXG1
14993

forkhead box H1
FOXH1
14994

forkhead box I1
FOXI1
14995

forkhead box I2
FOXI2
14996

forkhead box I3
FOXI3
14997

forkhead box J1
FOXJ1
14998

forkhead box J2
FOXJ2
14999

forkhead box J3
FOXJ3
15000

forkhead box K1
FOXK1
15001

forkhead box K2
FOXK2
15002

forkhead box L1
FOXL1
15003

forkhead box L2
FOXL2
15004

forkhead box M1
FOXM1
15005

forkhead box N1
FOXN1
15006

forkhead box N2
FOXN2
15007

forkhead box N3
FOXN3
15008

forkhead box N4
FOXN4
15009

forkhead box O1
FOXO1
15010

forkhead box O3
FOXO3
15011

forkhead box O4
FOXO4
15012

forkhead box O6
FOXO6
15013

forkhead box P1
FOXP1
15014

forkhead box P2
FOXP3
15015

forkhead box P3
FOXP4
15016

forkhead box P4
FOXQ1
15017

forkhead box Q1
FOXR1
15018

forkhead box R1
FOXR2
15019

forkhead box R2
FOXS1
15020

forkhead box S1
FOXP3
15021

far upstream element binding protein 1
FUBP1
15022

far upstream element (FUSE) binding protein 3
FUBP3
15023

GA binding protein transcription factor alpha subunit
GABPA
15024

GA binding protein transcription factor, beta subunit 1
GABPB1
15025

GA binding protein transcription factor, beta subunit 2
GABPB2
15026

GATA binding protein 1 (globin transcription factor 1)
GATA1
15027

GATA binding protein 2
GATA2
15028

GATA binding protein 3
GATA3
15029

GATA binding protein 4
GATA4
15030

GATA binding protein 5
GATA5
15031

GATA binding protein 6
GATA6
15032

GATA zinc finger domain containing 1
GATAD1
15033

GATA zinc finger domain containing 2 A
GATAD2A
15034

GATA zinc finger domain containing 2B
GATAD2B
15035

gastrulation brain homeobox 1
GBX1
15036

gastrulation brain homeobox 2
GBX2
15037

GC-rich sequence DNA-binding factor 2
GCFC2
15038

glial cells missing homolog 1
GCM1
15039

glial cells missing homolog 2
GCM2
15040

growth factor independent 1 transcription repressor
GFI1
15041

growth factor independent 1B transcription repressor
GFI1B
15042

GLI family zinc finger 1
GLI1
15043

GLI family zinc finger 2
GLI2
15044

GLI family zinc finger 3
GLI3
15045

GLI family zinc finger 4
GLI4
15046

GLIS family zinc finger 1
GLIS1
15047

GLIS family zinc finger 2
GLIS2
15048

GLIS family zinc finger 3
GLIS3
15049

glucocorticoid modulatory element binding protein 1
GMEB1
15050

glucocorticoid modulatory element binding protein 2
GMEB2
15051

gon-4-like (C. elegans)
GON4L
15052

grainyhead like transcription factor 1
GRHL1
15053

grainyhead like transcription factor 2
GRHL2
15054

grainyhead like transcription factor 3
GRHL3
15055

goosecoid homeobox
GSC
15056

goosecoid homeobox 2
GSC2
15057

GS homeobox 1
GSX1
15058

GS homeobox 2
GSX2
15059

general transcription factor IIi
GTF2I
15060

general transcription factor IIIA
GTF3A
15061

GDNF inducible zinc finger protein 1
GZF1
15062

heart and neural crest derivatives expressed 1
HAND1
15063

heart and neural crest derivatives expressed 2
HAND2
15064

HMG-box transcription factor 1
HBP1
15065-15066

highly divergent homeobox
HDX
15067

helt bHLH transcription factor
HELT
15068

hes family bHLH transcription factor 1
HES1
15069-15070

hes family bHLH transcription factor 2
HES2
15071

hes family bHLH transcription factor 3
HES3
15072

hes family bHLH transcription factor 4
HES4
15073

hes family bHLH transcription factor 5
HES5
15074

hes family bHLH transcription factor 6
HES6
15075

hes family bHLH transcription factor 7
HES7
15076

HESX homeobox 1
HESX1
15077

hes-related family bHLH transcription factor with
HEY1
15078

YRPW motif 1

hes-related family bHLH transcription factor with
HEY2
15079

YRPW motif 2

hes-related family bHLH transcription factor with
HEYL
15080

YRPW motif-like

hematopoietically expressed homeobox
HHEX
15081

hypermethylated in cancer 1
HIC1
15082

hypermethylated in cancer 2
HIC2
15083

hypoxia inducible factor 1, alpha subunit (basic helix-
HIF1A
15084

loop-helix transcription factor)

hypoxia inducible factor 3, alpha subunit
HIF3A
15085

histone H4 transcription factor
HINFP
15086

human immunodeficiency virus type I enhancer
HIVEP1
15087

binding protein 1

human immunodeficiency virus type I enhancer
HIVEP2
15088

binding protein 2

human immunodeficiency virus type I enhancer
HIVEP3
15089

binding protein 3

HKR1, GLI-Kruppel zinc finger family member
HKR1
15090

hepatic leukemia factor
HLF
15091

helicase-like transcription factor
HLTF
15092

H2.0-like homeobox
HLX
15093

homeobox containing 1
HMBOX1
15094

high mobility group 20A
HMG20A
15095

high mobility group 20B
HMG20B
15096

high mobility group AT-hook 1
HMGA1
15097

high mobility group AT-hook 2
HMGA2
15098

HMG-box containing 3
HMGXB3
15099

HMG-box containing 4
HMGXB4
15100

H6 family homeobox 1
HMX1
15101

H6 family homeobox 2
HMX2
15102

H6 family homeobox 3
HMX3
15103-15104

HNF1 homeobox A
HNF1A
15105

HNF1 homeobox B
HNF1B
15106

hepatocyte nuclear factor 4 alpha
HNF4A
15107

hepatocyte nuclear factor 4 gamma
HNF4G
15108

heterogeneous nuclear ribonucleoprotein K
HNRNPK
15109

homeobox and leucine zipper encoding
HOMEZ
15110

HOP homeobox
HOPX
15111

homeobox A1
HOXA1
15112

homeobox A10
HOXA10
15113

homeobox A11
HOXA11
15114

homeobox A13
HOXA13
15115

homeobox A2
HOXA2
15116

homeobox A3
HOXA3
15117

homeobox A4
HOXA4
15118

homeobox A5
HOXA5
15119

homeobox A6
HOXA6
15120

homeobox A7
HOXA7
15121

homeobox A9
HOXA9
15122

homeobox B1
HOXB1
15123

homeobox B13
HOXB13
15124

homeobox B2
HOXB2
15125

homeobox B3
HOXB3
15126

homeobox B4
HOXB4
15127

homeobox B5
HOXB5
15128

homeobox B6
HOXB6
15129

homeobox B7
HOXB7
15130

homeobox B8
HOXB8
15131

homeobox B9
HOXB9
15132

homeobox C10
HOXC10
15133

homeobox C11
HOXC11
15134

homeobox C12
HOXC12
15135

homeobox C13
HOXC13
15136

homeobox C4
HOXC4
15137

homeobox C5
HOXC5
15138

homeobox C6
HOXC6
15139

homeobox C8
HOXC8
15140

homeobox C9
HOXC9
15141

homeobox D1
HOXD1
15142

homeobox D10
HOXD10
15143

homeobox D11
HOXD11
15144

homeobox D12
HOXD12
15145

homeobox D13
HOXD13
15146

homeobox D3
HOXD3
15147

homeobox D4
HOXD4
15148

homeobox D8
HOXD8
15149

homeobox D9
HOXD9
15150

heat shock transcription factor 1
HSF1
15151

heat shock transcription factor 2
HSF2
15152

heat shock transcription factor 4
HSF4
15153

heat shock transcription factor family member 5
HSF5
15154

heat shock transcription factor family, X-linked 1
HSFX1
15155

heat shock transcription factor, Y-linked 1
HSFY1
15156

heat shock transcription factor, Y-linked 2
HSFY2
15156

inhibitor of DNA binding 1, dominant negative helix-
ID1
15157

loop-helix protein

inhibitor of DNA binding 2, dominant negative helix-
ID2
15158

loop-helix protein

inhibitor of DNA binding 3, dominant negative helix-
ID3
15159

loop-helix protein

inhibitor of DNA binding 4, dominant negative helix-
ID4
15160

loop-helix protein

interferon, gamma-inducible protein 16
IFI16
15161

IKAROS family zinc finger 1
IKZF1
15162

IKAROS family zinc finger 2
IKZF2
15163

IKAROS family zinc finger 3
IKZF3
15164

IKAROS family zinc finger 4
IKZF4
15165

IKAROS family zinc finger 5
IKZF5
15166

insulinoma associated 1
INSM1
15167

insulinoma-associated 2
INSM2
15168

interferon regulatory factor 1
IRF1
15169

interferon regulatory factor 2
IRF2
15170

interferon regulatory factor 3
IRF3
15171

interferon regulatory factor 4
IRF4
15172

interferon regulatory factor 5
IRF5
15173

interferon regulatory factor 6
IRF6
15174

interferon regulatory factor 7
IRF7
15175

interferon regulatory factor 8
IRF8
15176

interferon regulatory factor 9
IRF9
15177

iroquois homeobox 1
IRX1
15178

iroquois homeobox 2
IRX2
15179

iroquois homeobox 3
IRX3
15180

iroquois homeobox 4
IRX4
15181

iroquois homeobox 5
IRX5
15182

iroquois homeobox 6
IRX6
15183

ISL LIM homeobox 1
ISL1
15184

ISL LIM homeobox 2
ISL2
15185

intestine specific homeobox
ISX
15186

jumonji and AT-rich interaction domain containing 2
JARID2
15187

JAZF zinc finger 1
JAZF1
15188

Jun dimerization protein 2
JDP2
15189

jun proto-oncogene
JUN
15190

jun B proto-oncogene
JUNB
15191

jun D proto-oncogene
JUND
15192

K(lysine) acetyltransferase 5
KAT5
15193

lysine acetyltransferase 6A
KAT6A
15194

lysine acetyltransferase 6B
KAT6B
15195

lysine acetyltransferase 7
KAT7
15196

lysine acetyltransferase 8
KAT8
15197

potassium channel modulatory factor 1
KCMF1
15198

potassium voltage-gated channel interacting protein 3
KCNIP3
15199

lysine demethylase 2A
KDM2A
15200

lysine demethylase 5A
KDM5A
15201

lysine demethylase 5B
KDM5B
15202

lysine demethylase 5C
KDM5C
15203

lysine demethylase 5D
KDM5D
15204

KH-type splicing regulatory protein
KHSRP
15205

KIAA1549
KIAA1549
15206

Kruppel-like factor 1 (erythroid)
KLF1
15207

Kruppel-like factor 10
KLF10
15208

Kruppel-like factor 11
KLF11
15209

Kruppel-like factor 12
KLF12
15210

Kruppel-like factor 13
KLF13
15211

Kruppel-like factor 14
KLF14
15212

Kruppel-like factor 15
KLF15
15213

Kruppel-like factor 16
KLF16
15214

Kruppel-like factor 17
KLF17
15215

Kruppel-like factor 2
KLF2
15216

Kruppel-like factor 3 (basic)
KLF3
15217

Kruppel-like factor 4 (gut)
KLF4
15218

Kruppel-like factor 5 (intestinal)
KLF5
15219

Kruppel-like factor 6
KLF6
15220

Kruppel-like factor 7 (ubiquitous)
KLF7
15221

Kruppel-like factor 8
KLF8
15222

Kruppel-like factor 9
KLF9
15223

lysine methyltransferase 2A
KMT2A
15224

lysine methyltransferase 2B
KMT2B
15225

lysine methyltransferase 2C
KMT2C
15226

lysine methyltransferase 2E
KMT2E
15227

l(3)mbt-like 1 (Drosophila)
L3MBTL1
15228

l(3)mbt-like 2 (Drosophila)
L3MBTL2
15229

l(3)mbt-like 3 (Drosophila)
L3MBTL3
15230

l(3)mbt-like 4 (Drosophila)
L3MBTL4
15231

ladybird homeobox 1
LBX1
15232

ladybird homeobox 2
LBX2
15233

ligand dependent nuclear receptor corepressor
LCOR
15234

ligand dependent nuclear receptor corepressor like
LCORL
15235

lymphoid enhancer binding factor 1
LEF1
15236

leucine twenty homeobox
LEUTX
15237

LIM homeobox 1
LHX1
15238

LIM homeobox 2
LHX2
15239

LIM homeobox 3
LHX3
15240

LIM homeobox 4
LHX4
15241

LIM homeobox 5
LHX5
15242

LIM homeobox 6
LHX6
15243

LIM homeobox 8
LHX8
15244

LIM homeobox 9
LHX9
15245

LIM homeobox transcription factor 1, alpha
LMX1A
15246

LIM homeobox transcription factor 1, beta
LMX1B
15247

LOC730110
LOC730110

leucine rich repeat (in FLII) interacting protein 1
LRRFIP1
15248

leucine rich repeat (in FLII) interacting protein 2
LRRFIP2
15249

Ly 1 antibody reactive
LYAR
15250

lymphoblastic leukemia associated hematopoiesis
LYL1
15251

regulator 1

maelstrom spermatogenic transposon silencer
MAEL
15252

v-maf avian musculoaponeurotic fibrosarcoma
MAF
15253

oncogene homolog

MAF1 homolog, negative regulator of RNA
MAF1
15254

polymerase III

v-maf avian musculoaponeurotic fibrosarcoma
MAFA
15255-15256

oncogene homolog A

v-maf avian musculoaponeurotic fibrosarcoma
MAFB
15257

oncogene homolog B

v-maf avian musculoaponeurotic fibrosarcoma
MAFF
15258

oncogene homolog F

v-maf avian musculoaponeurotic fibrosarcoma
MAFG
15259

oncogene homolog G

v-maf avian musculoaponeurotic fibrosarcoma
MAFK
15260

oncogene homolog K

matrin 3
MATR3
15261

MYC associated factor X
MAX
15262

MYC associated zinc finger protein
MAZ
15263

methyl-CpG binding domain protein 1
MBD1
15264

methyl-CpG binding domain protein 2
MBD2
15265

methyl-CpG binding domain protein 3
MBD3
15266

methyl-CpG binding domain protein 3-like 1
MBD3L1
15267

methyl-CpG binding domain protein 3-like 2
MBD3L2
15268

methyl-CpG binding domain 4 DNA glycosylase
MBD4
15269

methyl-CpG binding domain protein 5
MBD5
15270

methyl-CpG binding domain protein 6
MBD6
15271

muscleblind like splicing regulator 3
MBNL3
15272

MDS1 and EVI1 complex locus
MECOM
15273

methyl-CpG binding protein 2
MECP2
15274

myocyte enhancer factor 2A
MEF2A
15275

myocyte enhancer factor 2B
MEF2B
15276

myocyte enhancer factor 2C
MEF2C
15277

myocyte enhancer factor 2D
MEF2D
15278

Meis homeobox 1
MEIS1
15279

Meis homeobox 2
MEIS2
15280

Meis homeobox 3
MEIS3
15281

Meis homeobox 3 pseudogene 1
MEIS3P1
15282

Meis homeobox 3 pseudogene 2
MEIS3P2
15283

mesenchyme homeobox 1
MEOX1
15284

mesenchyme homeobox 2
MEOX2
15285

mesoderm posterior bHLH transcription factor 1
MESP1
15286

mesoderm posterior bHLH transcription factor 2
MESP2
15287

MGA, MAX dimerization protein
MGA
15288-15289

MIER1 transcriptional regulator
MIER1
15290

MIER family member 2
MIER2
15291

MIER family member 3
MIER3
15292

MIS18 binding protein 1
MIS18BP1
15293

microphthalmia-associated transcription factor
MITF
15294

Mix paired-like homeobox
MIXL1
15295

mohawk homeobox
MKX
15296

myeloid/lymphoid or mixed-lineage leukemia;
MLLT1
15297

translocated to, 1

myeloid/lymphoid or mixed-lineage leukemia;
MLLT10
15298

translocated to, 10

myeloid/lymphoid or mixed-lineage leukemia;
MLLT11
15299

translocated to, 11

myeloid/lymphoid or mixed-lineage leukemia;
MLLT3
15300

translocated to, 3

myeloid/lymphoid or mixed-lineage leukemia;
MLLT4
15301

translocated to, 4

myeloid/lymphoid or mixed-lineage leukemia;
MLLT6
15302

translocated to, 6

MLX, MAX dimerization protein
MLX
15303

MLX interacting protein
MLXIP
15304

MLX interacting protein-like
MLXIPL
15305

MAX network transcriptional repressor
MNT
15306

motor neuron and pancreas homeobox 1
MNX1
15307

musculin
MSC
15308

mesogenin 1
MSGN1
15309

msh homeobox 1
MSX1
15310

msh homeobox 2
MSX2
15311

metastasis associated 1
MTA1
15312

metastasis associated 1 family member 2
MTA2
15313

metastasis associated 1 family member 3
MTA3
15314

metal-regulatory transcription factor 1
MTF1
15315

metal response element binding transcription factor 2
MTF2
15316

MAX dimerization protein 1
MXD1
15317

MAX dimerization protein 3
MXD3
15318

MAX dimerization protein 4
MXD4
15319

MAX interactor 1, dimerization protein
MXI1
15320

v-myb avian myeloblastosis viral oncogene homolog
MYB
15321

v-myb avian myeloblastosis viral oncogene homolog-
MYBL1
15322

like 1

v-myb avian myeloblastosis viral oncogene homolog-
MYBL2
15323

like 2

v-myc avian myelocytomatosis viral oncogene
MYC
15324

homolog

v-myc avian myelocytomatosis viral oncogene lung
MYCL
15325

carcinoma derived homolog

MYCL pseudogene 1
MYCLP1
15326

v-myc avian myelocytomatosis viral oncogene
MYCN
15327

neuroblastoma derived homolog

myogenic factor 5
MYF5
15328

myogenic factor 6
MYF6
15329

myoneurin
MYNN
15330

myogenic differentiation 1
MYOD1
15331

myogenin (myogenic factor 4)
MYOG
15332

myelin regulatory factor
MYRF
15333

Myb-like, SWIRM and MPN domains 1
MYSM1
15334

myelin transcription factor 1
MYT1
15335-15336

myelin transcription factor 1 like
MYT1L
15337

myeloid zinc finger 1
MZF1
15338

Nanog homeobox
NANOG
15339

NANOG neighbor homeobox
NANOGNB
15340

Nanog homeobox pseudogene 1
NANOGP1
15341

Nanog homeobox pseudogene 8
NANOGP8
15342

nuclear receptor coactivator 1
NCOA1
15343

nuclear receptor coactivator 2
NCOA2
15344

nuclear receptor coactivator 3
NCOA3
15345

nuclear receptor coactivator 4
NCOA4
15346

nuclear receptor coactivator 5
NCOA5
15347

nuclear receptor coactivator 6
NCOA6
15348

nuclear receptor coactivator 7
NCOA7
15349

nuclear receptor corepressor 1
NCOR1
15350

nuclear receptor corepressor 2
NCOR2
15351

neuronal differentiation 1
NEUROD1
15352

neuronal differentiation 2
NEUROD2
15353

neuronal differentiation 4
NEUROD4
15354

neuronal differentiation 6
NEUROD6
15355

neuro genin 1
NEUROG1
15356

neuro genin 2
NEUROG2
15357

neuro genin 3
NEUROG3
15358

nuclear factor of activated T-cells 5, tonicity-
NFAT5
15359

responsive

nuclear factor of activated T-cells, cytoplasmic,
NFATC1
15360

calcineurin-dependent 1

nuclear factor of activated T-cells, cytoplasmic,
NFATC2
15361

calcineurin-dependent 2

nuclear factor of activated T-cells, cytoplasmic,
NFATC3
15362

calcineurin-dependent 3

nuclear factor of activated T-cells, cytoplasmic,
NFATC4
15363

calcineurin-dependent 4

nuclear factor, erythroid 2
NFE2
15364

nuclear factor, erythroid 2 like 1
NFE2L1
15365

nuclear factor, erythroid 2 like 2
NFE2L2
15366

nuclear factor, erythroid 2 like 3
NFE2L3
15367

nuclear factor I/A
NFIA
15368

nuclear factor I/B
NFIB
15369

nuclear factor I/C (CCAAT-binding transcription
NFIC
15370

factor)

nuclear factor, interleukin 3 regulated
NFIL3
15371

nuclear factor I/X (CCAAT-binding transcription
NFIX
15372

factor)

nuclear factor of kappa light polypeptide gene
NFKB1
15373

enhancer in B-cells 1

nuclear factor of kappa light polypeptide gene
NFKB2
15374

enhancer in B-cells 2 (p49/p100)

nuclear factor of kappa light polypeptide gene
NFKBIA
15375

enhancer in B-cells inhibitor, alpha

nuclear factor of kappa light polypeptide gene
NFKBIB
15376

enhancer in B-cells inhibitor, beta

nuclear factor of kappa light polypeptide gene
NFKBID
15377

enhancer in B-cells inhibitor, delta

nuclear factor of kappa light polypeptide gene
NFKBIE
15378

enhancer in B-cells inhibitor, epsilon

nuclear factor of kappa light polypeptide gene
NFKBIL1
15379

enhancer in B-cells inhibitor-like 1

nuclear factor of kappa light polypeptide gene
NFKBIZ
15380

enhancer in B-cells inhibitor, zeta

nuclear factor related to kappaB binding protein
NFRKB
15381

nuclear transcription factor, X-box binding 1
NFX1
15382

nuclear transcription factor, X-box binding-like 1
NFXL1
15383

nuclear transcription factor Y subunit alpha
NFYA
15384

nuclear transcription factor Y subunit beta
NFYB
15385

nuclear transcription factor Y subunit gamma
NFYC
15386

nescient helix-loop-helix 1
NHLH1
15387

nescient helix-loop-helix 2
NHLH2
15388

NFKB repressing factor
NKRF
15389

NK1 homeobox 1
NKX1-1
15390

NK1 homeobox 2
NKX1-2
15391

NK2 homeobox 1
NKX2-1
15392

NK2 homeobox 2
NKX2-2
15393

NK2 homeobox 3
NKX2-3
15394

NK2 homeobox 4
NKX2-4
15395

NK2 homeobox 5
NKX2-5
15396

NK2 homeobox 6
NKX2-6
15397

NK2 homeobox 8
NKX2-8
15398

NK3 homeobox 1
NKX3-1
15399

NK3 homeobox 2
NKX3-2
15400

NK6 homeobox 1
NKX6-1
15401

NK6 homeobox 2
NKX6-2
15402

NK6 homeobox 3
NKX6-3
15403

NOBOX oogenesis homeobox
NOBOX
15404

NOC3 like DNA replication regulator
NOC3L
15405

nucleolar complex associated 4 homolog
NOC4L
15406

non-POU domain containing, octamer-binding
NONO
15407

notochord homeobox
NOTO
15408

neuronal PAS domain protein 1
NPAS1
15409

neuronal PAS domain protein 2
NPAS2
15410

neuronal PAS domain protein 3
NPAS3
15411

neuronal PAS domain protein 4
NPAS4
15412

nuclear receptor subfamily 0 group B member 1
NR0B1
15413

nuclear receptor subfamily 0 group B member 2
NR0B2
15414

nuclear receptor subfamily 1 group D member 1
NR1D1
15415

nuclear receptor subfamily 1 group D member 2
NR1D2
15416

nuclear receptor subfamily 1 group H member 2
NR1H2
15417

nuclear receptor subfamily 1 group H member 3
NR1H3
15418

nuclear receptor subfamily 1 group H member 4
NR1H4
15419

nuclear receptor subfamily 1 group I member 2
NR1I2
15420

nuclear receptor subfamily 1 group I member 3
NR1I3
15421

nuclear receptor subfamily 2 group C member 1
NR2C1
15422

nuclear receptor subfamily 2 group C member 2
NR2C2
15423

nuclear receptor subfamily 2 group E member 1
NR2E1
15424

nuclear receptor subfamily 2 group E member 3
NR2E3
15425

nuclear receptor subfamily 2 group F member 1
NR2F1
15426

nuclear receptor subfamily 2 group F member 2
NR2F2
15427

nuclear receptor subfamily 2 group F member 6
NR2F6
15428

nuclear receptor subfamily 3 group C member 1
NR3C1
15429

nuclear receptor subfamily 3 group C member 2
NR3C2
15430

nuclear receptor subfamily 4 group A member 1
NR4A1
15431

nuclear receptor subfamily 4 group A member 2
NR4A2
15432

nuclear receptor subfamily 4 group A member 3
NR4A3
15433

nuclear receptor subfamily 5 group A member 1
NR5A1
15434

nuclear receptor subfamily 5 group A member 2
NR5A2
15435

nuclear receptor subfamily 6 group A member 1
NR6A1
15436

nuclear respiratory factor 1
NRF1
15437-15438

neural retina leucine zipper
NRL
15439

oligodendrocyte transcription factor 1
OLIG1
15440

oligodendrocyte lineage transcription factor 2
OLIG2
15441

oligodendrocyte transcription factor 3
OLIG3
15442

one cut homeobox 1
ONECUT1
15443

one cut homeobox 2
ONECUT2
15444

one cut homeobox 3
ONECUT3
15445

odd-skipped related transciption factor 1
OSR1
15446

odd-skipped related transciption factor 2
OSR2
15447

orthopedia homeobox
OTP
15448

orthodenticle homeobox 1
OTX1
15449

orthodenticle homeobox 2
OTX2
15450

ovo like zinc finger 1
OVOL1
15451

ovo like zinc finger 2
OVOL2
15452

ovo like zinc finger 3
OVOL3
15453

poly(ADP-ribose) polymerase 1
PARP1
15454

poly(ADP-ribose) polymerase family member 12
PARP12
15455

POZ/BTB and AT hook containing zinc finger 1
PATZ1
15456

PRKC, apoptosis, WT1, regulator
PAWR
15457

paired box 1
PAX1
15458

paired box 2
PAX2
15459

paired box 3
PAX3
15460

paired box 4
PAX4
15461

paired box 5
PAX5
15462

paired box 6
PAX6
15463

paired box 7
PAX7
15464

paired box 8
PAX8
15465

paired box 9
PAX9
15466

PAX3 and PAX7 binding protein 1
PAXBP1
15467

polybromo 1
PBRM1
15468

pre-B-cell leukemia homeobox 1
PBX1
15469

pre-B-cell leukemia homeobox 2
PBX2
15470

pre-B-cell leukemia homeobox 3
PBX3
15471

pre-B-cell leukemia homeobox 4
PBX4
15472

poly(rC) binding protein 1
PCBP1
15473

poly(rC) binding protein 2
PCBP2
15474

poly(rC) binding protein 3
PCBP3
15475

poly(rC) binding protein 4
PCBP4
15476

poly comb group ring finger 6
PCGF6
15477

pancreatic and duodenal homeobox 1
PDX1
15478-15479

paternally expressed 3
PEG3
15480

progesterone receptor
PGR
15481

prohibitin
PHB
15482

prohibitin 2
PHB2
15483

PHD finger protein 20
PHF20
15484

PHD finger protein 5A
PHF5A
15485

paired like homeobox 2a
PHOX2A
15486

paired like homeobox 2b
PHOX2B
15487

putative homeodomain transcription factor 1
PHTF1
15488

putative homeodomain transcription factor 2
PHTF2
15489

paired like homeodomain 1
PITX1
15490

paired like homeodomain 2
PITX2
15491

paired like homeodomain 3
PITX3
15492

PBX/knotted 1 homeobox 1
PKNOX1
15493

PBX/knotted 1 homeobox 2
PKNOX2
15494

PLAG1 zinc finger
PLAG1
15495

PLAG1 like zinc finger 1
PLAGL1
15496

PLAG1 like zinc finger 2
PLAGL2
15497

pleckstrin
PLEK
15498

promyelocytic leukaemia zinc finger
PLZF
15499

pogo transposable element with ZNF domain
POGZ
15500

POU class 1 homeobox 1
POU1F1
15501

POU class 2 associating factor 1
POU2AF1
15502

POU class 2 homeobox 1
POU2F1
15503

POU class 2 homeobox 2
POU2F2
15504

POU class 2 homeobox 3
POU2F3
15505

POU class 3 homeobox 1
POU3F1
15506

POU class 3 homeobox 2
POU3F2
15507

POU class 3 homeobox 3
POU3F3
15508

POU class 3 homeobox 4
POU3F4
15509

POU class 4 homeobox 1
POU4F1
15510

POU class 4 homeobox 2
POU4F2
15511

POU class 4 homeobox 3
POU4F3
15512

POU class 5 homeobox 1
POU5F1
15513

POU class 5 homeobox 1B
POU5F1B
15514

POU domain class 5, transcription factor 2
POU5F2
15515

POU class 6 homeobox 1
POU6F1
15516

POU class 6 homeobox 2
POU6F2
15517

peroxisome proliferator activated receptor alpha
PPARA
15518

peroxisome proliferator activated receptor delta
PPARD
15519

peroxisome proliferator activated receptor gamma
PPARG
15520

protein phosphatase 1 regulatory subunit 13 like
PPP1R13L
15521

PR domain 1
PRDM1
15522

PR domain 10
PRDM10
15523

PR domain 11
PRDM11
15524

PR domain 12
PRDM12
15525

PR domain 13
PRDM13
15526

PR domain 14
PRDM14
15527

PR domain 15
PRDM15
15528

PR domain 16
PRDM16
15529

PR domain 2
PRDM2
15530

PR domain 4
PRDM4
15531

PR domain 5
PRDM5
15532

PR domain 6
PRDM6
15533

PR domain 7
PRDM7
15534

PR domain 8
PRDM8
15535

PR domain 9
PRDM9
15536

prolactin regulatory element binding
PREB
15537

PROP paired-like homeobox 1
PROP1
15538

prospero homeobox 1
PROX1
15539

prospero homeobox 2
PROX2
15540

paired related homeobox 1
PRRX1
15541

paired related homeobox 2
PRRX2
15542

paraspeckle component 1
PSPC1
15543

pancreas specific transcription factor, 1a
PTF1A
15544

purine-rich element binding protein A
PURA
15545

purine-rich element binding protein B
PURB
15546

purine-rich element binding protein G
PURG
15547

retinoic acid receptor alpha
RARA
15548

retinoic acid receptor beta
RARB
15549

retinoic acid receptor gamma
RARG
15550

retina and anterior neural fold homeobox
RAX
15551-15552

retina and anterior neural fold homeobox 2
RAX2
15553

RB associated KRAB zinc finger
RBAK
15554

RNA binding motif protein 22
RBM22
15555

recombination signal binding protein for
RBPJ
15556

immunoglobulin kappa J region

recombination signal binding protein for
RBPJL
15557

immunoglobulin kappa J region-like

ring finger and CCCH-type domains 1
RC3H1
15558

ring finger and CCCH-type domains 2
RC3H2
15559

REST corepressor 1
RCOR1
15560

REST corepressor 2
RCOR2
15561

REST corepressor 3
RCOR3
15562

v-rel avian reticuloendothcliosis viral oncogene
REL
15563

homolog

v-rel avian reticuloendothcliosis viral oncogene
RELA
15564

homolog A

v-rel avian reticuloendothcliosis viral oncogene
RELB
15565

homolog B

arginine-glutamic acid di peptide (RE) repeats
RERE
15566

RE1-silencing transcription factor
REST
15567

regulatory factor X1
RFX1
15568

regulatory factor X2
RFX2
15569

regulatory factor X3
RFX3
15570

regulatory factor X4
RFX4
15571

regulatory factor X5
RFX5
15572

regulatory factor X6
RFX6
15573

regulatory factor X7
RFX7
15574

RFX family member 8, lacking RFX DNA binding
RFX8
15575

domain

regulatory factor X associated ankyrin containing
RFXANK
15576

protein

regulatory factor X associated protein
RFXAP
15577

Rhox homeobox family member 1
RHOXF1
15578

Rhox homeobox family member 2
RHOXF2
15579

Rhox homeobox family member 2B
RHOXF2B
15580

rearranged L-myc fusion
RLF
15581-15582

RAR related orphan receptor A
RORA
15583

RAR related orphan receptor B
RORB
15584

RAR related orphan receptor C
RORC
15585

retinoic acid receptor-related orphan nuclear receptor
RORgT
15586

gamma

ras responsive element binding protein 1
RREB1
15587

runt related transcription factor 1
RUNX1
15588

runt related transcription factor 1; translocated to, 1
RUNX1T1
15589

(cyclin D related)

runt related transcription factor 2
RUNX2
15590

runt related transcription factor 3
RUNX3
15591

retinoid X receptor alpha
RXRA
15592

retinoid X receptor beta
RXRB
15593

retinoid X receptor gamma
RXRG
15594

spalt-like transcription factor 1
SALL1
15595

spalt-like transcription factor 2
SALL2
15596

spalt-like transcription factor 3
SALL3
15597

spalt-like transcription factor 4
SALL4
15598

SATB homeobox 1
SATB1
15599

SATB homeobox 2
SATB2
15600

S-phase cyclin A-associated protein in the ER
SCAPER
15601

scratch family zinc finger 1
SCRT1
15602

scratch family zinc finger 2
SCRT2
15603

scleraxis bHLH transcription factor
SCX
15604

SEBOX homeobox
SEBOX
15605

SET binding protein 1
SETBP1
15606

splicing factor proline/glutamine-rich
SFPQ
15607

short stature homeobox
SHOX
15608

short stature homeobox 2
SHOX2
15609

single-minded family bHLH transcription factor 1
SIM1
15610

single-minded family bHLH transcription factor 2
SIM2
15611

SIX homeobox 1
SIX1
15612

SIX homeobox 2
SIX2
15613

SIX homeobox 3
SIX3
15614

SIX homeobox 4
SIX4
15615

SIX homeobox 5
SIX5
15616

SIX homeobox 6
SIX6
15617

SKI proto-oncogene
SKI
15618

SKI-like proto-oncogene
SKIL
15619

SKI family transcriptional corepressor 1
SKOR1
15620

SKI family transcriptional corepressor 2
SKOR2
15621

solute carrier family 30 (zinc transporter), member 9
SLC30A9
15622

SMAD family member 1
SMAD1
15623

SMAD family member 2
SMAD2
15624

SMAD family member 3
SMAD3
15625

SMAD family member 4
SMAD4
15626

SMAD family member 5
SMAD5
15627

SMAD family member 6
SMAD6
15628

SMAD family member 7
SMAD7
15629

SMAD family member 9
SMAD9
15630

SWI/SNF related, matrix associated, actin dependent
SMARCA1
15631

regulator of chromatin, subfamily a, member 1

SWI/SNF related, matrix associated, actin dependent
SMARCA2
15632

regulator of chromatin, subfamily a, member 2

SWI/SNF related, matrix associated, actin dependent
SMARCA4
15633

regulator of chromatin, subfamily a, member 4

SWI/SNF related, matrix associated, actin dependent
SMARCA5
15634

regulator of chromatin, subfamily a, member 5

SWI/SNF-related, matrix-associated actin-dependent
SMARCAD1
15635

regulator of chromatin, subfamily a, containing

DEAD/H box 1

SWI/SNF related, matrix associated, actin dependent
SMARCAL1
15636

regulator of chromatin, subfamily a-like 1

SWI/SNF related, matrix associated, actin dependent
SMARCB1
15637

regulator of chromatin, subfamily b, member 1

SWI/SNF related, matrix associated, actin dependent
SMARCC1
15638

regulator of chromatin, subfamily c, member 1

SWI/SNF related, matrix associated, actin dependent
SMARCC2
15639

regulator of chromatin, subfamily c, member 2

SWI/SNF related, matrix associated, actin dependent
SMARCD1
15640

regulator of chromatin, subfamily d, member 1

SWI/SNF related, matrix associated, actin dependent
SMARCD2
15641

regulator of chromatin, subfamily d, member 2

SWI/SNF related, matrix associated, actin dependent
SMARCD3
15642

regulator of chromatin, subfamily d, member 3

SWI/SNF related, matrix associated, actin dependent
SMARCE1
15643

regulator of chromatin, subfamily e, member 1

snail family zinc finger 1
SNAI1
15644

snail family zinc finger 2
SNAI2
15645

snail family zinc finger 3
SNAI3
15646

small nuclear RNA activating complex polypeptide 4
SNAPC4
15647

spermatogenesis and oogenesis specific basic helix-
SOHLH1
15648

loop-helix 1

spermatogenesis and oogenesis specific basic helix-
SOHLH2
15649

loop-helix 2

SRY-box 1
SOX1
15650

SRY-box 10
SOX10
15651

SRY-box 11
SOX11
15652

SRY-box 12
SOX12
15653

SRY-box 13
SOX13
15654

SRY-box 14
SOX14
15655

SRY-box 15
SOX15
15656

SRY-box 17
SOX17
15657

SRY-box 18
SOX18
15658

SRY-box 2
SOX2
15659

SRY-box 21
SOX21
15660

SRY-box 3
SOX3
15661

SRY-box 30
SOX30
15662

SRY-box 4
SOX4
15663

SRY-box 5
SOX5
15664

SRY-box 6
SOX6
15665

SRY-box 7
SOX7
15666

SRY-box 8
SOX8
15667

SRY-box 9
SOX9
15668

Sp1 transcription factor
SP1
15669-15670

SP100 nuclear antigen
SP100
15671

SP110 nuclear body protein
SP110
15672

SP140 nuclear body protein
SP140
15673

SP140 nuclear body protein like
SP140L
15674

Sp2 transcription factor
SP2
15675

Sp3 transcription factor
SP3
15676

Sp4 transcription factor
SP4
15677

Sp5 transcription factor
SP5
15678

Sp6 transcription factor
SP6
15679

Sp7 transcription factor
SP7
15680

Sp8 transcription factor
SP8
15681

Sp9 transcription factor
SP9
15682

SAM pointed domain containing ETS transcription
SPDEF
15683

factor

Spi-1 proto-oncogene
SPI1
15684

Spi-B transcription factor (Spi-1/PU.1 related)
SPIB
15685

Spi-C transcription factor (Spi-1/PU.1 related)
SPIC
15686

spermatogenic leucine zipper 1
SPZ1
15687

sterol regulatory element binding transcription factor 1
SREBF1
15688

sterol regulatory element binding transcription factor 2
SREBF2
15689

serum response factor
SRF
15690

sex determining region Y
SRY
15691

structure specific recognition protein 1
SSRP1
15692

suppression of tumorigenicity 18, zinc finger
ST18
15693

signal transducer and activator of transcription 1
STAT1
15694

signal transducer and activator of transcription 2
STAT2
15695

signal transducer and activator of transcription 3
STAT3
15696

(acute-phase response factor)

signal transducer and activator of transcription 4
STAT4
15697

signal transducer and activator of transcription 5
STAT5
15698

signal transducer and activator of transcription 5A
STAT5A
15699

signal transducer and activator of transcription 5B
STAT5B
15700

signal transducer and activator of transcription 6,
STAT6
15701

interleukin-4 induced

transcriptional adaptor 2A
TADA2A
15702

transcriptional adaptor 2B
TADA2B
15703

TATA-box binding protein associated factor 1
TAF1
15704

T-cell acute lymphocytic leukemia 1
TAL1
15705

T-cell acute lymphocytic leukemia 2
TAL2
15706

Taxi (human T-cell leukemia virus type I) binding
TAX1BP1
15707

protein 1

Taxi (human T-cell leukemia virus type I) binding
TAX1BP3
15708

protein 3

T-box transcription factor T-bet
Tbet
15709

TATA-box binding protein
TBP
15710

TATA-box binding protein like 1
TBPL1
15711

TATA-box binding protein like 2
TBPL2
15712

T-box, brain 1
TBR1
15713

T-box 1
TBX1
15714

T-box 10
TBX10
15715

T-box 15
TBX15
15716

T-box 18
TBX18
15717

T-box 19
TBX19
15718

T-box 2
TBX2
15719

T-box 20
TBX20
15720

T-box 21
TBX21
15721

T-box 22
TBX22
15722

T-box 3
TBX3
15723

T-box 4
TBX4
15724

T-box 5
TBX5
15725

T-box 6
TBX6
15726

transcription factor 12
TCF12
15727

transcription factor 15 (basic helix-loop-helix)
TCF15
15728

transcription factor 19
TCF19
15729

transcription factor 20 (AR1)
TCF20
15730

transcription factor 21
TCF21
15731

transcription factor 23
TCF23
15732

transcription factor 24
TCF24
15733

transcription factor 25 (basic helix-loop-helix)
TCF25
15734

transcription factor 3
TCF3
15735

transcription factor 4
TCF4
15736

transcription factor 7 (T-cell specific, HMG-box,
TCF7
15737

TCF1)

transcription factor 7 like 1
TCF7L1
15738

transcription factor 7 like 2
TCF7L2
15739

transcription factor-like 5 (basic helix-loop-helix)
TCFL5
15740

TEA domain transcription factor 1
TEAD1
15741

TEA domain transcription factor 2
TEAD2
15742

TEA domain transcription factor 3
TEAD3
15743

TEA domain transcription factor 4
TEAD4
15744

thyrotrophic embryonic factor
TEF
15745

telomeric repeat binding factor (NIMA-interacting) 1
TERF1
15746

telomeric repeat binding factor 2
TERF2
15747

tet methylcytosine dioxygenase 1
TET1
15748

tet methylcytosine dioxygenase 2
TET2
15749

tet methylcytosine dioxygenase 3
TET3
15750

transcription factor A, mitochondrial
TFAM
15751

transcription factor AP-2 alpha (activating enhancer
TFAP2A
15752

binding protein 2 alpha)

transcription factor AP-2 beta (activating enhancer
TFAP2B
15753

binding protein 2 beta)

transcription factor AP-2 gamma (activating enhancer
TFAP2C
15754

binding protein 2 gamma)

transcription factor AP-2 delta (activating enhancer
TFAP2D
15755

binding protein 2 delta)

transcription factor AP-2 epsilon (activating enhancer
TFAP2E
15756

binding protein 2 epsilon)

transcription factor AP-4 (activating enhancer binding
TFAP4
15757

protein 4)

transcription factor B1, mitochondrial
TFB1M
15758

transcription factor B2, mitochondrial
TFB2M
15759

transcription factor CP2
TFCP2
15760

transcription factor CP2-like 1
TFCP2L1
15761

transcription factor Dp-1
TFDP1
15762

transcription factor Dp-2 (E2F dimerization partner 2)
TFDP2
15763

transcription factor Dp family member 3
TFDP3
15764

transcription factor binding to IGHM enhancer 3
TFE3
15765

transcription factor EB
TFEB
15766

transcription factor EC
TFEC
15767

TGFB induced factor homeobox 1
TGIF1
15768

TGFB induced factor homeobox 2
TGIF2
15769

TGFB induced factor homeobox 2 like, X-linked
TGIF2LX
15770

TGFB induced factor homeobox 2 like, Y-linked
TGIF2LY
15771

THAP domain containing, apoptosis associated protein
THAP1
15772

1

THAP domain containing 10
THAP10
15773

THAP domain containing 11
THAP11
15774

THAP domain containing 12
THAP12
15775

THAP domain containing, apoptosis associated protein
THAP2
15776

2

THAP domain containing, apoptosis associated protein
THAP3
15777

3

THAP domain containing 4
THAP4
15778

THAP domain containing 5
THAP5
15779

THAP domain containing 6
THAP6
15780

THAP domain containing 7
THAP7
15781

THAP domain containing 8
THAP8
15782

THAP domain containing 9
THAP9
15783

Th inducing POZ-Kruppel Factor
ThPOK
15784

thyroid hormone receptor, alpha
THRA
15785

thyroid hormone receptor, beta
THRB
15786

T-cell leukemia homeobox 1
TLX1
15787

T-cell leukemia homeobox 2
TLX2
15788

T-cell leukemia homeobox 3
TLX3
15789

target of EGR1, member 1 (nuclear)
TOE1
15790

tonsoku-like, DNA repair protein
TONSL
15791

topoisomerase I binding, arginine/serine-rich, E3
TOPORS
15792

ubiquitin protein ligase

thymocyte selection associated high mobility group
TOX
15793

box

TOX high mobility group box family member 2
TOX2
15794

TOX high mobility group box family member 3
TOX3
15795

TOX high mobility group box family member 4
TOX4
15796

tumor protein p53
TP53
15797

tumor protein p63
TP63
15798

tumor protein p73
TP73
15799

tetra-peptide repeat homeobox 1
TPRX1
15800

tetra-peptide repeat homeobox-like
TPRXL
15801

transcriptional regulating factor 1
TRERF1
15802

trichorhinophalangeal syndrome I
TRPS1
15803

TSC22 domain family member 1
TSC22D1
15804

TSC22 domain family member 2
TSC22D2
15805

TSC22 domain family member 3
TSC22D3
15806

TSC22 domain family member 4
TSC22D4
15807

teashirt zinc finger homeobox 1
TSHZ1
15808

teashirt zinc finger homeobox 2
TSHZ2
15809

teashirt zinc finger homeobox 3
TSHZ3
15810

transcription termination factor, RNA polymerase I
TTF1
15811-15812

transcription termination factor, RNA polymerase II
TTF2
15813-15814

tubby bipartite transcription factor
TUB
15815

twist family bHLH transcription factor 1
TWIST1
15816

twist family bHLH transcription factor 2
TWIST2
15817

upstream binding protein 1 (LBP-1a)
UBP1
15818

upstream binding transcription factor, RNA
UBTF
15819

polymerase I

upstream binding transcription factor, RNA
UBTFL1
15820

polymerase I-like 1

upstream binding transcription factor, RNA
UBTFL6
15821

polymerase I-like 6 (pseudogene)

UNC homeobox
UNCX
15822

unkempt family zinc finger
UNK
15823

unkempt family like zinc finger
UNKL
15824

upstream transcription factor 1
USF1
15825

upstream transcription factor 2, c-fos interacting
USF2
15826

upstream transcription factor family member 3
USF3
15827

undifferentiated embryonic cell transcription factor 1
UTF1
15828

ventral anterior homeobox 1
VAX1
15829

ventral anterior homeobox 2
VAX2
15830

vitamin D (1,25-dihydroxyvitamin D3) receptor
VDR
15831

VENT homeobox
VENTX
15832

vascular endothelial zinc finger 1
VEZF1
15833

visual system homeobox 1
VSX1
15834

visual system homeobox 2
VSX2
15835

WD repeat and HMG-box DNA binding protein 1
WDHD1
15836

Wolf-Hirschhorn syndrome candidate 1
WHSC1
15837

widely interspaced zinc finger motifs
WIZ
15838

Wilms tumor 1
WT1
15839

X-box binding protein 1
XBP1
15840

Y-box binding protein 1
YBX1
15841

Y-box binding protein 2
YBX2
15842

Y-box binding protein 3
YBX3
15843

YEATS domain containing 2
YEATS2
15844

YEATS domain containing 4
YEATS4
15845

YY1 transcription factor
YY1
15846

YY2 transcription factor
YY2
15847

zinc finger BED-type containing 1
ZBED1
15848

zinc finger BED-type containing 2
ZBED2
15849

zinc finger BED-type containing 3
ZBED3
15850

zinc finger BED-type containing 4
ZBED4
15851

zinc finger BED-type containing 5
ZBED5
15852

zinc finger, BED-type containing 6
ZBED6
15853

Z-DNA binding protein 1
ZBP1
15854-15855

zinc finger and BTB domain containing 1
ZBTB1
15856

zinc finger and BTB domain containing 10
ZBTB10
15857

zinc finger and BTB domain containing 11
ZBTB11
15858

zinc finger and BTB domain containing 12
ZBTB12
15859

zinc finger and BTB domain containing 14
ZBTB14
15860

zinc finger and BTB domain containing 16
ZBTB16
15861

zinc finger and BTB domain containing 17
ZBTB17
15862

zinc finger and BTB domain containing 18
ZBTB18
15863

zinc finger and BTB domain containing 2
ZBTB2
15864

zinc finger and BTB domain containing 20
ZBTB20
15865

zinc finger and BTB domain containing 21
ZBTB21
15866

zinc finger and BTB domain containing 22
ZBTB22
15867

zinc finger and BTB domain containing 24
ZBTB24
15868

zinc finger and BTB domain containing 25
ZBTB25
15869

zinc finger and BTB domain containing 26
ZBTB26
15870

zinc finger and BTB domain containing 3
ZBTB3
15871

zinc finger and BTB domain containing 32
ZBTB32
15872

zinc finger and BTB domain containing 33
ZBTB33
15873

zinc finger and BTB domain containing 34
ZBTB34
15874

zinc finger and BTB domain containing 37
ZBTB37
15875

zinc finger and BTB domain containing 38
ZBTB38
15876

zinc finger and BTB domain containing 39
ZBTB39
15877

zinc finger and BTB domain containing 4
ZBTB4
15878

zinc finger and BTB domain containing 40
ZBTB40
15879

zinc finger and BTB domain containing 41
ZBTB41
15880

zinc finger and BTB domain containing 42
ZBTB42
15881

zinc finger and BTB domain containing 43
ZBTB43
15882

zinc finger and BTB domain containing 44
ZBTB44
15883

zinc finger and BTB domain containing 45
ZBTB45
15884

zinc finger and BTB domain containing 46
ZBTB46
15885

zinc finger and BTB domain containing 47
ZBTB47
15886

zinc finger and BTB domain containing 48
ZBTB48
15887

zinc finger and BTB domain containing 49
ZBTB49
15888

zinc finger and BTB domain containing 5
ZBTB5
15889

zinc finger and BTB domain containing 6
ZBTB6
15890

zinc finger and BTB domain containing 7A
ZBTB7A
15891

zinc finger and BTB domain containing 7B
ZBTB7B
15892

zinc finger and BTB domain containing 7C
ZBTB7C
15893

zinc finger and BTB domain containing 8A
ZBTB8A
15894

zinc finger and BTB domain containing 9
ZBTB9
15895

zinc finger CCCH-type containing 10
ZC3H10
15896

zinc finger CCCH-type containing 11A
ZC3H11A
15897

zinc finger CCCH-type containing 12A
ZC3H12A
15898

zinc finger CCCH-type containing 12B
ZC3H12B
15899

zinc finger CCCH-type containing 13
ZC3H13
15900

zinc finger CCCH-type containing 14
ZC3H14
15901

zinc finger CCCH-type containing 15
ZC3H15
15902

zinc finger CCCH-type containing 18
ZC3H18
15903

zinc finger CCCH-type containing 3
ZC3H3
15904

zinc finger CCCH-type containing 4
ZC3H4
15905

zinc finger CCCH-type containing 6
ZC3H6
15906

zinc finger CCCH-type containing 7A
ZC3H7A
15907

zinc finger CCCH-type containing 7B
ZC3H7B
15908

zinc finger CCCH-type containing 8
ZC3H8
15909

zinc finger CCHC-type containing 11
ZCCHC11
15910

zinc finger CCHC-type containing 6
ZCCHC6
15911

zinc finger E-box binding homeobox 1
ZEB1
15912

zinc finger E-box binding homeobox 2
ZEB2
15913

zinc finger and AT-hook domain containing
ZFAT
15914

zinc finger homeobox 2
ZFHX2
15915

zinc finger homeobox 3
ZFHX3
15916

zinc finger homeobox 4
ZFHX4
15917

ZFP1 zinc finger protein
ZFP1
15918

ZFP14 zinc finger protein
ZFP14
15919

ZFP2 zinc finger protein
ZFP2
15920

ZFP28 zinc finger protein
ZFP28
15921

ZFP3 zinc finger protein
ZFP3
15922

ZFP30 zinc finger protein
ZFP30
15923

ZFP36 ring finger protein-like 1
ZFP36L1
15924

ZFP36 ring finger protein-like 2
ZFP36L2
15925

ZFP37 zinc finger protein
ZFP37
15926

ZFP41 zinc finger protein
ZFP41
15927

ZFP42 zinc finger protein
ZFP42
15928

ZFP57 zinc finger protein
ZFP57
15929

ZFP62 zinc finger protein
ZFP62
15930

ZFP64 zinc finger protein
ZFP64
15931

ZFP69 zinc finger protein
ZFP69
15932-15933

ZFP69 zinc finger protein B
ZFP69B
15934

ZFP82 zinc finger protein
ZFP82
15935

ZFP90 zinc finger protein
ZFP90
15936

ZFP91 zinc finger protein
ZFP91
15937

ZFP92 zinc finger protein
ZFP92
15938

zinc finger protein, FOG family member 1
ZFPM1
15939

zinc finger protein, FOG family member 2
ZFPM2
15940

zinc finger protein, X-linked
ZFX
15941

zinc finger protein, Y-linked
ZFY
15942

zinc finger, FYVE domain containing 26
ZFYVE26
15943

zinc finger, GATA-like protein 1
ZGLP1
15944

zinc finger CCCH-type and G-patch domain
ZGPAT
15945

containing

zinc fingers and homeoboxes 1
ZHX1
15946

zinc fingers and homeoboxes 2
ZHX2
15947

zinc fingers and homeoboxes 3
ZHX3
15948

Zic family member 1
ZIC1
15949

Zic family member 2
ZIC2
15950

Zic family member 3
ZIC3
15951

Zic family member 4
ZIC4
15952

Zic family member 5
ZIC5
15953

zinc finger protein interacting with K protein 1
ZIK1
15954

zinc finger, imprinted 2
ZIM2
15955

zinc finger, imprinted 3
ZIM3
15956

zinc finger with KRAB and SCAN domains 1
ZKSCAN1
15957

zinc finger with KRAB and SCAN domains 2
ZKSCAN2
15958

zinc finger with KRAB and SCAN domains 3
ZKSCAN3
15959

zinc finger with KRAB and SCAN domains 4
ZKSCAN4
15960

zinc finger with KRAB and SCAN domains 5
ZKSCAN5
15961

zinc finger with KRAB and SCAN domains 7
ZKSCAN7
15962

zinc finger with KRAB and SCAN domains 8
ZKSCAN8
15963

zinc finger matrin-type 1
ZMAT1
15964

zinc finger matrin-type 2
ZMAT2
15965

zinc finger matrin-type 3
ZMAT3
15966

zinc finger matrin-type 4
ZMAT4
15967

zinc finger matrin-type 5
ZMAT5
15968

zinc finger protein 10
ZNF10
15969

zinc finger protein 100
ZNF100
15970

zinc finger protein 101
ZNF101
15971

zinc finger protein 106
ZNF106
15972

zinc finger protein 107
ZNF107
15973

zinc finger protein 112
ZNF112
15974

zinc finger protein 114
ZNF114
15975

zinc finger protein 117
ZNF117
15976

zinc finger protein 12
ZNF12
15977

zinc finger protein 121
ZNF121
15978

zinc finger protein 124
ZNF124
15979

zinc finger protein 131
ZNF131
15980

zinc finger protein 132
ZNF132
15981

zinc finger protein 133
ZNF133
15982

zinc finger protein 134
ZNF134
15983

zinc finger protein 135
ZNF135
15984

zinc finger protein 136
ZNF136
15985

zinc finger protein 137, pseudogene
ZNF137P
15986

zinc finger protein 138
ZNF138
15987

zinc finger protein 14
ZNF14
15988

zinc finger protein 140
ZNF140
15989

zinc finger protein 141
ZNF141
15990

zinc finger protein 142
ZNF142
15991

zinc finger protein 143
ZNF143
15992

zinc finger protein 146
ZNF146
15993

zinc finger protein 148
ZNF148
15994

zinc finger protein 154
ZNF154
15995

zinc finger protein 155
ZNF155
15996

zinc finger protein 157
ZNF157
15997

zinc finger protein 16
ZNF16
15998

zinc finger protein 160
ZNF160
15999

zinc finger protein 165
ZNF165
16000

zinc finger protein 169
ZNF169
16001

zinc finger protein 17
ZNF17
16002

zinc finger protein 174
ZNF174
16003

zinc finger protein 175
ZNF175
16004

zinc finger protein 18
ZNF18
16005

zinc finger protein 180
ZNF180
16006

zinc finger protein 181
ZNF181
16007

zinc finger protein 182
ZNF182
16008

zinc finger protein 184
ZNF184
16009

zinc finger protein 189
ZNF189
16010

zinc finger protein 19
ZNF19
16011

zinc finger protein 195
ZNF195
16012

zinc finger protein 197
ZNF197
16013

zinc finger protein 2
ZNF2
16014

zinc finger protein 20
ZNF20
16015-16016

zinc finger protein 200
ZNF200
16017

zinc finger protein 202
ZNF202
16018

zinc finger protein 205
ZNF205
16019

zinc finger protein 207
ZNF207
16020

zinc finger protein 208
ZNF208
16021

zinc finger protein 211
ZNF211
16022

zinc finger protein 212
ZNF212
16023

zinc finger protein 213
ZNF213
16024

zinc finger protein 214
ZNF214
16025

zinc finger protein 215
ZNF215
16026

zinc finger protein 217
ZNF217
16027

zinc finger protein 219
ZNF219
16028

zinc finger protein 22
ZNF22
16029

zinc finger protein 221
ZNF221
16030

zinc finger protein 223
ZNF223
16031

zinc finger protein 224
ZNF224
16032

zinc finger protein 225
ZNF225
16033-16034

zinc finger protein 226
ZNF226
16035

zinc finger protein 227
ZNF227
16036

zinc finger protein 229
ZNF229
16037

zinc finger protein 23
ZNF23
16038

zinc finger protein 230
ZNF230
16039-16040

zinc finger protein 232
ZNF232
16041

zinc finger protein 233
ZNF233
16042-16043

zinc finger protein 234
ZNF234
16044

zinc finger protein 235
ZNF235
16045

zinc finger protein 236
ZNF236
16046

zinc finger protein 239
ZNF239
16047

zinc finger protein 24
ZNF24
16048

zinc finger protein 248
ZNF248
16049

zinc finger protein 25
ZNF25
16050

zinc finger protein 250
ZNF250
16051

zinc finger protein 251
ZNF251
16052

zinc finger protein 252, pseudogene
ZNF252P
16053

zinc finger protein 253
ZNF253
16054

zinc finger protein 254
ZNF254
16055

zinc finger protein 256
ZNF256
16056

zinc finger protein 257
ZNF257
16057

zinc finger protein 26
ZNF26
16058

zinc finger protein 260
ZNF260
16059

zinc finger protein 263
ZNF263
16060

zinc finger protein 264
ZNF264
16061

zinc finger protein 266
ZNF266
16062

zinc finger protein 267
ZNF267
16063

zinc finger protein 268
ZNF268
16064

zinc finger protein 273
ZNF273
16065

zinc finger protein 274
ZNF274
16066

zinc finger protein 275
ZNF275
16067

zinc finger protein 276
ZNF276
16068

zinc finger protein 277
ZNF277
16069

zinc finger protein 28
ZNF28
16070

zinc finger protein 280A
ZNF280A
16071

zinc finger protein 280B
ZNF280B
16072

zinc finger protein 280C
ZNF280C
16073

zinc finger protein 280D
ZNF280D
16074

zinc finger protein 281
ZNF281
16075

zinc finger protein 282
ZNF282
16076

zinc finger protein 283
ZNF283
16077

zinc finger protein 284
ZNF284
16078

zinc finger protein 285
ZNF285
16079

zinc finger protein 286A
ZNF286A
16080

zinc finger protein 286B
ZNF286B
16081

zinc finger protein 287
ZNF287
16082

zinc finger protein 292
ZNF292
16083

zinc finger protein 296
ZNF296
16084

zinc finger protein 3
ZNF3
16085

zinc finger protein 30
ZNF30
16086

zinc finger protein 300
ZNF300
16087

zinc finger protein 302
ZNF302
16088

zinc finger protein 304
ZNF304
16089

zinc finger protein 311
ZNF311
16090

zinc finger protein 316
ZNF316
16091

zinc finger protein 317
ZNF317
16092

zinc finger protein 318
ZNF318
16093

zinc finger protein 319
ZNF319
16094

zinc finger protein 32
ZNF32
16095

zinc finger protein 320
ZNF320
16096

zinc finger protein 322
ZNF322
16097

zinc finger protein 324
ZNF324
16098

zinc finger protein 324B
ZNF324B
16099

zinc finger protein 326
ZNF326
16100

zinc finger protein 329
ZNF329
16101

zinc finger protein 331
ZNF331
16102

zinc finger protein 333
ZNF333
16103

zinc finger protein 334
ZNF334
16104

zinc finger protein 335
ZNF335
16105

zinc finger protein 337
ZNF337
16106

zinc finger protein 33A
ZNF33A
16107

zinc finger protein 33B
ZNF33B
16108

zinc finger protein 34
ZNF34
16109

zinc finger protein 341
ZNF341
16110

zinc finger protein 343
ZNF343
16111

zinc finger protein 345
ZNF345
16112

zinc finger protein 346
ZNF346
16113

zinc finger protein 347
ZNF347
16114

zinc finger protein 35
ZNF35
16115

zinc finger protein 350
ZNF350
16116

zinc finger protein 354A
ZNF354A
16117

zinc finger protein 354B
ZNF354B
16118

zinc finger protein 354C
ZNF354C
16119

zinc finger protein 355, pseudogene
ZNF355P
16120

zinc finger protein 358
ZNF358
16121

zinc finger protein 362
ZNF362
16122

zinc finger protein 365
ZNF365
16123-16124

zinc finger protein 366
ZNF366
16125

zinc finger protein 367
ZNF367
16126

zinc finger protein 37A
ZNF37A
16127

zinc finger protein 382
ZNF382
16128

zinc finger protein 383
ZNF383
16129

zinc finger protein 384
ZNF384
16130

zinc finger protein 385A
ZNF385A
16131

zinc finger protein 385B
ZNF385B
16132

zinc finger protein 385C
ZNF385C
16133

zinc finger protein 385D
ZNF385D
16134

zinc finger protein 391
ZNF391
16135

zinc finger protein 394
ZNF394
16136

zinc finger protein 395
ZNF395
16137

zinc finger protein 396
ZNF396
16138

zinc finger protein 397
ZNF397
16139

zinc finger protein 398
ZNF398
16140

zinc finger protein 404
ZNF404
16141

zinc finger protein 407
ZNF407
16142

zinc finger protein 408
ZNF408
16143

zinc finger protein 41
ZNF41
16144

zinc finger protein 410
ZNF410
16145

zinc finger protein 414
ZNF414
16146

zinc finger protein 415
ZNF415
16147

zinc finger protein 416
ZNF416
16148

zinc finger protein 417
ZNF417
16149

zinc finger protein 418
ZNF418
16150

zinc finger protein 419
ZNF419
16151

zinc finger protein 420
ZNF420
16152

zinc finger protein 423
ZNF423
16153

zinc finger protein 425
ZNF425
16154

zinc finger protein 426
ZNF426
16155

zinc finger protein 428
ZNF428
16156

zinc finger protein 429
ZNF429
16157

zinc finger protein 43
ZNF43
16158

zinc finger protein 430
ZNF430
16159

zinc finger protein 431
ZNF431
16160

zinc finger protein 432
ZNF432
16161

zinc finger protein 433
ZNF433
16162

zinc finger protein 436
ZNF436
16163

zinc finger protein 438
ZNF438
16164

zinc finger protein 439
ZNF439
16165

zinc finger protein 44
ZNF44
16166

zinc finger protein 440
ZNF440
16167

zinc finger protein 441
ZNF441
16168

zinc finger protein 442
ZNF442
16169

zinc finger protein 443
ZNF443
16170

zinc finger protein 444
ZNF444
16171

zinc finger protein 445
ZNF445
16172

zinc finger protein 446
ZNF446
16173

zinc finger protein 449
ZNF449
16174

zinc finger protein 45
ZNF45
16175

zinc finger protein 451
ZNF451
16176

zinc finger protein 454
ZNF454
16177

zinc finger protein 460
ZNF460
16178

zinc finger protein 461
ZNF461
16179

zinc finger protein 462
ZNF462
16180

zinc finger protein 467
ZNF467
16181

zinc finger protein 468
ZNF468
16182

zinc finger protein 469
ZNF469
16183

zinc finger protein 470
ZNF470
16184

zinc finger protein 471
ZNF471
16185

zinc finger protein 473
ZNF473
16186

zinc finger protein 474
ZNF474
16187-16188

zinc finger protein 479
ZNF479
16189

zinc finger protein 48
ZNF48
16190

zinc finger protein 480
ZNF480
16191

zinc finger protein 483
ZNF483
16192

zinc finger protein 484
ZNF484
16193

zinc finger protein 485
ZNF485
16194

zinc finger protein 486
ZNF486
16195

zinc finger protein 487
ZNF487
16196

zinc finger protein 488
ZNF488
16197

zinc finger protein 490
ZNF490
16198

zinc finger protein 491
ZNF491
16199

zinc finger protein 492
ZNF492
16200

zinc finger protein 493
ZNF493
16201

zinc finger protein 496
ZNF496
16202

zinc finger protein 497
ZNF497
16203

zinc finger protein 500
ZNF500
16204

zinc finger protein 501
ZNF501
16205

zinc finger protein 502
ZNF502
16206

zinc finger protein 503
ZNF503
16207

zinc finger protein 506
ZNF506
16208

zinc finger protein 507
ZNF507
16209

zinc finger protein 510
ZNF510
16210

zinc finger protein 511
ZNF511
16211

zinc finger protein 512
ZNF512
16212

zinc finger protein 512B
ZNF512B
16213

zinc finger protein 513
ZNF513
16214

zinc finger protein 514
ZNF514
16215

zinc finger protein 516
ZNF516
16216

zinc finger protein 517
ZNF517
16217

zinc finger protein 518A
ZNF518A
16218

zinc finger protein 518B
ZNF518B
16219

zinc finger protein 519
ZNF519
16220

zinc finger protein 521
ZNF521
16221

zinc finger protein 524
ZNF524
16222

zinc finger protein 526
ZNF526
16223

zinc finger protein 527
ZNF527
16224

zinc finger protein 528
ZNF528
16225

zinc finger protein 529
ZNF529
16226

zinc finger protein 530
ZNF530
16227

zinc finger protein 532
ZNF532
16228

zinc finger protein 534
ZNF534
16229

zinc finger protein 536
ZNF536
16230

zinc finger protein 540
ZNF540
16231

zinc finger protein 541
ZNF541
16232

zinc finger protein 542, pseudogene
ZNF542P
16233

zinc finger protein 543
ZNF543
16234

zinc finger protein 544
ZNF544
16235

zinc finger protein 546
ZNF546
16236

zinc finger protein 547
ZNF547
16237

zinc finger protein 548
ZNF548
16238

zinc finger protein 549
ZNF549
16239

zinc finger protein 550
ZNF550
16240

zinc finger protein 552
ZNF552
16241

zinc finger protein 554
ZNF554
16242

zinc finger protein 555
ZNF555
16243

zinc finger protein 556
ZNF556
16244

zinc finger protein 557
ZNF557
16245

zinc finger protein 558
ZNF558
16246

zinc finger protein 559
ZNF559
16247

zinc finger protein 56
ZNF56
16248

zinc finger protein 560
ZNF560
16249

zinc finger protein 561
ZNF561
16250

zinc finger protein 562
ZNF562
16251

zinc finger protein 563
ZNF563
16252

zinc finger protein 564
ZNF564
16253

zinc finger protein 565
ZNF565
16254

zinc finger protein 566
ZNF566
16255

zinc finger protein 567
ZNF567
16256

zinc finger protein 568
ZNF568
16257

zinc finger protein 569
ZNF569
16258

zinc finger protein 57
ZNF57
16259

zinc finger protein 570
ZNF570
16260

zinc finger protein 571
ZNF571
16261

zinc finger protein 572
ZNF572
16262

zinc finger protein 573
ZNF573
16263

zinc finger protein 574
ZNF574
16264

zinc finger protein 575
ZNF575
16265

zinc finger protein 576
ZNF576
16266-16267

zinc finger protein 577
ZNF577
16268

zinc finger protein 578
ZNF578
16269

zinc finger protein 579
ZNF579
16270

zinc finger protein 580
ZNF580
16271

zinc finger protein 581
ZNF581
16272

zinc finger protein 582
ZNF582
16273

zinc finger protein 583
ZNF583
16274

zinc finger protein 584
ZNF584
16275

zinc finger protein 585A
ZNF585A
16276

zinc finger protein 585B
ZNF585B
16277

zinc finger protein 586
ZNF586
16278

zinc finger protein 587
ZNF587
16279

zinc finger protein 589
ZNF589
16280

zinc finger protein 592
ZNF592
16281

zinc finger protein 593
ZNF593
16282

zinc finger protein 594
ZNF594
16283

zinc finger protein 595
ZNF595
16284

zinc finger protein 596
ZNF596
16285

zinc finger protein 597
ZNF597
16286

zinc finger protein 598
ZNF598
16287

zinc finger protein 599
ZNF599
16288

zinc finger protein 600
ZNF600
16289

zinc finger protein 605
ZNF605
16290

zinc finger protein 606
ZNF606
16291

zinc finger protein 607
ZNF607
16292

zinc finger protein 608
ZNF608
16293

zinc finger protein 609
ZNF609
16294

zinc finger protein 610
ZNF610
16295

zinc finger protein 611
ZNF611
16296

zinc finger protein 613
ZNF613
16297

zinc finger protein 614
ZNF614
16298

zinc finger protein 615
ZNF615
16299

zinc finger protein 616
ZNF616
16300

zinc finger protein 618
ZNF618
16301

zinc finger protein 619
ZNF619
16302

zinc finger protein 620
ZNF620
16303

zinc finger protein 621
ZNF621
16304

zinc finger protein 622
ZNF622
16305

zinc finger protein 623
ZNF623
16306

zinc finger protein 624
ZNF624
16307

zinc finger protein 625
ZNF625
16308

zinc finger protein 626
ZNF626
16309

zinc finger protein 627
ZNF627
16310

zinc finger protein 628
ZNF628
16311

zinc finger protein 629
ZNF629
16312

zinc finger protein 639
ZNF639
16313

zinc finger protein 641
ZNF641
16314

zinc finger protein 644
ZNF644
16315

zinc finger protein 645
ZNF645
16316

zinc finger protein 646
ZNF646
16317

zinc finger protein 648
ZNF648
16318

zinc finger protein 649
ZNF649
16319

zinc finger protein 652
ZNF652
16320

zinc finger protein 653
ZNF653
16321

zinc finger protein 654
ZNF654
16322

zinc finger protein 655
ZNF655
16323

zinc finger protein 658
ZNF658
16324

zinc finger protein 658B (pseudogene)
ZNF658B
16325

zinc finger protein 66
ZNF66
16326

zinc finger protein 660
ZNF660
16327

zinc finger protein 662
ZNF662
16328

zinc finger protein 664
ZNF664
16329

zinc finger protein 665
ZNF665
16330

zinc finger protein 667
ZNF667
16331

zinc finger protein 668
ZNF668
16332

zinc finger protein 669
ZNF669
16333

zinc finger protein 670
ZNF670
16334

zinc finger protein 671
ZNF671
16335

zinc finger protein 672
ZNF672
16336

zinc finger protein 674
ZNF674
16337

zinc finger protein 675
ZNF675
16338

zinc finger protein 676
ZNF676
16339

zinc finger protein 677
ZNF677
16340

zinc finger protein 678
ZNF678
16341

zinc finger protein 679
ZNF679
16342

zinc finger protein 680
ZNF680
16343

zinc finger protein 681
ZNF681
16344

zinc finger protein 682
ZNF682
16345

zinc finger protein 683
ZNF683
16346

zinc finger protein 684
ZNF684
16347

zinc finger protein 687
ZNF687
16348

zinc finger protein 688
ZNF688
16349

zinc finger protein 689
ZNF689
16350

zinc finger protein 69
ZNF69
16351

zinc finger protein 691
ZNF691
16352

zinc finger protein 692
ZNF692
16353

zinc finger protein 695
ZNF695
16354

zinc finger protein 696
ZNF696
16355

zinc finger protein 697
ZNF697
16356

zinc finger protein 699
ZNF699
16357

zinc finger protein 7
ZNF7
16358

zinc finger protein 70
ZNF70
16359

zinc finger protein 701
ZNF701
16360

zinc finger protein 702, pseudogene
ZNF702P
16361

zinc finger protein 703
ZNF703
16362

zinc finger protein 704
ZNF704
16363

zinc finger protein 705A
ZNF705A
16364

zinc finger protein 705D
ZNF705D
16365

zinc finger protein 705E
ZNF705E
16366

zinc finger protein 705G
ZNF705G
16367

zinc finger protein 706
ZNF706
16368

zinc finger protein 707
ZNF707
16369

zinc finger protein 708
ZNF708
16370

zinc finger protein 709
ZNF709
16371

zinc finger protein 71
ZNF71
16372

zinc finger protein 710
ZNF710
16373

zinc finger protein 711
ZNF711
16374

zinc finger protein 713
ZNF713
16375

zinc finger protein 714
ZNF714
16376

zinc finger protein 716
ZNF716
16377

zinc finger protein 717
ZNF717
16378

zinc finger protein 718
ZNF718
16379

zinc finger protein 720
ZNF720
16380

zinc finger protein 721
ZNF721
16381

zinc finger protein 724, pseudogene
ZNF724P
16382

zinc finger protein 726
ZNF726
16383

zinc finger protein 727
ZNF727
16384

zinc finger protein 729
ZNF729
16385

zinc finger protein 730
ZNF730
16386

zinc finger protein 732
ZNF732
16387

zinc finger protein 735
ZNF735
16388

zinc finger protein 737
ZNF737
16389

zinc finger protein 74
ZNF74
16390

zinc finger protein 740
ZNF740
16391

zinc finger protein 746
ZNF746
16392

zinc finger protein 747
ZNF747
16393

zinc finger protein 749
ZNF749
16394

zinc finger protein 750
ZNF750
16395

zinc finger protein 75a
ZNF75A
16396

zinc finger protein 75D
ZNF75D
16397

zinc finger protein 76
ZNF76
16398

zinc finger protein 761
ZNF761
16399

zinc finger protein 763
ZNF763
16400

zinc finger protein 764
ZNF764
16401

zinc finger protein 765
ZNF765
16402

zinc finger protein 766
ZNF766
16403

zinc finger protein 768
ZNF768
16404

zinc finger protein 77
ZNF77
16405

zinc finger protein 770
ZNF770
16406

zinc finger protein 771
ZNF771
16407

zinc finger protein 772
ZNF772
16408

zinc finger protein 773
ZNF773
16409

zinc finger protein 774
ZNF774
16410

zinc finger protein 775
ZNF775
16411

zinc finger protein 776
ZNF776
16412

zinc finger protein 777
ZNF777
16413

zinc finger protein 778
ZNF778
16414

zinc finger protein 780A
ZNF780A
16415

zinc finger protein 780B
ZNF780B
16416

zinc finger protein 781
ZNF781
16417

zinc finger protein 782
ZNF782
16418

zinc finger family member 783
ZNF783
16419

zinc finger protein 784
ZNF784
16420

zinc finger protein 785
ZNF785
16421

zinc finger protein 786
ZNF786
16422

zinc finger protein 787
ZNF787
16423

zinc finger family member 788
ZNF788
16424

zinc finger protein 789
ZNF789
16425

zinc finger protein 79
ZNF79
16426

zinc finger protein 790
ZNF790
16427

zinc finger protein 791
ZNF791
16428

zinc finger protein 792
ZNF792
16429

zinc finger protein 793
ZNF793
16430

zinc finger protein 799
ZNF799
16431

zinc finger protein 8
ZNF8
16432

zinc finger protein 80
ZNF80
16433

zinc finger protein 800
ZNF800
16434

zinc finger protein 804A
ZNF804A
16435

zinc finger protein 804B
ZNF804B
16436

zinc finger protein 805
ZNF805
16437

zinc finger protein 806
ZNF806
16438

zinc finger protein 808
ZNF808
16439

zinc finger protein 81
ZNF81
16440

zinc finger protein 813
ZNF813
16441

zinc finger protein 814
ZNF814
16442

zinc finger protein 816
ZNF816
16443

zinc finger protein 821
ZNF821
16444

zinc finger protein 823
ZNF823
16445

zinc finger protein 827
ZNF827
16446

zinc finger protein 829
ZNF829
16447

zinc finger protein 83
ZNF83
16448

zinc finger protein 830
ZNF830
16449

zinc finger protein 831
ZNF831
16450

zinc finger protein 833, pseudogene
ZNF833P
16451

zinc finger protein 835
ZNF835
16452

zinc finger protein 836
ZNF836
16453

zinc finger protein 837
ZNF837
16454

zinc finger protein 839
ZNF839
16455

zinc finger protein 84
ZNF84
16456

zinc finger protein 840, pseudogene
ZNF840P
16457

zinc finger protein 841
ZNF841
16458

zinc finger protein 843
ZNF843
16459

zinc finger protein 844
ZNF844
16460

zinc finger protein 845
ZNF845
16461

zinc finger protein 846
ZNF846
16462

zinc finger protein 85
ZNF85
16463

zinc finger protein 853
ZNF853
16464

zinc finger protein 860
ZNF860
16465

zinc finger protein 876, pseudogene
ZNF876P
16466

zinc finger protein 878
ZNF878
16467

zinc finger protein 879
ZNF879
16468

zinc finger protein 880
ZNF880
16469

zinc finger protein 891
ZNF891
16470

zinc finger protein 90
ZNF90
16471

zinc finger protein 91
ZNF91
16472

zinc finger protein 92
ZNF92
16473

zinc finger protein 93
ZNF93
16474

zinc finger protein 98
ZNF98
16475

zinc finger protein 99
ZNF99
16476

zinc finger, NFX1-type containing 1
ZNFX1
16477

zinc finger and SCAN domain containing 1
ZSCAN1
16478

zinc finger and SCAN domain containing 10
ZSCAN10
16479

zinc finger and SCAN domain containing 12
ZSCAN12
16480

zinc finger and SCAN domain containing 16
ZSCAN16
16481

zinc finger and SCAN domain containing 18
ZSCAN18
16482

zinc finger and SCAN domain containing 2
ZSCAN2
16483

zinc finger and SCAN domain containing 20
ZSCAN20
16484

zinc finger and SCAN domain containing 21
ZSCAN21
16485

zinc finger and SCAN domain containing 22
ZSCAN22
16486

zinc finger and SCAN domain containing 23
ZSCAN23
16487

zinc finger and SCAN domain containing 25
ZSCAN25
16488

zinc finger and SCAN domain containing 26
ZSCAN26
16489

zinc finger and SCAN domain containing 29
ZSCAN29
16490

zinc finger and SCAN domain containing 30
ZSCAN30
16491

zinc finger and SCAN domain containing 31
ZSCAN31
16492

zinc finger and SCAN domain containing 32
ZSCAN32
16493

zinc finger and SCAN domain containing 4
ZSCAN4
16494

zinc finger and SCAN domain containing 5A
ZSCAN5A
16495

zinc finger and SCAN domain containing 5B
ZSCAN5B
16496

zinc finger and SCAN domain containing 5C,
ZSCAN5CP
16497

pseudogene

zinc finger and SCAN domain containing 9
ZSCAN9
16498

zinc finger with UFM1-specific peptidase domain
ZUFSP
16499

zinc finger, X-linked, duplicated A
ZXDA
16500

zinc finger, X-linked, duplicated B
ZXDB
16501

ZXD family zinc finger C
ZXDC
16502

zinc finger ZZ-type containing 3
ZZZ3
16503

In some embodiments, a T-cell of the disclosure is modified to silence or reduce expression of one or more gene(s) encoding a cell death or cell apoptosis receptor to produce an armored T-cell of the disclosure. Interaction of a death receptor and its endogenous ligand results in the initiation of apoptosis. Disruption of an expression an activity, or an interaction of a cell death and/or cell apoptosis receptor and/or ligand render an armored T-cell of the disclosure less receptive to death signals, consequently, making the armored T cell of the disclosure more efficacious in a tumor environment. An exemplary cell death receptor which may be modified in an armored T cell of the disclosure is Fas (CD95). Exemplary cell death and/or cell apoptosis receptors and ligands of the disclosure include, but are not limited to, the exemplary receptors and ligands provided in Table 4.

TABLE 4

Exemplary Cell Death and/or Cell

Apoptosis Receptors and Ligands.

Full Name
Abbreviation
SEQ ID NO:

Cluster of Differentiation 120
CD120a
16504-16505

Death receptor 3
DR3
16506

Death receptor 6
DR6
16507

first apoptosis signal (Fas) receptor
Fas
16508-16509

(CD95/APO-1)

Fas Ligand
FasL
16510

cellular tumor antigen p53
p53
16511

Tumor necrosis factor receptor 1
TNF-R1
16512

Tumor necrosis factor receptor 2
TNF-R2
16513

Tumor necrosis factor-related apoptosis-
TRAIL-R1
16514

inducing ligand receptor 1
(DR4)

Tumor necrosis factor-related apoptosis-
TRAIL-R2
16515

inducing ligand receptor 2
(DR5)

Fas-associated protein with death domain
FADD
16516

Tumor necrosis factor receptor type 1-
TRADD
16517

associated DEATH domain protein

Bcl-2-associatcd X protein
Bax
16518

Bcl-2 homologous killer
BAK
16519

14-3-3 protein
14-3-3
16520

B-cell lymphoma 2
Bcl-2
16521

Cytochrome C
CytC
16522

Second mitochondria-derived activator of
Smac/Diablo
16523

caspase

High temperature requirement protein A2
HTRA2/Omi
16524

Apoptosis inducing factor
AIF
16525

Endonuclease G
EXOG
16526

Caspase 9
Cas9
16527

Caspase 2
Cas2
16528

Caspase 8
Cas8
16529

Caspase 10
Cas10
16530

Caspase 3
Cas3
16531

Caspase 6
Cas6
16532

Caspase 7
Cas7
16533

Tumor Necrosis Factor alpha
TNF-alpha
16534

TNF-related weak inducer of apoptosis
TWEAK
16535

TNF-related weak inducer of apoptosis
TWEAK -R
16536

receptor

Tumor necrosis factor-related apoptosis-
TRAIL
16537

inducing ligand

TNF ligand-related molecule 1
TL1A
16538

Receptor-interacting serine/threonine-
RIP1
16539

protein kinase 1

Cellular inhibitor of apoptosis 1
cIAP-1
16540

TNF receptor-associated factor 2
TRAF-2
16541

In some embodiments, a T-cell of the disclosure is modified to silence or reduce expression of one or more gene(s) encoding a metabolic sensing protein to produce an armored T-cell of the disclosure. Disruption to the metabolic sensing of the immunosuppressive tumor microenvironment (characterized by low levels of oxygen pH, glucose and other molecules) by an armored T-cell of the disclosure leads to extended retention of T-cell function and, consequently, more tumor cells killed per amored T cell. For example, HIF1a and VHL play a role in T-cell function while in a hypoxic environment. An armored T-cell of the disclosure may have silenced or reduced expression of one or more genes encoding HIF1a or VHL. Genes and proteins involved in metabolic sensing include, but are not limited to, the exemplary genes and proteins provided in Table 5.

TABLE 5

Exemplary Metabolic Sensing Genes (and encoded Proteins).

Full Name
Metabolite
Abbreviation
SEQ ID NO:

hypoxia-inducible factor 1α
Low oxygen
HIF-1α
16542

von Hippel-Lindau tumor suppressor
Low oxygen
VHL
16543

Prolyl-hydroxylase domain proteins
High oxygen
PHD proteins

Glucose transporter 1
glucose
GLUT1
16544

Linker of Activated T cells
Amino acid (leucine)
LAT
16545

CD98 glycoprotein
Amino acid (leucine)
CD98
16546

Alanine, serine, cysteine-preferring
Cationic Amino acid
ASCT2/Slc1a5
16547

transporter 2
(glutamine)

Solute carrier family 7 member 1
Cationic Amino acids
Slc7a1
16548

Solute carrier family 7 member 2
Cationic Amino acids
Slc7a2
16549

Solute carrier family 7 member 3
Cationic Amino acids
Slc7a3
16550

Solute carrier family 7 member 4
Cationic Amino acids
Slc7a4
16551

Solute carrier family 7 member 5
Glycoprotein
Slc7a5
16552

associated Amino

acids

Solute carrier family 7 member 6
Glycoprotein
Slc7a6
16553

associated Amino

acids

Solute carrier family 7 member 7
Glycoprotein
Slc7a7
16554

associated Amino

acids

Solute carrier family 7 member 8
Glycoprotein
Slc7a8
16555

associated Amino

acids

Solute carrier family 7 member 9
Glycoprotein
Slc7a9
16556

associated Amino

acids

Solute carrier family 7 member 10
Glycoprotein
Slc7a10
16557

associated Amino

acids

Solute carrier family 7 member 11
Glycoprotein
Slc7a11
16558

associated Amino

acids

Solute carrier family 7 member 13
Glycoprotein
Slc7a13
16559

associated Amino

acids

Solute carrier family 7 member 14
Cationic Amino acids
Slc7a14
16560

Solute carrier family 3 member 2
Amino acid
Slc3a2
16561

Calcium transport protein 2
Cationic Amino acid
CAT2
16562

(arginine)

Calcium transport protein 3
Cationic Amino acid
CAT3
16563

(arginine)

Calcium transport protein 4
Cationic Amino acid
CAT4
16564

(arginine)

Bromodomain adjacent to zinc finger
Amino acid (arginine)
BAZ1B
16565

domain protein 1B

PC4 and SFRS1-interacting protein
Amino acid (arginine)
PSIP1
16566

Translin
Amino acid (arginine)
TSN
16567

G-protein-coupled receptors
Fatty Acid and
GPCRs

Cholesterol

T-cell Receptor, subunit alpha
Fatty Acid and
TCR alpha
16568

Cholesterol

T-cell Receptor, subunit beta
Fatty Acid and
TCR beta
16569

Cholesterol

T-cell Receptor, subunit zeta
Fatty Acid and
TCR zeta
16570

Cholesterol

T-cell Receptor, subunit CD3 epsilon
Fatty Acid and
TCR CD3 epsilon
16571

Cholesterol

T-cell Receptor, subunit CD3
Fatty Acid and
TCR CD3 gamma
16572

gamma
Cholesterol

T-cell Receptor, subunit CD3 delta
Fatty Acid and
TCR CD3 delta
16573

Cholesterol

peroxisome proliferator-activated
Fatty Acid and
PPARs

receptors
Cholesterol

AMP-activated protein kinase
Energy homeostasis
AMPK
16574-16575

(intracellular AMP to

ATP ratio)

P2X purinoceptor 7
Redox homeostasis
P2X7
16576

In some embodiments, a T-cell of the disclosure is modified to silence or reduce expression of one or more gene(s) encoding proteins that that confer sensitivity to a cancer therapy, including a monoclonal antibody, to produce an armored T-cell of the disclosure. Thus, an armored T-cell of the disclosure can function and may demonstrate superior function or efficacy whilst in the presence of a cancer therapy (e.g. a chemotherapy, a monoclonal antibody therapy, or another anti-tumor treatment). Proteins involved in conferring sensitivity to a cancer therapy include, but are not limited to, the exemplary proteins provided in Table 6.

TABLE 6

Exemplary Proteins that Confer Sensitivity to a Cancer Therapeutic.

Full Name
Abbreviation
SEQ ID NO:

Copper-transporting ATPase 2
ATP7B
16577

Breakpoint cluster region protein
BCR
16578

Abelson tyrosine-protein kinase 1
ABL
16579

Breast cancer resistance protein
BCRP
16580

Breast cancer type 1 susceptibility protein
BRCA1
16581

Breast cancer type 2 susceptibility protein
BRCA2
16582

CAMPATH-1 antigen
CD52
16583

Cytochrome P450 2D6
CYP2D6
16584

Deoxycytidine kinase
dCK
16585

Dihydrofolate reductase
DHFR
16586

Dihydropyrimidine dehydrogenase [NADP(+)]
DPYD
16587

Epidermal growth factor receptor
EGFR
16588

DNA excision repair protein ERCC-1
ERCC1
16589

Estrogen Receptor
ESR
16590

Low affinity immunoglobulin gamma Fc region
FCGR3A
16591

receptor III-A

Receptor tyrosine-protein kinase erbB-2
HER2 or ERBB2
16592

Insulin-like growth factor 1 receptor
IGF1R
16593

GTPase KRas
KRAS
16594

Multidrug resistance protein 1
MDR1 or ABCB1
16595

Methylated-DNA--protein-cysteine methyltransferase
MGMT
16596

Multidrug resistance-associated protein 1
MRP1 or ABCC1
16597

Progesterone Receptor
PGR
16598

Regulator of G-protein signaling 10
RGS10
16599

Suppressor of cytokine signaling 3
SOCS-3
16600

Thymidylate synthase
TYMS
16601

UDP-glucuronosyltransferase 1-1
UGT1A1
16602

In some embodiments, a T-cell of the disclosure is modified to silence or reduce expression of one or more gene(s) encoding a growth advantage factor to produce an armored T-cell. Silencing or reducing expression of an oncogene can confer a growth advantage for an armored T-cell of the disclosure. For example, silencing or reducing expression (e.g. disrupting expression) of a TET2 gene during a CAR-T manufacturing process results in the generation of an armored CAR-T with a significant capacity for expansion and subsequent eradication of a tumor when compared to a non-armored CAR-T lacking this capacity for expansion. This strategy may be coupled to a safety switch (e.g. an iC9 safety switch of the disclosure), which allows for the targeted disruption of an armored CAR-T-cell in the event of an adverse reaction from a subject or uncontrolled growth of the armored CAR-T. Exemplary growth advantage factors include, but are not limited to, the factors provided in Table 7.

TABLE 7

Exemplary Growth Advantage Factors.

Full Name
Abbreviation
SEQ ID NO:

Ten Eleven Translocation 2
TET2
16603

DNA (cytosine-5)-methyltransferase 3A
DNMT3A
16604

Transforming protein RhoA
RHOA
16605

Proto-oncogene vav
VAV1
16606

Rhombotin-2
LMO2
16607

T-cell acute lymphocytic leukemia
TALI
16608

protein 1

Suppressor of cytokine signaling 1
SOCS1
16609

herpes virus entry mediator
HVEM
16610

T cell death-associated gene 8
TDAG8
16611

BCL6 corepressor
BCOR
16612

B and T cell attenuator
BTLA
16613

SPARC-like protein 1
SPARCL1
16614

Msh homeobox 1-like protein
MSX1
16615

Armored T-Cells “Null or Switch Receptor” Strategy

In some embodiments, a T-cell of the disclosure is modified to express a modified/chimeric checkpoint receptor to produce an armored T-cell of the disclosure.

In some embodiments, the modified/chimeric checkpoint receptor comprises a null receptor, decoy receptor or dominant negative receptor. A null receptor, decoy receptor or dominant negative receptor of the disclosure may be modified/chimeric receptor/protein. A null receptor, decoy receptor or dominant negative receptor of the disclosure may be truncated for expression of the intracellular signaling domain. Alternatively, or in addition, a null receptor, decoy receptor or dominant negative receptor of the disclosure may be mutated within an intracellular signaling domain at one or more amino acid positions that are determinative or required for effective signaling. Truncation or mutation of null receptor, decoy receptor or dominant negative receptor of the disclosure may result in loss of the receptor's capacity to convey or transduce a checkpoint signal to the cell or within the cell.

For example, a dilution or a blockage of an immunosuppressive checkpoint signal from a PD-L1 receptor expressed on the surface of a tumor cell may be achieved by expressing a modified/chimeric PD-1 null receptor on the surface of an armored T-cell of the disclosure, which effectively competes with the endogenous (non-modified) PD-1 receptors also expressed on the surface of the armored T-cell to reduce or inhibit the transduction of the immunosuppressive checkpoint signal through endogenous PD-1 receptors of the armored T cell. In this exemplary embodiment, competition between the two different receptors for binding to PD-L1 expressed on the tumor cell reduces or diminishes a level of effective checkpoint signaling, thereby enhancing a therapeutic potential of the armored T-cell expressing the PD-1 null receptor.

In some embodiments, the modified/chimeric checkpoint receptor comprises a null receptor, decoy receptor or dominant negative receptor that is a transmembrane receptor.

In some embodiments, the modified/chimeric checkpoint receptor comprises a null receptor, decoy receptor or dominant negative receptor that is a membrane-associated or membrane-linked receptor/protein.

In some embodiments, the modified/chimeric checkpoint receptor comprises a null receptor, decoy receptor or dominant negative receptor that is an intracellular receptor/protein.

In some embodiments, the modified/chimeric checkpoint receptor comprises a null receptor, decoy receptor or dominant negative receptor that is an intracellular receptor/protein. Exemplary null, decoy, or dominant negative intracellular receptors/proteins of the disclosure include, but are not limited to, signaling components downstream of an inhibitory checkpoint signal (as provided, for example, in Tables 1 and 2), a transcription factor (as provided, for example, in Table 3), a cytokine or a cytokine receptor, a chemokine or a chemokine receptor, a cell death or apoptosis receptor/ligand (as provided, for example, in Table 4), a metabolic sensing molecule (as provided, for example, in Table 5), a protein conferring sensitivity to a cancer therapy (as provided, for example, in Table 6), and an oncogene or a tumor suppressor gene (as provided, for example, in Table 7). Exemplary cytokines, cytokine receptors, chemokines and chemokine receptors of the disclosure include, but are not limited to, the cytokines and cytokine receptors as well as chemokines and chemokine receptors provided in Table 8.

TABLE 8

Exemplary Cytokines, Cytokine receptors,

Chemokines and Chemokine Receptors.

Full Name
Abbreviation
SEQ ID NO:

4-1BB Ligand
4-1BBL
16616

Tumor necrosis factor receptor
Apo3 or TNFRSF25
16617

superfamily member 25

Tumor necrosis factor receptor
APRIL or TNFRSF13
16618

superfamily member 13

Bcl2-associated agonist of cell death
Bcl-xL or BAD
16619

Tumor necrosis factor receptor
BCMA or TNFRSF17
16620

superfamily member 17

C-C motif chemokine 1
CCL1
16621

C-C motif chemokine 11
CCL11
16622

C-C motif chemokine 13
CCL13
16623

C-C motif chemokine 14
CCL14
16624

C-C motif chemokine 15
CCL15
16625

C-C motif chemokine 16
CCL16
16626

C-C motif chemokine 17
CCL17
16627

C-C motif chemokine 18
CCL18
16628

C-C motif chemokine 19
CCL19
16629

C-C motif chemokine 2
CCL2
16630

C-C motif chemokine 20
CCL20
16631

C-C motif chemokine 21
CCL21
16632

C-C motif chemokine 22
CCL22
16633

C-C motif chemokine 23
CCL23
16634

C-C motif chemokine 24
CCL24
16635

C-C motif chemokine 25
CCL25
16636

C-C motif chemokine 26
CCL26
16637

C-C motif chemokine 27
CCL27
16638

C-C motif chemokine 28
CCL28
16639

C-C motif chemokine 3
CCL3
16640

C-C motif chemokine 4
CCL4
16641

C-C motif chemokine 5
CCL5
16642

C-C motif chemokine 7
CCL7
16643

C-C motif chemokine 8
CCL8
16644

C-C chemokine receptor type 1
CCR1
16645

C-C chemokine receptor type 10
CCR10
16646

C-C chemokine receptor type 11
CCR11
16647

C-C chemokine receptor type 2
CCR2
16648

C-C chemokine receptor type 3
CCR3
16649

C-C chemokine receptor type 4
CCR4
16650

C-C chemokine receptor type 5
CCR5
16651

C-C chemokine receptor type 6
CCR6
16652

C-C chemokine receptor type 7
CCR7
16653

C-C chemokine receptor type 8
CCR8
16654

C-C chemokine receptor type 9
CCR9
16655

Granulocyte colony-stimulating factor
CD114 or CSF3R
16656

receptor

Macrophage colony-stimulating factor 1
CD115 or CSFIR
16657

receptor

Granulocyte-macrophage colony-
CD116 or CSF2RA
16658

stimulating factor receptor subunit alpha

Mast/stem cell growth factor receptor
CD117 or KIT
16659

Kit

Leukemia inhibitory factor receptor
CD118 or LIFR
16660

Tumor necrosis factor receptor
CD120a or TNFRSF1A
16661

superfamily member 1A

Tumor necrosis factor receptor
CD120b or TNFRSF1B
16662

superfamily member 1B

Interleukin-1 receptor type 1
CD121a or IL1R1
16663

Interleukin-2 receptor subunit beta
CD122 or IL2RB
16664

Interleukin-3 receptor subunit alpha
CD123 or IL3RA
16665

Interleukin-4 receptor subunit alpha
CD124 or IL4R
16666

Interleukin-6 receptor subunit alpha
CD126 or IL6R
16667

Interleukin-7 receptor subunit alpha
CD127 or IL7R
16668

Interleukin-6 receptor subunit beta
CD130 or IL6ST
16669

Cytokine receptor common subunit
CD132 or IL2RG
16670

gamma

Tumor necrosis factor ligand
CD153 or TNFSF8
16671

superfamily member 8

CD40 ligand
CD154 or CD40L
16672

Tumor necrosis factor ligand
CD178 or FASLG
16673

superfamily member 6

Interleukin-12 receptor subunit beta-1
CD212 or IL12RB1
16674

Interleukin-13 receptor subunit alpha-1
CD213a1 or IL13RA1
16675

Interleukin-13 receptor subunit alpha-2
CD213a2 or IL13RA2
16676

Interleukin-2 receptor subunit alpha
CD25 or IL2RA
16677

CD27 antigen
CD27
16678

Tumor necrosis factor receptor
CD30 or TNFRSF8
16679

superfamily member 8

T-cell surface glycoprotein CD4
CD4
16680

Tumor necrosis factor receptor
CD40 or TNFRSF5
16681

superfamily member 5

CD70 antigen
CD70
16682

Tumor necrosis factor receptor
CD95 or FAS or
16683

superfamily member 6
FNFRSF6

Granulocyte-macrophage colony-
CDw116 or CSF2RA
16684

stimulating factor receptor subunit alpha

Interferon gamma receptor 1
CDw119 or IFNGR1
16685

Interleukin-1 receptor type 2
CDw121b or IL1R2
16686

Interleukin-5 receptor subunit alpha
CDw125 or IL5RA
16687

Cytokine receptor common subunit beta
CDw131 or CSF2RB
16688

Tumor necrosis factor receptor
CDw137 or TNFRSF9
16689

superfamily member 9

Interleukin-10 receptor
CDw210 or IL10R
16690

Interleukin-17 receptor A
CDw217 or IL17RA
16691

C-X3-C motif chemokine 1
CX3CL1
16692

CX3C chemokine receptor 1
CX3CR1
16693

C-X-C motif chemokine 1
CXCL1
16694

C-X-C motif chemokine 10
CXCL10
16695

C-X-C motif chemokine 11
CXCL11
16696

C-X-C motif chemokine 12
CXCL12
16697

C-X-C motif chemokine 13
CXCL13
16698

C-X-C motif chemokine 14
CXCL14
16699

C-X-C motif chemokine 16
CXCL16
16700

C-X-C motif chemokine 2
CXCL2
16701

C-X-C motif chemokine 3
CXCL3
16702

C-X-C motif chemokine 4
CXCL4
16703

C-X-C motif chemokine 5
CXCL5
16704

C-X-C motif chemokine 6
CXCL6
16705

C-X-C motif chemokine 7
CXCL7
16706

C-X-C motif chemokine 8
CXCL8
16707

C-X-C motif chemokine 9
CXCL9
16708

C-X-C chemokine receptor type 1
CXCR1
16709

C-X-C chemokine receptor type 2
CXCR2
16710

C-X-C chemokine receptor type 3
CXCR3
16711

C-X-C chemokine receptor type 4
CXCR4
16712

C-X-C chemokine receptor type 5
CXCR5
16713

C-X-C chemokine receptor type 6
CXCR6
16714

C-X-C chemokine receptor type 7
CXCR7
16715

Atypical chemokine receptor 1
DARC or ACKR1
16716

Erythropoietin
Epo
16717

Erythropoietin receptor
EpoR
16718

Receptor-type tyrosine-protein kinase
Flt-3
16719

FLT3

FLT3 Ligand
Flt-3L
16720

Granulocyte colony-stimulating factor
G-CSF or GSF3R
16721

receptor

Tumor necrosis factor receptor
GITR or TNFRSF18
16722

superfamily member 18

GITR Ligand
GITRL
16723

Cytokine receptor common subunit beta
GM-CSF or CSF2RB
16724

Interleukin-6 receptor subunit beta
gp130 or IL6ST
16725

Tumor necrosis factor receptor
HVEM or TNFRSF14
16726

superfamily member 14

Interferon gamma
IENγ
16727

Interferon gamma receptor 2
IFNGR2
16728

Interferon-alpha
IFN-α
16729

Interferon-beta
IFN-β
16730

Interleukin-1 alpha
IL1
16731

Interleukin-10
IL10
16732

Interleukin-10 receptor
IL10R
16733

Interleukin-11
IL-11
16734

Interleukin-11 receptor alpha
IL-11Ra
16735

Interleukin-12
IL12
16736

Interleukin-13
IL13
16737

Interleukin-13 receptor
IL13R
16738

Interleukin-14
IL-14
16739

Interleukin-15
IL15
16740

Interleukin-15 receptor alpha
IL-15Ra
16741

Interleukin-16
IL-16
16742

Interleukin-17
IL17
16743

Interleukin-17 receptor
IL17R
16744

Interleukin-18
IL18
16745

Interleukin-1 receptor alpha
IL-1RA
16746

Interleukin-1 alpha
IL-1α
16747

Interleukin-1beta
IL-1β
16748

Interleukin-2
IL2
16749

Interleukin-20
IL-20
16750

Interleukin-20 receptor alpha
IL-20Rα
16751

Interleukin-20 receptor beta
IL-20Rβ
16752

Interleukin-21
IL21
16753

Interleukin-3
IL-3
16754

Interleukin-35
IL35
16755

Interleukin-4
IL4
16756

Interleukin-4 receptor
IL4R
16757

Interleukin-5
IL5
16758

Interleukin-5 receptor
IL5R
16759

Interleukin-6
IL6
16760

Interleukin-6 receptor
IL6R
16761

Interleukin-7
IL7
16762

Interleukin-9 receptor
IL-9R
16763

Leukemia inhibitory factor
LIF
16764

Leukemia inhibitory factor receptor
LIFR
16765

tumor necrosis factor superfamily
LIGHT or TNFSF14
16766

member 14

Tumor necrosis factor receptor
LTβR or TNFRSF3
16767

superfamily member 3

Lymphotoxin-beta
LT-β
16768

Macrophage colony-stimulating factor 1
M-CSF
16769

Tumor necrosis factor receptor
OPG or TNFRSF11B
16770

superfamily member 11B

Oncostatin-M
OSM
16771

Oncostatin-M receptor
OSMR
16772

Tumor necrosis factor receptor
OX40 or TNFRSF4
16773

superfamily member 4

Tumor necrosis factor ligand
OX40L or TNFSF4
16774

superfamily member 4

Tumor necrosis factor receptor
RANK or TNFRSF11A
16775

superfamily member 11A

Kit Ligand
SCF or KITLG
16776

Tumor necrosis factor receptor
TACI or TNFRSF13B
16777

superfamily member 13B

Tumor necrosis factor ligand
TALL-1 or TNFSF13B
16778

superfamily member 13B

TGF-beta receptor type-1
TGF-βR1
16779

TGF-beta receptor type-2
TGF-βR2
16780

TGF-beta receptor typc-3
TGF-βR3
16781

Transforming growth factor beta-1
TGF-β1
16782

Transforming growth factor beta-2
TGF-β2
16783

Transforming growth factor beta-3
TGF-β3
16784

Tumor necrosis factor alpha
TNF or TNF-α
16785

Tumor necrosis factor beta
TNF-β
16786

Thyroid peroxidase
Tpo
16787

Thyroid peroxidase receptor
TpoR
16788

Tumor necrosis factor ligand
TRAIL or TNFSF10
16789

superfamily member 10

Tumor necrosis factor receptor
TRAILR1 or
16790

superfamily member 10A
TNFRSF10A

Tumor necrosis factor receptor
TRAILR2 or
16791

superfamily member 10B
TNFRSF10B

Tumor necrosis factor ligand
TRANCE or TNFSF11
16792

superfamily member 11

Tumor necrosis factor ligand
TWEAK or TNFSF11
16793

superfamily member 12

Lymphotactin
XCL1
16794

Cytokine SCM-1 beta
XCL2
16795

In some embodiments, the modified/chimeric checkpoint receptor comprises a switch receptor. Exemplary switch receptors may comprise a modified/chimeric receptor/protein of the disclosure wherein a native or wild type intracellular signaling domain is switched or replaced with a different intracellular signaling domain that is either non-native to the protein and/or not a wild-type domain. For example, replacement of an inhibitory signaling domain with a stimulatory signaling domain would switch an immunosuppressive signal into an immunostimulatory signal. Alternatively, replacement of an inhibitory signaling domain with a different inhibitory domain can reduce or enhance the level of inhibitory signaling. Expression or overexpression, of a switch receptor can result in the dilution and/or blockage of a cognate checkpoint signal via competition with an endogenous wildtype checkpoint receptor (not a switch receptor) for binding to the cognate checkpoint receptor expressed within the immunosuppressive tumor microenvironment. Armored T cells of the disclosure may comprise a sequence encoding switch receptors of the disclosure, leading to the expression of one or more switch receptors of the disclosure, and consequently, altering an activity of an armored T-cell of the disclosure. Armored T cells of the disclosure may express a switch receptor of the disclosure that targets an intracellularly expressed protein downstream of a checkpoint receptor, a transcription factor, a cytokine receptor, a death receptor, a metabolic sensing molecule, a cancer therapy, an oncogene, and/or a tumor suppressor protein or gene of the disclosure.

Exemplary switch receptors of the disclosure may comprise or may be derived from a protein including, but are not limited to, the signaling components downstream of an inhibitory checkpoint signal (as provided, for example, in Tables 1 and 2), a transcription factor (as provided, for example, in Table 3), a cytokine or a cytokine receptor, a chemokine or a chemokine receptor, a cell death or apoptosis receptor/ligand (as provided, for example, in Table 4), a metabolic sensing molecule (as provided, for example, in Table 5), a protein conferring sensitivity to a cancer therapy (as provided, for example, in Table 6), and an oncogene or a tumor suppressor gene (as provided, for example, in Table 7). Exemplary cytokines, cytokine receptors, chemokines and chemokine receptors of the disclosure include, but are not limited to, the cytokines and cytokine receptors as well as chemokines and chemokine receptors provided in Table 8.

Armored T-Cells “Synthetic Gene Expression” Strategy

In some embodiments, a T-cell of the disclosure is modified to express chimeric ligand receptor (CLR) or a chimeric antigen receptor (CAR) that mediates conditional gene expression to produce an armored T-cell of the disclosure. The combination of the CLR/CAR and the condition gene expression system in the nucleus of the armored T cell constitutes a synthetic gene expression system that is conditionally activated upon binding of cognate ligand(s) with CLR or cognate antigen(s) with CAR. This system may help to ‘armor’ or enhance therapeutic potential of modified T cells by reducing or limiting synthetic gene expression at the site of ligand or antigen binding, at or within the tumor environment for example.

Exogenous Receptors

In some embodiments, the armored T-cell comprises a composition comprising (a) an inducible transgene construct, comprising a sequence encoding an inducible promoter and a sequence encoding a transgene, and (b) a receptor construct, comprising a sequence encoding a constitutive promoter and a sequence encoding an exogenous receptor, such as a CLR or CAR, wherein, upon integration of the construct of (a) and the construct of (b) into a genomic sequence of a cell, the exogenous receptor is expressed, and wherein the exogenous receptor, upon binding a ligand or antigen, transduces an intracellular signal that targets directly or indirectly the inducible promoter regulating expression of the inducible transgene (a) to modify gene expression.

In some embodiments of a synthetic gene expression system of the disclosure, the composition modifies gene expression by decreasing gene expression. In some embodiments, the composition modifies gene expression by transiently modifying gene expression (e.g. for the duration of binding of the ligand to the exogenous receptor). In some embodiments, the composition modifies gene expression acutely (e.g. the ligand reversibly binds to the exogenous receptor). In some embodiments, the composition modifies gene expression chronically (e.g. the ligand irreversibly binds to the exogenous receptor).

In some embodiments of the compositions of the disclosure, the exogenous receptor of (b) comprises an endogenous receptor with respect to the genomic sequence of the cell. Exemplary receptors include, but are not limited to, intracellular receptors, cell-surface receptors, transmembrane receptors, ligand-gated ion channels, and G-protein coupled receptors.

In some embodiments of the compositions of the disclosure, the exogenous receptor of (b) comprises a non-naturally occurring receptor. In some embodiments, the non-naturally occurring receptor is a synthetic, modified, recombinant, mutant or chimeric receptor. In some embodiments, the non-naturally occurring receptor comprises one or more sequences isolated or derived from a T-cell receptor (TCR). In some embodiments, the non-naturally occurring receptor comprises one or more sequences isolated or derived from a scaffold protein. In some embodiments, including those wherein the non-naturally occurring receptor does not comprise a transmembrane domain, the non-naturally occurring receptor interacts with a second transmembrane, membrane-bound and/or an intracellular receptor that, following contact with the non-naturally occurring receptor, transduces an intracellular signal.

In some embodiments of the compositions of the disclosure, the exogenous receptor of (b) comprises a non-naturally occurring receptor. In some embodiments, the non-naturally occurring receptor is a synthetic, modified, recombinant, mutant or chimeric receptor. In some embodiments, the non-naturally occurring receptor comprises one or more sequences isolated or derived from a T-cell receptor (TCR). In some embodiments, the non-naturally occurring receptor comprises one or more sequences isolated or derived from a scaffold protein. In some embodiments, the non-naturally occurring receptor comprises a transmembrane domain. In some embodiments, the non-naturally occurring receptor interacts with an intracellular receptor that transduces an intracellular signal. In some embodiments, the non-naturally occurring receptor comprises an intracellular signalling domain. In some embodiments, the non-naturally occurring receptor is a chimeric ligand receptor (CLR). In some embodiments, the CLR is a chimeric antigen receptor (CAR).

In some embodiments of the CLR/CARs of the disclosure, the signal peptide comprises a sequence encoding a human CD2, CD3δ, CD3ε, CD3γ, CD3ζ, CD4, CD8α, CD19, CD28, 4-1BB or GM-CSFR signal peptide. In some embodiments, the signal peptide comprises a sequence encoding a human CD8α signal peptide. In some embodiments, the signal peptide comprises an amino acid sequence comprising MALPVTALLLPLALLLHAARP (SEQ ID NO: 17037). In some embodiments, the signal peptide is encoded by a nucleic acid sequence comprising atggcactgccagtcaccgccctgctgctgcctctggctctgctgctgcacgcagctagacca (SEQ ID NO: 17039).

In some embodiments of the CLR/CARs of the disclosure, the transmembrane domain comprises a sequence encoding a human CD2, CD3δ, CD3, CD3γ, CD3ζ, CD4, CD8α, CD19, CD28, 4-1BB or GM-CSFR transmembrane domain. In some embodiments, the transmembrane domain comprises a sequence encoding a human CD8α transmembrane domain. In some embodiments, the transmembrane domain comprises an amino acid sequence comprising IYIWAPLAGTCGVLLLSLVITLYC (SEQ ID NO: 17038). In some embodiments, the transmembrane domain is encoded by a nucleic acid sequence comprising atctacatttgggcaccactggccgggacctgtggagtgctgctgctgagcctggtcatcacactgtactgc (SEQ ID NO: 17040).

In some embodiments of the CLR/CARs of the disclosure, the endodomain comprises a human CD3ζ endodomain. In some embodiments, the at least one costimulatory domain comprises a human 4-1BB, CD28, CD40, ICOS, MyD88, OX-40 intracellular segment, or any combination thereof. In some embodiments, the at least one costimulatory domain comprises a human CD28 and/or a 4-1BB costimulatory domain. In some embodiments, the CD3ζ costimulatory domain comprises an amino acid sequence comprising RVKFSRSADAPAYKQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQ EGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALP PR (SEQ ID NO: 14477). In some embodiments, the CD3ζ costimulatory domain is encoded by a nucleic acid sequence comprising cgcgtgaagtttagtcgatcagcagatgccccagcttacaaacagggacagaaccagctgtataacgagctgaatctgggccgccga gaggaatatgacgtgctggataagcggagaggacgcgaccccgaaatgggaggcaagcccaggcgcaaaaaccctcaggaagg cctgtataacgagctgcagaaggacaaaatggcagaagcctattctgagatcggcatgaagggggagcgacggagaggcaaagg gcacgatgggctgtaccagggactgagcaccgccacaaaggacacctatgatgctctgcatatgcaggcactgcctccaagg (SEQ ID NO: 14478). In some embodiments, the 4-1BB costimulatory domain comprises an amino acid sequence comprising KRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCEL (SEQ ID NO: 14479). In some embodiments, the 4-1BB costimulatory domain is encoded by a nucleic acid sequence comprising aagagaggcaggaagaaactgctgtatattttcaaacagcccttcatgcgccccgtgcagactacccaggaggaagacgggtgctcc tgtcgattccctgaggaagaggaaggcgggtgtgagctg (SEQ ID NO: 14480). In some embodiments, the 4-1BB costimulatory domain is located between the transmembrane domain and the CD28 costimulatory domain.

In some embodiments of the CLR/CARs of the disclosure, the hinge comprises a sequence derived from a human CD8α, IgG4, and/or CD4 sequence. In some embodiments, the hinge comprises a sequence derived from a human CD8α sequence. In some embodiments, the hinge comprises an amino acid sequence comprising TTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACD (SEQ ID NO: 14481). In some embodiments, the hinge is encoded by a nucleic acid sequence comprising actaccacaccagcacctagaccaccaactccagctccaaccatcgcgagtcagcccctgagtctgagacctgaggcctgcaggcc agctgcaggaggagctgtgcacaccaggggcctggacttcgcctgcgac (SEQ ID NO: 14482) or ACCACAACCCCTGCCCCCAGACCTCCCACACCCGCCCCTACCATCGCGAGTCAGC CCCTGAGTCTGAGACCTGAGGCCTGCAGGCCAGCTGCAGGAGGAGCTGTGCACA CCAGGGGCCTGGACTTCGCCTGCGAC (SEQ ID NO: 17047). In some embodiments, the at least one protein scaffold specifically binds the ligand.

In some embodiments of the compositions of the disclosure, the exogenous receptor of (b) comprises a non-naturally occurring receptor. In some embodiments, the CLR is a chimeric antigen receptor (CAR). In some embodiments, the chimeric ligand receptor comprises (a) an ectodomain comprising a ligand recognition region, wherein the ligand recognition region comprises at least scaffold protein; (b) a transmembrane domain, and (c) an endodomain comprising at least one costimulatory domain. In some embodiments, the Centyrin comprises or consists of a consensus sequence of at least one fibronectin type III (FN3) domain. In some embodiments, the at least one fibronectin type III (FN3) domain is derived from a human protein. In some embodiments, the human protein is Tenascin-C. In some embodiments, the consensus sequence comprises LPAPKNLVVSEVTEDSLRLSWTAPDAAFDSFLIQYQESEKVGEAINLTVPGSERSYDL TGLKPGTEYTVSIYGVKGGHRSNPLSAEFTT (SEQ ID NO: 14488). In some embodiments, the consensus sequence comprises MLPAPKNLVVSEVTEDSLRLSWTAPDAAFDSFLIQYQESEKVGEAINLTVPGSERSY DLTGLKPGTEYTVSIYGVKGGHRSNPLSAEFTT (SEQ ID NO: 14489). In some embodiments, the consensus sequence is modified at one or more positions within (a) a A-B loop comprising or consisting of the amino acid residues TEDS at positions 13-16 of the consensus sequence; (b) a B-C loop comprising or consisting of the amino acid residues TAPDAAF at positions 22-28 of the consensus sequence; (c) a C-D loop comprising or consisting of the amino acid residues SEKVGE at positions 38-43 of the consensus sequence; (d) a D-E loop comprising or consisting of the amino acid residues GSER at positions 51-54 of the consensus sequence; (e) a E-F loop comprising or consisting of the amino acid residues GLKPG at positions 60-64 of the consensus sequence; (f) a F-G loop comprising or consisting of the amino acid residues KGGHRSN at positions 75-81 of the consensus sequence; or (g) any combination of (a)-(f). In some embodiments, the Centyrin comprises a consensus sequence of at least 5 fibronectin type III (FN3) domains. In some embodiments, the Centyrin comprises a consensus sequence of at least 10 fibronectin type III (FN3) domains. In some embodiments, the Centyrin comprises a consensus sequence of at least 15 fibronectin type III (FN3) domains. In some embodiments, the scaffold binds an antigen with at least one affinity selected from a K_Dof less than or equal to 10⁻⁹M, less than or equal to 10⁻¹⁰M, less than or equal to 10⁻¹¹M, less than or equal to 10⁻¹²M, less than or equal to 10⁻¹³M, less than or equal to 10⁻¹⁴M, and less than or equal to 10⁻¹⁵M. In some embodiments, the K_Dis determined by surface plasmon resonance.

Inducible Promoters

In some embodiments of the compositions of the disclosure, the sequence encoding the inducible promoter of (a) comprises a sequence encoding an NFκB promoter. In some embodiments of the compositions of the disclosure, the sequence encoding the inducible promoter of (a) comprises a sequence encoding an interferon (IFN) promoter or a sequence encoding an interleukin-2 promoter. In some embodiments, the interferon (IFN) promoter is an IFNγ promoter. In some embodiments of the compositions of the disclosure, the inducible promoter is isolated or derived from the promoter of a cytokine or a chemokine. In some embodiments, the cytokine or chemokine comprises IL2, IL3, IL4, IL5, IL6, IL10. IL12, IL13, IL17A/F, IL21, IL22, IL23, transforming growth factor beta (TGFβ), colony stimulating factor 2 (GM-CSF), interferon gamma (IFNγ), Tumor necrosis factor (TNFα), LTα, perforin, Granzyme C (Gzmc), Granzyme B (Gzmb). C-C motif chemokine ligand 5 (CCL5), C-C motif chemokine ligand 4 (Ccl4), C-C motif chemokine ligand 3 (Ccl3), X-C motif chemokine ligand 1 (Xcl1) and LIF interleukin 6 family cytokine (Lif).

In some embodiments of the compositions of the disclosure, the inducible promoter is isolated or derived from the promoter of a gene comprising a surface protein involved in cell differentiation, activation, exhaustion and function. In some embodiments, the gene comprises CD69, CD71, CTLA4, PD-1, TIG1T, LAG3, TIM-3, GITR, MHCII, COX-2, FASL and 4-1BB.

In some embodiments of the compositions of the disclosure, the inducible promoter is isolated or derived from the promoter of a gene involved in CD metabolism and differentiation. In some embodiments of the compositions of the disclosure, the inducible promoter is isolated or derived from the promoter of Nr4a1, Nr4a3, Tnfrsf9 (4-1BB), Sema7a, Zfp3612, Gadd45b, Dusp5, Dusp6 and Neto2.

Inducible Transgene

In some embodiments, the inducible transgene construct comprises or drives expression of a signaling component downstream of an inhibitory checkpoint signal (as provided, for example, in Tables 1 and 2), a transcription factor (as provided, for example, in Table 3), a cytokine or a cytokine receptor, a chemokine or a chemokine receptor, a cell death or apoptosis receptor/ligand (as provided, for example, in Table 4), a metabolic sensing molecule (as provided, for example, in Table 5), a protein conferring sensitivity to a cancer therapy (as provided, for example, in Table 6 and/or 9), and an oncogene or a tumor suppressor gene (as provided, for example, in Table 7). Exemplary cytokines, cytokine receptors, chemokines and chemokine receptors of the disclosure include, but are not limited to, the cytokines and cytokine receptors as well as chemokines and chemokine receptors provided in Table 8.

TABLE 9

Exemplary therapeutic proteins (and proteins to enhance CAR-T efficacy).

Gene Name
Gene Description
Protein SEQ ID NO

A1BG
Alpha-1-B glycoprotein
SEQ ID NOS: 1-2

A2M
Alpha-2-macroglobulin
SEQ ID NOS: 3-6

A2ML1
Alpha-2-macroglobulin-like 1
SEQ ID NOS: 7-12

A4GNT
Alpha-1,4-N-acetylglucosaminyltransferase
SEQ ID NO: 13

AADACL2
Arylacetamide deacetylase-like 2
SEQ ID NOS: 14-15

AANAT
Aralkylamine N-acetyltransferase
SEQ ID NOS: 16-19

ABCG1
ATP-binding cassette, sub-family G
SEQ ID NOS: 20-26

(WHITE), member 1

ABHD1
Abhydrolase domain containing 1
SEQ ID NOS: 27-31

ABHD10
Abhydrolase domain containing 10
SEQ ID NOS: 32-35

ABHD14A
Abhydrolase domain containing 14A
SEQ ID NOS: 36-40

ABHD15
Abhydrolase domain containing 15
SEQ ID NO: 41

ABI3BP
ABI family, member 3 (NESH) binding
SEQ ID NOS: 42-63

protein

AC008641.1

SEQ ID NO: 73

AC009133.22

SEQ ID NO: 76

AC009491.2

SEQ ID NO: 77

AC011513.3

SEQ ID NOS: 92-93

AC136352.5

SEQ ID NO: 88

AC145212.4
MaFF-interacting protein
SEQ ID NO: 90

AC233755.1

SEQ ID NO: 91

ACACB
Acetyl-CoA carboxylase beta
SEQ ID NOS: 94-100

ACAN
Aggrecan
SEQ ID NOS: 101-108

ACE
Angiotensin I converting enzyme
SEQ ID NOS: 109-121

ACHE
Acetylcholinesterase (Yt blood group)
SEQ ID NOS: 122-134

ACP2
Acid phosphatase 2, lysosomal
SEQ ID NOS: 135-142

ACP5
Acid phosphatase 5, tartrate resistant
SEQ ID NOS: 143-151

ACP6
Acid phosphatase 6, lysophosphatidic
SEQ ID NOS: 152-158

ACPP
Acid phosphatase, prostate
SEQ ID NOS: 163-167

ACR
Acrosin
SEQ ID NOS: 168-169

ACRBP
Acrosin binding protein
SEQ ID NOS: 170-174

ACRV1
Acrosomal vesicle protein 1
SEQ ID NOS: 175-178

ACSF2
Acyl-CoA synthetase family member 2
SEQ ID NOS: 179-187

ACTL10
Actin-like 10
SEQ ID NO: 188

ACVR1
Activin A receptor, type I
SEQ ID NOS: 189-197

ACVR1C
Activin A receptor, type IC
SEQ ID NOS: 198-201

ACVRL1
Activin A receptor type II-like 1
SEQ ID NOS: 202-207

ACYP1
Acylphosphatase 1, erythrocyte (common)
SEQ ID NOS: 208-213

type

ACYP2
Acylphosphatase 2, muscle type
SEQ ID NOS: 214-221

ADAM10
ADAM metallopeptidase domain 10
SEQ ID NOS: 230-237

ADAM12
ADAM metallopeptidase domain 12
SEQ ID NOS: 238-240

ADAM15
ADAM metallopeptidase domain 15
SEQ ID NOS: 241-252

ADAM17
ADAM metallopeptidase domain 17
SEQ ID NOS: 253-255

ADAM18
ADAM metallopeptidase domain 18
SEQ ID NOS: 256-260

ADAM22
ADAM metallopeptidase domain 22
SEQ ID NOS: 261-269

ADAM28
ADAM metallopeptidase domain 28
SEQ ID NOS: 270-275

ADAM29
ADAM metallopeptidase domain 29
SEQ ID NOS: 276-284

ADAM32
ADAM metallopeptidase domain 32
SEQ ID NOS: 285-291

ADAM33
ADAM metallopeptidase domain 33
SEQ ID NOS: 292-296

ADAM7
ADAM metallopeptidase domain 7
SEQ ID NOS: 297-300

ADAM8
ADAM metallopeptidase domain 8
SEQ ID NOS: 301-305

ADAM9
ADAM metallopeptidase domain 9
SEQ ID NOS: 306-311

ADAMDEC1
ADAM-like, decysin 1
SEQ ID NOS: 312-314

ADAMTS1
ADAM metallopeptidase with
SEQ ID NOS: 315-318

thrombospondin type 1 motif, 1

ADAMTS10
ADAM metallopeptidase with
SEQ ID NOS: 319-324

thrombospondin type 1 motif, 10

ADAMTS12
ADAM metallopeptidase with
SEQ ID NOS: 325-327

thrombospondin type 1 motif, 12

ADAMTS13
ADAM metallopeptidase with
SEQ ID NOS: 328-335

thrombospondin type 1 motif, 13

ADAMTS14
ADAM metallopeptidase with
SEQ ID NOS: 336-337

thrombospondin type 1 motif, 14

ADAMTS15
ADAM metallopeptidase with
SEQ ID NO: 338

thrombospondin type 1 motif, 15

ADAMTS16
ADAM metallopeptidase with
SEQ ID NOS: 339-340

thrombospondin type 1 motif, 16

ADAMTS17
ADAM metallopeptidase with
SEQ ID NOS: 341-344

thrombospondin type 1 motif, 17

ADAMTS18
ADAM metallopeptidase with
SEQ ID NOS: 345-348

thrombospondin type 1 motif, 18

ADAMTS19
ADAM metallopeptidase with
SEQ ID NOS: 349-352

thrombospondin type 1 motif, 19

ADAMTS2
ADAM metallopeptidase with
SEQ ID NOS: 353-355

thrombospondin type 1 motif, 2

ADAMTS20
ADAM metallopeptidase with
SEQ ID NOS: 356-359

thrombospondin type 1 motif, 20

ADAMTS3
ADAM metallopeptidase with
SEQ ID NOS: 360-361

thrombospondin type 1 motif, 3

ADAMTS5
ADAM metallopeptidase with
SEQ ID NO: 362

thrombospondin type 1 motif, 5

ADAMTS6
ADAM metallopeptidase with
SEQ ID NOS: 363-364

thrombospondin type 1 motif, 6

ADAMTS7
ADAM metallopeptidase with
SEQ ID NO: 365

thrombospondin type 1 motif, 7

ADAMTS8
ADAM metallopeptidase with
SEQ ID NO: 366

thrombospondin type 1 motif, 8

ADAMTS9
ADAM metallopeptidase with
SEQ ID NOS: 367-371

thrombospondin type 1 motif, 9

ADAMTSL1
ADAMTS-like 1
SEQ ID NOS: 372-382

ADAMTSL2
ADAMTS-like 2
SEQ ID NOS: 383-385

ADAMTSL3
ADAMTS-like 3
SEQ ID NOS: 386-387

ADAMTSL4
ADAMTS-like 4
SEQ ID NOS: 388-391

ADAMTSL5
ADAMTS-like 5
SEQ ID NOS: 392-397

ADCK1
AarF domain containing kinase 1
SEQ ID NOS: 398-402

ADCYAP1
Adenylate cyclase activating polypeptide 1
SEQ ID NOS: 403-404

(pituitary)

ADCYAP1R1
Adenylate cyclase activating polypeptide 1
SEQ ID NOS: 405-411

(pituitary) receptor type I

ADGRA3
Adhesion G protein-coupled receptor A3
SEQ ID NOS: 412-416

ADGRB2
Adhesion G protein-coupled receptor B2
SEQ ID NOS: 417-425

ADGRD1
Adhesion G protein-coupled receptor D1
SEQ ID NOS: 426-431

ADGRE3
Adhesion G protein-coupled receptor E3
SEQ ID NOS: 432-436

ADGRE5
Adhesion G protein-coupled receptor E5
SEQ ID NOS: 437-442

ADGRF1
Adhesion G protein-coupled receptor F1
SEQ ID NOS: 443-447

ADGRG1
Adhesion G protein-coupled receptor G1
SEQ ID NOS: 448-512

ADGRG5
Adhesion G protein-coupled receptor G5
SEQ ID NOS: 513-515

ADGRG6
Adhesion G protein-coupled receptor G6
SEQ ID NOS: 516-523

ADGRV1
Adhesion G protein-coupled receptor V1
SEQ ID NOS: 524-540

ADI1
Acireductone dioxygenase 1
SEQ ID NOS: 541-543

ADIG
Adipogenin
SEQ ID NOS: 544-547

ADIPOQ
Adiponectin, C1Q and collagen domain
SEQ ID NOS: 548-549

containing

ADM
Adrenomedullin
SEQ ID NOS: 550-557

ADM2
Adrenomedullin 2
SEQ ID NOS: 558-559

ADM5
Adrenomedullin 5 (putative)
SEQ ID NO: 560

ADPGK
ADP-dependent glucokinase
SEQ ID NOS: 561-570

ADPRHL2
ADP-ribosylhydrolase like 2
SEQ ID NO: 571

AEBP1
AE binding protein 1
SEQ ID NOS: 572-579

AFM
Afamin
SEQ ID NO: 584

AFP
Alpha-fetoprotein
SEQ ID NOS: 585-586

AGA
Aspartylglucosaminidase
SEQ ID NOS: 587-589

AGER
Advanced glycosylation end product-
SEQ ID NOS: 590-600

specific receptor

AGK
Acylglycerol kinase
SEQ ID NOS: 601-606

AGPS
Alkylglycerone phosphate synthase
SEQ ID NOS: 607-610

AGR2
Anterior gradient 2, protein disulphide
SEQ ID NOS: 611-614

isomerase family member

AGR3
Anterior gradient 3, protein disulphide
SEQ ID NOS: 615-617

isomerase family member

AGRN
Agrin
SEQ ID NOS: 618-621

AGRP
Agouti related neuropeptide
SEQ ID NO: 622

AGT
Angiotensinogen (serpin peptidase inhibitor,
SEQ ID NO: 623

clade A, member 8)

AGTPBP1
ATP/GTP binding protein 1
SEQ ID NOS: 624-627

AGTRAP
Angiotensin II receptor-associated protein
SEQ ID NOS: 628-635

AHCYL2
Adenosylhomocysteinase-like 2
SEQ ID NOS: 636-642

AHSG
Alpha-2-HS-glycoprotein
SEQ ID NOS: 643-644

AIG1
Androgen-induced 1
SEQ ID NOS: 645-653

AK4
Adenylate kinase 4
SEQ ID NOS: 654-657

AKAP10
A kinase (PRKA) anchor protein 10
SEQ ID NOS: 658-666

AKR1C1
Aldo-keto reductase family 1, member C1
SEQ ID NOS: 667-669

AL356289.1

SEQ ID NO: 677

AL589743.1

SEQ ID NO: 678

ALAS2
5′-aminolevulinate synthase 2
SEQ ID NOS: 684-691

ALB
Albumin
SEQ ID NOS: 692-701

ALDH9A1
Aldehyde dehydrogenase 9 family, member
SEQ ID NO: 702

A1

ALDOA
Aldolase A, fructose-bisphosphate
SEQ ID NOS: 703-717

ALG1
ALG1, chitobiosyldiphosphodolichol beta-
SEQ ID NOS: 718-723

mannosyltransferase

ALG5
ALG5, dolichyl-phosphate beta-
SEQ ID NOS: 724-725

glucosyltransferase

ALG9
ALG9, alpha-1,2-mannosyltransferase
SEQ ID NOS: 726-736

ALKBH1
AlkB homolog 1, histone H2A dioxygenase
SEQ ID NOS: 746-748

ALKBH5
AlkB homolog 5, RNA demethylase
SEQ ID NOS: 749-750

ALPI
Alkaline phosphatase, intestinal
SEQ ID NOS: 751-752

ALPL
Alkaline phosphatase, liver/bone/kidney
SEQ ID NOS: 753-757

ALPP
Alkaline phosphatase, placental
SEQ ID NO: 758

ALPPL2
Alkaline phosphatase, placental-like 2
SEQ ID NO: 759

AMBN
Ameloblastin (enamel matrix protein)
SEQ ID NOS: 760-762

AMBP
Alpha-1-microglobulin/bikunin precursor
SEQ ID NOS: 763-765

AMELX
Amelogenin, X-linked
SEQ ID NOS: 766-768

AMELY
Amelogenin, Y-linked
SEQ ID NOS: 769-770

AMH
Anti-Mullerian hormone
SEQ ID NO: 771

AMICA1
Adhesion molecule, interacts with CXADR
SEQ ID NOS: 7348-

antigen 1
7356

AMPD1
Adenosine monophosphate deaminase 1
SEQ ID NOS: 772-774

AMTN
Amelotin
SEQ ID NOS: 775-776

AMY1A
Amylase, alpha 1A (salivary)
SEQ ID NOS: 777-779

AMY1B
Amylase, alpha 1B (salivary)
SEQ ID NOS: 780-783

AMY1C
Amylase, alpha 1C (salivary)
SEQ ID NO: 784

AMY2A
Amylase, alpha 2A (pancreatic)
SEQ ID NOS: 785-787

AMY2B
Amylase, alpha 2B (pancreatic)
SEQ ID NOS: 788-792

ANG
Angiogenin, ribonuclease, RNase A family,
SEQ ID NOS: 793-794

5

ANGEL1
Angel homolog 1 (Drosophila)
SEQ ID NOS: 795-798

ANGPT1
Angiopoietin 1
SEQ ID NOS: 799-803

ANGPT2
Angiopoietin 2
SEQ ID NOS: 804-807

ANGPT4
Angiopoietin 4
SEQ ID NO: 808

ANGPTL1
Angiopoietin-like 1
SEQ ID NOS: 809-811

ANGPTL2
Angiopoietin-like 2
SEQ ID NOS: 812-813

ANGPTL3
Angiopoietin-like 3
SEQ ID NO: 814

ANGPTL4
Angiopoietin-like 4
SEQ ID NOS: 815-822

ANGPTL5
Angiopoietin-like 5
SEQ ID NOS: 823-824

ANGPTL6
Angiopoietin-like 6
SEQ ID NOS: 825-827

ANGPTL7
Angiopoietin-like 7
SEQ ID NO: 828

ANK1
Ankyrin 1, erythrocytic
SEQ ID NOS: 833-843

ANKDD1A
Ankyrin repeat and death domain containing
SEQ ID NOS: 844-850

1A

ANKRD54
Ankyrin repeat domain 54
SEQ ID NOS: 851-859

ANKRD60
Ankyrin repeat domain 60
SEQ ID NO: 860

ANO7
Anoctamin 7
SEQ ID NOS: 861-864

ANO1
#N/A
SEQ ID NO: 865

ANTXR1
Anthrax toxin receptor 1
SEQ ID NOS: 866-869

AOAH
Acyloxyacyl hydrolase (neutrophil)
SEQ ID NOS: 870-874

AOC1
Amine oxidase, copper containing 1
SEQ ID NOS: 875-880

AOC2
Amine oxidase, copper containing 2 (retina-
SEQ ID NOS: 881-882

specific)

AOC3
Amine oxidase, copper containing 3
SEQ ID NOS: 883-889

AP000721.4

SEQ ID NO: 890

APBB1
Amyloid beta (A4) precursor protein-
SEQ ID NOS: 891-907

binding, family B, member 1 (Fe65)

APCDD1
Adenomatosis polyposis coli down-
SEQ ID NOS: 908-913

regulated 1

APCS
Amyloid P component, serum
SEQ ID NO: 914

APELA
Apelin receptor early endogenous ligand
SEQ ID NOS: 915-917

APLN
Apelin
SEQ ID NO: 918

APLP2
Amyloid beta (A4) precursor-like protein 2
SEQ ID NOS: 919-928

APOA1BP
Apolipoprotein A-I
SEQ ID NOS: 929-933

APOA1BP
Apolipoprotein A-I binding protein
SEQ ID NOS: 9177-

9179

APOA2
Apolipoprotein A-II
SEQ ID NOS: 934-942

APOA4
Apolipoprotein A-IV
SEQ ID NO: 943

APOA5
Apolipoprotein A-V
SEQ ID NOS: 944-946

APOB
Apolipoprotein B
SEQ ID NOS: 947-948

APOC1
Apolipoprotein C-I
SEQ ID NOS: 949-957

APOC2
Apolipoprotein C-II
SEQ ID NOS: 958-962

APOC3
Apolipoprotein C-III
SEQ ID NOS: 963-966

APOC4
Apolipoprotein C-IV
SEQ ID NOS: 967-968

APOC4-
APOC4-APOC2 readthrough (NMD
SEQ ID NOS: 969-970

APOC2
candidate)

APOD
Apolipoprotein D
SEQ ID NOS: 971-974

APOE
Apolipoprotein E
SEQ ID NOS: 975-978

APOF
Apolipoprotein F
SEQ ID NO: 979

APOH
Apolipoprotein H (beta-2-glycoprotein I)
SEQ ID NOS: 980-983

APOL1
Apolipoprotein L, 1
SEQ ID NOS: 984-994

APOL3
Apolipoprotein L, 3
SEQ ID NOS: 995-1009

APOM
Apolipoprotein M
SEQ ID NOS: 1010-

1012

APOOL
Apolipoprotein O-like
SEQ ID NOS: 1013-

1015

ARCN1
Archain 1
SEQ ID NOS: 1016-

1020

ARFIP2
ADP-ribosylation factor interacting protein
SEQ ID NOS: 1021-

2
1027

ARHGAP36
Rho GTPase activating protein 36
SEQ ID NOS: 1028-

1033

ARHGAP6
Rho GTPase activating protein 6
SEQ ID NOS: 1043-

1048

ARHGEF4
Rho guanine nucleotide exchange factor
SEQ ID NOS: 1049-

(GEF) 4
1059

ARL16
ADP-ribosylation factor-like 16
SEQ ID NOS: 1060-

1068

ARMC5
Armadillo repeat containing 5
SEQ ID NOS: 1069-

1075

ARNTL
Aryl hydrocarbon receptor nuclear
SEQ ID NOS: 1076-

translocator-like
1090

ARSA
Arylsulfatase A
SEQ ID NOS: 1091-

1096

ARSB
Arylsulfatase B
SEQ ID NOS: 1097-

1100

ARSE
Arylsulfatase E (chondrodysplasia punctata
SEQ ID NOS: 1101-

1)
1104

ARSG
Arylsulfatase G
SEQ ID NOS: 1105-

1108

ARSI
Arylsulfatase family, member I
SEQ ID NOS: 1109-

1111

ARSK
Arylsulfatase family, member K
SEQ ID NOS: 1112-

1116

ART3
ADP-ribosyltransferase 3
SEQ ID NOS: 1117-

1124

ART4
ADP-ribosyltransferase 4 (Dombrock blood
SEQ ID NOS: 1125-

group)
1128

ART5
ADP-ribosyltransferase 5
SEQ ID NOS: 1129-

1133

ARTN
Artemin
SEQ ID NOS: 1134-

1144

ASAH1
N-acylsphingosine amidohydrolase (acid
SEQ ID NOS: 1145-

ceramidase) 1
1195

ASAH2
N-acylsphingosine amidohydrolase (non-
SEQ ID NOS: 1196-

lysosomal ceramidase) 2
1201

ASCL1
Achaete-scute family bHLH transcription
SEQ ID NO: 1202

factor 1

ASIP
Agouti signaling protein
SEQ ID NOS: 1203-

1204

ASPN
Asporin
SEQ ID NOS: 1205-

1206

ASTL
Astacin-like metallo-endopeptidase (M12
SEQ ID NO: 1207

family)

ATAD5
ATPase family, AAA domain containing 5
SEQ ID NOS: 1208-

1209

ATAT1
Alpha tubulin acetyltransferase 1
SEQ ID NOS: 1210-

1215

ATG2A
Autophagy related 2A
SEQ ID NOS: 1216-

1218

ATG5
Autophagy related 5
SEQ ID NOS: 1219-

1227

ATMIN
ATM interactor
SEQ ID NOS: 1228-

1231

ATP13A1
ATPase type 13A1
SEQ ID NOS: 1232-

1234

ATP5F1
ATP synthase, H+ transporting,
SEQ ID NOS: 1235-

mitochondrial Fo complex, subunit Bl
1236

ATP6AP1
ATPase, H+ transporting, lysosomal
SEQ ID NOS: 1237-

accessory protein 1
1244

ATP6AP2
ATPase, H+ transporting, lysosomal
SEQ ID NOS: 1245-

accessory protein 2
1267

ATPAF1
ATP synthase mitochondrial F1 complex
SEQ ID NOS: 1268-

assembly factor 1
1278

AUH
AU RNA binding protein/enoyl-CoA
SEQ ID NOS: 1279-

hydratase
1280

AVP
Arginine vasopressin
SEQ ID NO: 1281

AXIN2
Axin 2
SEQ ID NOS: 1282-

1289

AZGP1
Alpha-2-glycoprotein 1, zinc-binding
SEQ ID NOS: 1290-

1292

AZU1
Azurocidin 1
SEQ ID NOS: 1293-

1294

B2M
Beta-2-microglobulin
SEQ ID NOS: 1295-

1301

B3GALNT1
Beta-1,3-N-acetylgalactosaminyltransferase
SEQ ID NOS: 1302-

1 (globoside blood group)
1314

B3GALNT2
Beta-1,3-N-acetylgalactosaminvltransferase
SEQ ID NOS: 1315-

2
1317

B3GALT1
UDP-Gal:betaGlcNAc beta 1,3-
SEQ ID NO: 1318

galactosyltransferase, polypeptide 1

B3GALT4
UDP-Gal:betaGlcNAc beta 1,3-
SEQ ID NO: 1319

galactosyltransferase, polypeptide 4

B3GALT5
UDP-Gal:betaGlcNAc beta 1,3-
SEQ ID NOS: 1320-

galactosyltransferase, polypeptide 5
1324

B3GALT6
UDP-Gal:betaGal beta 1,3-
SEQ ID NO: 1325

galactosyltransferase polypeptide 6

B3GAT3
Beta-1,3-glucuronyltransferase 3
SEQ ID NOS: 1326-

1330

B3GLCT
Beta 3-glucosvltransferase
SEQ ID NO: 1331

B3GNT3
UDP-GlcNAc:betaGal beta-1,3-N-
SEQ ID NOS: 1332-

acetylglucosaminyltransferase 3
1335

B3GNT4
UDP-GlcNAc:betaGal beta-1,3-N-
SEQ ID NOS: 1336-

acetylglucosaminyltransferase 4
1339

B3GNT6
UDP-GlcNAc:betaGal beta-1,3-N-
SEQ ID NOS: 1340-

acetylglucosaminyltransferase 6
1341

B3GNT7
UDP-GlcNAc:betaGal beta-1,3-N-
SEQ ID NO: 1342

acetylglucosaminyltransferase 7

B3GNT8
UDP-GlcNAc:betaGal beta-1,3-N-
SEQ ID NO: 1343

acetylglucosaminyltransferase 8

B3GNT9
UDP-GlcNAc:betaGal beta-1,3-N-
SEQ ID NO: 1344

acetylglucosaminyltransferase 9

B4GALNT1
Beta-1,4-N-acetyl-galactosaminyl
SEQ ID NOS: 1345-

transferase 1
1356

B4GALNT3
Beta-1,4-N-acetyl-galactosaminyl
SEQ ID NOS: 1357-

transferase 3
1358

B4GALNT4
Beta-1,4-N-acetyl-galactosaminyl
SEQ ID NOS: 1359-

transferase 4
1361

B4GALT4
UDP-Gal:betaGlcNAc beta 1,4-
SEQ ID NOS: 1362-

galactosyltransferase, polypeptide 4
1374

B4GALT5
UDP-Gal:betaGlcNAc beta 1,4-
SEQ ID NO: 1375

galactosyltransferase, polypeptide 5

B4GALT6
UDP-Gal:betaGlcNAc beta 1,4-
SEQ ID NOS: 1376-

galactosyltransferase, polypeptide 6
1379

B4GAT1
Beta-1,4-glucuronyltransferase 1
SEQ ID NO: 1380

B9D1
B9 protein domain 1
SEQ ID NOS: 1381-

1397

BACE2
Beta-site APP-cleaving enzyme 2
SEQ ID NOS: 1398-

1400

BAGE5
B melanoma antigen family, member 5
SEQ ID NO: 1401

BCAM
Basal cell adhesion molecule (Lutheran
SEQ ID NOS: 1402-

blood group)
1405

BCAN
Brevican
SEQ ID NOS: 1406-

1412

BCAP29
B-cell receptor-associated protein 29
SEQ ID NOS: 1413-

1425

BCAR1
Breast cancer anti-estrogen resistance 1
SEQ ID NOS: 1426-

1443

BCHE
Butyrylcholinesterase
SEQ ID NOS: 1444-

1448

BCKDHB
Branched chain keto acid dehydrogenase
SEQ ID NOS: 1449-

E1, beta polypeptide
1451

BDNF
Brain-derived neurotrophic factor
SEQ ID NOS: 1452-

1469

BGLAP
Bone gamma-carboxyglutamate (gla)
SEQ ID NO: 1470

protein

BGN
Biglycan
SEQ ID NOS: 1471-

1472

BLVRB
Biliverdin reductase B
SEQ ID NOS: 1473-

1477

BMP1
Bone morphogenetic protein 1
SEQ ID NOS: 1478-

1489

BMP10
Bone morphogenetic protein 10
SEQ ID NO: 1490

BMP15
Bone morphogenetic protein 15
SEQ ID NO: 1491

BMP2
Bone morphogenetic protein 2
SEQ ID NO: 1492

BMP3
Bone morphogenetic protein 3
SEQ ID NO: 1493

BMP4
Bone morphogenetic protein 4
SEQ ID NOS: 1494-

1501

BMP6
Bone morphogenetic protein 6
SEQ ID NO: 1502

BMP7
Bone morphogenetic protein 7
SEQ ID NOS: 1503-

1506

BMP8A
Bone morphogenetic protein 8a
SEQ ID NO: 1507

BMP8B
Bone morphogenetic protein 8b
SEQ ID NO: 1508

BMPER
BMP binding endothelial regulator
SEQ ID NOS: 1509-

1512

BNC1
Basonuclin 1
SEQ ID NOS: 1513-

1514

BOC
BOC cell adhesion associated, oncogene
SEQ ID NOS: 1515-

regulated
1525

BOD1
Biorientation of chromosomes in cell
SEQ ID NOS: 1526-

division 1
1530

BOLA1
BolA family member 1
SEQ ID NOS: 1531-

1533

BPI
Bactericidal/permeability-increasing protein
SEQ ID NOS: 1534-

1537

BPIFA1
BPI fold containing family A, member 1
SEQ ID NOS: 1538-

1541

BPIFA2
BPI fold containing family A, member 2
SEQ ID NOS: 1542-

1543

BPIFA3
BPI fold containing family A, member 3
SEQ ID NOS: 1544-

1545

BPIFB1
BPI fold containing family B, member 1
SEQ ID NOS: 1546-

1547

BPIFB2
BPI fold containing family B, member 2
SEQ ID NO: 1548

BPIFB3
BPI fold containing family B, member 3
SEQ ID NO: 1549

BPIFB4
BPI fold containing family B, member 4
SEQ ID NOS: 1550-

1551

BPIFB6
BPI fold containing family B, member 6
SEQ ID NOS: 1552-

1553

BPIFC
BPI fold containing family C
SEQ ID NOS: 1554-

1557

BRF1
BRF1, RNA polymerase III transcription
SEQ ID NOS: 1558-

initiation factor 90 kDa subunit
1573

BRINP1
Bone morphogenetic protein/retinoic acid
SEQ ID NOS: 1574-

inducible neural-specific 1
1575

BRINP2
Bone morphogenetic protein/retinoic acid
SEQ ID NO: 1576

inducible neural-specific 2

BRINP3
Bone morphogenetic protein/retinoic acid
SEQ ID NOS: 1577-

inducible neural-specific 3
1579

BSG
Basigin (Ok blood group)
SEQ ID NOS: 1580-

1590

BSPH1
Binder of sperm protein homolog 1
SEQ ID NO: 1591

BST1
Bone marrow stromal cell antigen 1
SEQ ID NOS: 1592-

1596

BTBD17
BTB (POZ) domain containing 17
SEQ ID NO: 1597

BTD
Biotinidase
SEQ ID NOS: 1598-

1607

BTN2A2
Butyrophilin, subfamily 2, member A2
SEQ ID NOS: 1608-

1621

BTN3A1
Butyrophilin, subfamily 3, member A1
SEQ ID NOS: 1622-

1628

BTN3A2
Butyrophilin, subfamily 3, member A2
SEQ ID NOS: 1629-

1639

BTN3A3
Butyrophilin, subfamily 3, member A3
SEQ ID NOS: 1640-

1648

C10orf10
Chromosome 10 open reading frame 10
SEQ ID NOS: 4169-

4170

C10orf99
Chromosome 10 open reading frame 99
SEQ ID NO: 1650

C11orf1
Chromosome 11 open reading frame 1
SEQ ID NOS: 1651-

1655

C11orf24
Chromosome 11 open reading frame 24
SEQ ID NOS: 1656-

1658

C11orf45
Chromosome 11 open reading frame 45
SEQ ID NOS: 1659-

1660

C11orf94
Chromosome 11 open reading frame 94
SEQ ID NO: 1661

C12orf10
Chromosome 12 open reading frame 10
SEQ ID NOS: 1662-

1665

C12orf49
Chromosome 12 open reading frame 49
SEQ ID NOS: 1666-

1669

C12orf73
Chromosome 12 open reading frame 73
SEQ ID NOS: 1670-

1679

C12orf76
Chromosome 12 open reading frame 76
SEQ ID NOS: 1680-

1687

C14orf80
Chromosome 14 open reading frame 80
SEQ ID NOS: 13083-

13096

C14orf93
Chromosome 14 open reading frame 93
SEQ ID NOS: 1688-

1703

C16orf89
Chromosome 16 open reading frame 89
SEQ ID NOS: 1704-

1706

C16orf90
Chromosome 16 open reading frame 90
SEQ ID NOS: 1707-

1708

C17orf67
Chromosome 17 open reading frame 67
SEQ ID NO: 1709

C17orf75
Chromosome 17 open reading frame 75
SEQ ID NOS: 1710-

1718

C17orf99
Chromosome 17 open reading frame 99
SEQ ID NOS: 1719-

1721

C18orf54
Chromosome 18 open reading frame 54
SEQ ID NOS: 1722-

1726

C19orf47
Chromosome 19 open reading frame 47
SEQ ID NOS: 1727-

1734

C19orf70
Chromosome 19 open reading frame 70
SEQ ID NOS: 1735-

1738

C19orf80
Chromosome 19 open reading frame 80
SEQ ID NOS: 829-832

C1GALT1
Core 1 synthase, glycoprotein-N-
SEQ ID NOS: 1739-

acetylgalactosamine 3-beta-
1743

galactosyltransferase 1

C1orf127
Chromosome 1 open reading frame 127
SEQ ID NOS: 1744-

1747

C1orf159
Chromosome 1 open reading frame 159
SEQ ID NOS: 1748-

1760

C1orf198
Chromosome 1 open reading frame 198
SEQ ID NOS: 1761-

1765

C1orf234
Chromosome 1 open reading frame 234
SEQ ID NOS: 13118-

13120

C1orf54
Chromosome 1 open reading frame 54
SEQ ID NOS: 1766-

1768

C1orf56
Chromosome 1 open reading frame 56
SEQ ID NO: 1769

C1QA
Complement component 1, q
SEQ ID NOS: 1770-

subcomponent, A chain
1772

C1QB
Complement component 1, q
SEQ ID NOS: 1773-

subcomponent, B chain
1776

C1QC
Complement component 1, q
SEQ ID NOS: 1777-

subcomponent, C chain
1779

C1QL1
Complement component 1, q
SEQ ID NO: 1780

subcomponent-like 1

C1QL2
Complement component 1, q
SEQ ID NO: 1781

subcomponent-like 2

C1QL3
Complement component 1, q
SEQ ID NOS: 1782-

subcomponent-like 3
1783

C1QL4
Complement component 1, q
SEQ ID NO: 1784

subcomponent-like 4

C1QTNF1
C1q and tumor necrosis factor related
SEQ ID NOS: 1785-

protein 1
1794

C1QTNF2
C1q and tumor necrosis factor related
SEQ ID NO: 1796

protein 2

C1QTNF3
C1q and tumor necrosis factor related
SEQ ID NOS: 1797-

protein 3
1798

C1QTNF4
C1q and tumor necrosis factor related
SEQ ID NOS: 1799-

protein 4
1800

C1QTNF5
C1q and tumor necrosis factor related
SEQ ID NOS: 1801-

protein 5
1803

C1QTNF7
C1q and tumor necrosis factor related
SEQ ID NOS: 1804-

protein 7
1808

C1QTNF8
C1q and tumor necrosis factor related
SEQ ID NOS: 1809-

protein 8
1810

C1QTNF9
C1q and tumor necrosis factor related
SEQ ID NOS: 1811-

protein 9
1812

C1QTNF9B
C1q and tumor necrosis factor related
SEQ ID NOS: 1813-

protein 9B
1815

C1R
Complement component 1, r subcomponent
SEQ ID NOS: 1816-

1824

C1RL
Complement component 1, r subcomponent-
SEQ ID NOS: 1825-

like
1833

C1S
Complement component 1, s subcomponent
SEQ ID NOS: 1834-

1843

C2
Complement component 2
SEQ ID NOS: 1844-

1858

C21orf33
Chromosome 21 open reading frame 33
SEQ ID NOS: 1859-

1867

C21orf62
Chromosome 21 open reading frame 62
SEQ ID NOS: 1868-

1871

C22orf15
Chromosome 22 open reading frame 15
SEQ ID NOS: 1872-

1874

C22orf46
Chromosome 22 open reading frame 46
SEQ ID NO: 1875

C2CD2
C2 calcium-dependent domain containing 2
SEQ ID NOS: 1876-

1878

C2orf40
Chromosome 2 open reading frame 40
SEQ ID NOS: 1879-

1881

C2orf66
Chromosome 2 open reading frame 66
SEQ ID NO: 1882

C2orf69
Chromosome 2 open reading frame 69
SEQ ID NO: 1883

C2orf78
Chromosome 2 open reading frame 78
SEQ ID NO: 1884

C3
Complement component 3
SEQ ID NOS: 1885-

1889

C3orf33
Chromosome 3 open reading frame 33
SEQ ID NOS: 1890-

1894

C3orf58
Chromosome 3 open reading frame 58
SEQ ID NOS: 1895-

1898

C4A
Complement component 4A (Rodgers blood
SEQ ID NOS: 1899-

group)
1900

C4B
Complement component 4B (Chido blood
SEQ ID NOS: 1901-

group)
1902

C4BPA
Complement component 4 binding protein,
SEQ ID NOS: 1903-

alpha
1905

C4BPB
Complement component 4 binding protein,
SEQ ID NOS: 1906-

beta
1910

C4orf26
Chromosome 4 open reading frame 26
SEQ ID NOS: 9751-

9754

C4orf48
Chromosome 4 open reading frame 48
SEQ ID NOS: 1911-

1912

C5
Complement component 5
SEQ ID NO: 1913

C5orf46
Chromosome 5 open reading frame 46
SEQ ID NOS: 1914-

1915

C6
Complement component 6
SEQ ID NOS: 1916-

1919

C6orf120
Chromosome 6 open reading frame 120
SEQ ID NO: 1920

C6orf15
Chromosome 6 open reading frame 15
SEQ ID NO: 1921

C6orf25
Chromosome 6 open reading frame 25
SEQ ID NOS: 8832-

8839

C6orf58
Chromosome 6 open reading frame 58
SEQ ID NO: 1922

C7
Complement component 7
SEQ ID NO: 1923

C7orf57
Chromosome 7 open reading frame 57
SEQ ID NOS: 1924-

1928

C7orf73
Chromosome 7 open reading frame 73
SEQ ID NOS: 12924-

12925

C8A
Complement component 8, alpha
SEQ ID NO: 1929

polypeptide

C8B
Complement component 8, beta polypeptide
SEQ ID NOS: 1930-

1932

C8G
Complement component 8, gamma
SEQ ID NOS: 1933-

polypeptide
1934

C9
Complement component 9
SEQ ID NO: 1935

C9orf47
Chromosome 9 open reading frame 47
SEQ ID NOS: 1936-

1938

CA10
Carbonic anhydrase X
SEQ ID NOS: 1939-

1945

CA11
Carbonic anhydrase XI
SEQ ID NOS: 1946-

1947

CA6
Carbonic anhydrase VI
SEQ ID NOS: 1948-

1952

CA9
Carbonic anhydrase IX
SEQ ID NOS: 1953-

1954

CABLES1
Cdk5 and Abl enzyme substrate 1
SEQ ID NOS: 1955-

1960

CABP1
Calcium binding protein 1
SEQ ID NOS: 1961-

1964

CACNA2D1
Calcium channel, voltage-dependent, alpha
SEQ ID NOS: 1965-

2/delta subunit 1
1968

CACNA2D4
Calcium channel, voltage-dependent, alpha
SEQ ID NOS: 1969-

2/delta subunit 4
1982

CADM3
Cell adhesion molecule 3
SEQ ID NOS: 1983-

1985

CALCA
Calcitonin-related polypeptide alpha
SEQ ID NOS: 1986-

1990

CALCB
Calcitonin-related polypeptide beta
SEQ ID NOS: 1991-

1993

CALCR
Calcitonin receptor
SEQ ID NOS: 1994-

2000

CALCRL
Calcitonin receptor-like
SEQ ID NOS: 2001-

2005

CALR
Calreticulin
SEQ ID NOS: 2011-

2014

CALR3
Calreticulin 3
SEQ ID NOS: 2015-

2016

CALU
Calumenin
SEQ ID NOS: 2017-

2022

CAMK2D
Calcium/calmodulin-dependent protein
SEQ ID NOS: 2023-

kinase II delta
2034

CAMP
Cathelicidin antimicrobial peptide
SEQ ID NO: 2035

CANX
Calnexin
SEQ ID NOS: 2036-

2050

CARKD
Carbohydrate kinase domain containing
SEQ ID NOS: 9175-

9176

CARM1
Coactivator-associated arginine
SEQ ID NOS: 2051-

methyltransferase 1
2058

CARNS1
Carnosine synthase 1
SEQ ID NOS: 2059-

2061

CARTPT
CART prepropeptide
SEQ ID NO: 2062

CASQ1
Calsequestrin 1 (fast-twitch, skeletal
SEQ ID NOS: 2063-

muscle)
2064

CASQ2
Calsequestrin 2 (cardiac muscle)
SEQ ID NO: 2065

CATSPERG
Catsper channel auxiliary subunit gamma
SEQ ID NOS: 2066-

2073

CBLN1
Cerebellin 1 precursor
SEQ ID NOS: 2074-

2076

CBLN2
Cerebellin 2 precursor
SEQ ID NOS: 2077-

2080

CBLN3
Cerebellin 3 precursor
SEQ ID NOS: 2081-

2082

CBLN4
Cerebellin 4 precursor
SEQ ID NO: 2083

CCBE1
Collagen and calcium binding EGF domains
SEQ ID NOS: 2084-

1
2086

CCDC108
Coiled-coil domain containing 108
SEQ ID NOS: 2659-

2668

CCDC112
Coiled-coil domain containing 112
SEQ ID NOS: 2087-

2090

CCDC129
Coiled-coil domain containing 129
SEQ ID NOS: 2091-

2098

CCDC134
Coiled-coil domain containing 134
SEQ ID NOS: 2099-

2100

CCDC149
Coiled-coil domain containing 149
SEQ ID NOS: 2101-

2104

CCDC3
Coiled-coil domain containing 3
SEQ ID NOS: 2105-

2106

CCDC80
Coiled-coil domain containing 80
SEQ ID NOS: 2107-

2110

CCDC85A
Coiled-coil domain containing 85A
SEQ ID NO: 2111

CCDC88B
Coiled-coil domain containing 88B
SEQ ID NOS: 2112-

2114

CCER2
Coiled-coil glutamate-rich protein 2
SEQ ID NOS: 2115-

2116

CCK
Cholecystokinin
SEQ ID NOS: 2117-

2119

CCL1
Chemokine (C-C motif) ligand 1
SEQ ID NO: 2120

CCL11
Chemokine (C-C motif) ligand 11
SEQ ID NO: 2121

CCL13
Chemokine (C-C motif) ligand 13
SEQ ID NOS: 2122-

2123

CCL14
Chemokine (C-C motif) ligand 14
SEQ ID NOS: 2124-

2127

CCL15
Chemokine (C-C motif) ligand 15
SEQ ID NOS: 2128-

2129

CCL16
Chemokine (C-C motif) ligand 16
SEQ ID NOS: 2130-

2132

CCL17
Chemokine (C-C motif) ligand 17
SEQ ID NOS: 2133-

2134

CCL18
Chemokine (C-C motif) ligand 18
SEQ ID NO: 2135

(pulmonary and activation-regulated)

CCL19
Chemokine (C-C motif) ligand 19
SEQ ID NOS: 2136-

2137

CCL2
Chemokine (C-C motif) ligand 2
SEQ ID NOS: 2138-

2139

CCL20
Chemokine (C-C motif) ligand 20
SEQ ID NOS: 2140-

2142

CCL21
Chemokine (C-C motif) ligand 21
SEQ ID NOS: 2143-

2144

CCL22
Chemokine (C-C motif) ligand 22
SEQ ID NO: 2145

CCL23
Chemokine (C-C motif) ligand 23
SEQ ID NOS: 2146-

2148

CCL24
Chemokine (C-C motif) ligand 24
SEQ ID NOS: 2149-

2150

CCL25
Chemokine (C-C motif) ligand 25
SEQ ID NOS: 2151-

2154

CCL26
Chemokine (C-C motif) ligand 26
SEQ ID NOS: 2155-

2156

CCL27
Chemokine (C-C motif) ligand 27
SEQ ID NO: 2157

CCL28
Chemokine (C-C motif) ligand 28
SEQ ID NOS: 2158-

2160

CCL3
Chemokine (C-C motif) ligand 3
SEQ ID NO: 2161

CCL3L3
Chemokine (C-C motif) ligand 3-like 3
SEQ ID NO: 2162

CCL4
Chemokine (C-C motif) ligand 4
SEQ ID NOS: 2163-

2164

CCL4L2
Chemokine (C-C motif) ligand 4-like 2
SEQ ID NOS: 2165-

2174

CCL5
Chemokine (C-C motif) ligand 5
SEQ ID NOS: 2175-

2177

CCL7
Chemokine (C-C motif) ligand 7
SEQ ID NOS: 2178-

2180

CCL8
Chemokine (C-C motif) ligand 8
SEQ ID NO: 2181

CCNB1IP1
Cyclin Bl interacting protein 1, E3
SEQ ID NOS: 2182-

ubiquitin protein ligase
2193

CCNL1
Cyclin L1
SEQ ID NOS: 2194-

2202

CCNL2
Cyclin L2
SEQ ID NOS: 2203-

2210

CD14
CD14 molecule
SEQ ID NOS: 2211-

2215

CD160
CD160 molecule
SEQ ID NOS: 2216-

2220

CD164
CD164 molecule, sialomucin
SEQ ID NOS: 2221-

2226

CD177
CD177 molecule
SEQ ID NOS: 2227-

2229

CD1E
CD1e molecule
SEQ ID NOS: 2230-

2243

CD2
CD2 molecule
SEQ ID NOS: 2244-

2245

CD200
CD200 molecule
SEQ ID NOS: 2246-

2252

CD200R1
CD200 receptor 1
SEQ ID NOS: 2253-

2257

CD22
CD22 molecule
SEQ ID NOS: 2258-

2275

CD226
CD226 molecule
SEQ ID NOS: 2276-

2283

CD24
CD24 molecule
SEQ ID NOS: 2284-

2290

CD276
CD276 molecule
SEQ ID NOS: 2291-

2306

CD300A
CD300a molecule
SEQ ID NOS: 2307-

2311

CD300LB
CD300 molecule-like family member b
SEQ ID NOS: 2312-

2313

CD300LF
CD300 molecule-like family member f
SEQ ID NOS: 2314-

2322

CD300LG
CD300 molecule-like family member g
SEQ ID NOS: 2323-

2328

CD3D
CD3d molecule, delta (CD3-TCR complex)
SEQ ID NOS: 2329-

2332

CD4
CD4 molecule
SEQ ID NOS: 2333-

2335

CD40
CD40 molecule, TNF receptor superfamily
SEQ ID NOS: 2336-

member 5
2339

CD44
CD44 molecule (Indian blood group)
SEQ ID NOS: 2340-

2366

CD48
CD48 molecule
SEQ ID NOS: 2367-

2369

CD5
CD5 molecule
SEQ ID NOS: 2370-

2371

CD55
CD55 molecule, decay accelerating factor
SEQ ID NOS: 2372-

for complement (Cromer blood group)
2382

CD59
CD59 molecule, complement regulatory
SEQ ID NOS: 2383-

protein
2393

CD5L
CD5 molecule-like
SEQ ID NO: 2394

CD6
CD6 molecule
SEQ ID NOS: 2395-

2402

CD68
CD68 molecule
SEQ ID NOS: 2403-

2406

CD7
CD7 molecule
SEQ ID NOS: 2407-

2412

CD79A
CD79a molecule, immunoglobulin-
SEQ ID NOS: 2413-

associated alpha
2415

CD80
CD80 molecule
SEQ ID NOS: 2416-

2418

CD86
CD86 molecule
SEQ ID NOS: 2419-

2425

CD8A
CD8a molecule
SEQ ID NOS: 2426-

2429

CD8B
CD8b molecule
SEQ ID NOS: 2430-

2435

CD99
CD99 molecule
SEQ ID NOS: 2436-

2444

CDC23
Cell division cycle 23
SEQ ID NOS: 2445-

2449

CDC40
Cell division cycle 40
SEQ ID NOS: 2450-

2452

CDC45
Cell division cycle 45
SEQ ID NOS: 2453-

2459

CDCP1
CUB domain containing protein 1
SEQ ID NOS: 2460-

2461

CDCP2
CUB domain containing protein 2
SEQ ID NOS: 2462-

2463

CDH1
Cadherin 1, type 1
SEQ ID NOS: 2464-

2471

CDH11
Cadherin 11, type 2, OB-cadherin
SEQ ID NOS: 2472-

(osteoblast)
2481

CDH13
Cadherin 13
SEQ ID NOS: 2482-

2491

CDH17
Cadherin 17, LI cadherin (liver-intestine)
SEQ ID NOS: 2492-

2496

CDH18
Cadherin 18, type 2
SEQ ID NOS: 2497-

2503

CDH19
Cadherin 19, type 2
SEQ ID NOS: 2504-

2508

CDH23
Cadherin-related 23
SEQ ID NOS: 2509-

2524

CDH5
Cadherin 5, type 2 (vascular endothelium)
SEQ ID NOS: 2525-

2532

CDHR1
Cadherin-related family member 1
SEQ ID NOS: 2533-

2538

CDHR4
Cadherin-related family member 4
SEQ ID NOS: 2539-

2543

CDHR5
Cadherin-related family member 5
SEQ ID NOS: 2544-

2550

CDKN2A
Cyclin-dependent kinase inhibitor 2A
SEQ ID NOS: 2551-

2561

CDNF
Cerebral dopamine neurotrophic factor
SEQ ID NOS: 2562-

2563

CDON
Cell adhesion associated, oncogene
SEQ ID NOS: 2564-

regulated
2571

CDSN
Corneodesmosin
SEQ ID NO: 2572

CEACAM16
Carcinoembryonic antigen-related cell
SEQ ID NOS: 2573-

adhesion molecule 16
2574

CEACAM18
Carcinoembryonic antigen-related cell
SEQ ID NO: 2575

adhesion molecule 18

CEACAM19
Carcinoembryonic antigen-related cell
SEQ ID NOS: 2576-

adhesion molecule 19
2582

CEACAM5
Carcinoembryonic antigen-related cell
SEQ ID NOS: 2583-

adhesion molecule 5
2590

CEACAM7
Carcinoembryonic antigen-related cell
SEQ ID NOS: 2591-

adhesion molecule 7
2593

CEACAM8
Carcinoembryonic antigen-related cell
SEQ ID NOS: 2594-

adhesion molecule 8
2595

CECR1
Cat eye syndrome chromosome region,
SEQ ID NOS: 222-229

candidate 1

CECR5
Cat eye syndrome chromosome region,
SEQ ID NOS: 6411-

candidate 5
6413

CEL
Carboxyl ester lipase
SEQ ID NO: 2596

CELA2A
Chymotrypsin-like elastase family, member
SEQ ID NO: 2597

2A

CELA2B
Chymotrypsin-like elastase family, member
SEQ ID NOS: 2598-

2B
2599

CELA3A
Chymotrypsin-like elastase family, member
SEQ ID NOS: 2600-

3A
2602

CELA3B
Chymotrypsin-like elastase family, member
SEQ ID NOS: 2603-

3B
2605

CEMIP
Cell migration inducing protein, hyaluronan
SEQ ID NOS: 2606-

binding
2610

CEP89
Centrosomal protein 89 kDa
SEQ ID NOS: 2611-

2616

CER1
Cerberus 1, DAN family BMP antagonist
SEQ ID NO: 2617

CERCAM
Cerebral endothelial cell adhesion molecule
SEQ ID NOS: 2618-

2625

CERS1
Ceramide synthase 1
SEQ ID NOS: 2626-

2630

CES1
Carboxylesterase 1
SEQ ID NOS: 2631-

2636

CES3
Carboxylesterase 3
SEQ ID NOS: 2637-

2641

CES4A
Carboxylesterase 4A
SEQ ID NOS: 2642-

2647

CES5A
Carboxylesterase 5A
SEQ ID NOS: 2648-

2655

CETP
Cholesteryl ester transfer protein, plasma
SEQ ID NOS: 2656-

2658

CFB
Complement factor B
SEQ ID NOS: 2669-

2673

CFC1
Cripto, FRL-1, cryptic family 1
SEQ ID NOS: 2674-

2676

CFC1B
Cripto, FRL-1, cryptic family 1B
SEQ ID NOS: 2677-

2679

CFD
Complement factor D (adipsin)
SEQ ID NOS: 2680-

2681

CFDP1
Craniofacial development protein 1
SEQ ID NOS: 2682-

2685

CFH
Complement factor H
SEQ ID NOS: 2686-

2688

CFHR1
Complement factor H-related 1
SEQ ID NOS: 2689-

2690

CFHR2
Complement factor H-related 2
SEQ ID NOS: 2691-

2692

CFHR3
Complement factor H-related 3
SEQ ID NOS: 2693-

2697

CFHR4
Complement factor H-related 4
SEQ ID NOS: 2698-

2701

CFHR5
Complement factor H-related 5
SEQ ID NO: 2702

CFI
Complement factor I
SEQ ID NOS: 2703-

2707

CFP
Complement factor properdin
SEQ ID NOS: 2708-

2711

CGA
Glycoprotein hormones, alpha polypeptide
SEQ ID NOS: 2712-

2716

CGB
Chorionic gonadotropin, beta polypeptide
SEQ ID NO: 2721

CGB1
Chorionic gonadotropin, beta polypeptide 1
SEQ ID NOS: 2717-

2718

CGB2
Chorionic gonadotropin, beta polypeptide 2
SEQ ID NOS: 2719-

2720

CGB5
Chorionic gonadotropin, beta polypeptide 5
SEQ ID NO: 2722

CGB7
Chorionic gonadotropin, beta polypeptide 7
SEQ ID NOS: 2723-

2725

CGB8
Chorionic gonadotropin, beta polypeptide 8
SEQ ID NO: 2726

CGREF1
Cell growth regulator with EF-hand domain
SEQ ID NOS: 2727-

1
2734

CH507-9B2.3

SEQ ID NOS: 5532-

5538

CHAD
Chondroadherin
SEQ ID NOS: 2735-

2737

CHADL
Chondroadherin-like
SEQ ID NOS: 2738-

2740

CHEK2
Checkpoint kinase 2
SEQ ID NOS: 2741-

2762

CHGA
Chromogranin A
SEQ ID NOS: 2763-

2765

CHGB
Chromogranin B
SEQ ID NOS: 2766-

2767

CHI3L1
Chitinase 3-like 1 (cartilage glycoprotein-
SEQ ID NOS: 2768-

39)
2769

CHI3L2
Chitinase 3-like 2
SEQ ID NOS: 2770-

2783

CHIA
Chitinase, acidic
SEQ ID NOS: 2784-

2792

CHID1
Chitinase domain containing 1
SEQ ID NOS: 2793-

2811

CHIT1
Chitinase 1 (chitotriosidase)
SEQ ID NOS: 2812-

2815

CHL1
Cell adhesion molecule L1-like
SEQ ID NOS: 2816-

2824

CHN1
Chimerin 1
SEQ ID NOS: 2825-

2835

CHPF
Chondroitin polymerizing factor
SEQ ID NOS: 2836-

2838

CHPF2
Chondroitin polymerizing factor 2
SEQ ID NOS: 2839-

2842

CHRD
Chordin
SEQ ID NOS: 2843-

2848

CHRDL1
Chordin-like 1
SEQ ID NOS: 2849-

2853

CHRDL2
Chordin-like 2
SEQ ID NOS: 2854-

2862

CHRNA2
Cholinergic receptor, nicotinic, alpha 2
SEQ ID NOS: 2863-

(neuronal)
2871

CHRNA5
Cholinergic receptor, nicotinic, alpha 5
SEQ ID NOS: 2872-

(neuronal)
2875

CHRNB1
Cholinergic receptor, nicotinic, beta 1
SEQ ID NOS: 2876-

(muscle)
2881

CHRND
Cholinergic receptor, nicotinic, delta
SEQ ID NOS: 2882-

(muscle)
2887

CHST1
Carbohydrate (keratan sulfate Gal-6)
SEQ ID NO: 2888

sulfotransferase 1

CHST10
Carbohydrate sulfotransferase 10
SEQ ID NOS: 2889-

2896

CHST11
Carbohydrate (chondroitin 4)
SEQ ID NOS: 2897-

sulfotransferase 11
2901

CHST13
Carbohydrate (chondroitin 4)
SEQ ID NOS: 2902-

sulfotransferase 13
2903

CHST4
Carbohydrate (N-acetylglucosamine 6-O)
SEQ ID NOS: 2904-

sulfotransferase 4
2905

CHST5
Carbohydrate (N-acetylglucosamine 6-O)
SEQ ID NOS: 2906-

sulfotransferase 5
2907

CHST6
Carbohydrate (N-acetylglucosamine 6-O)
SEQ ID NOS: 2908-

sulfotransferase 6
2909

CHST7
Carbohydrate (N-acetylglucosamine 6-O)
SEQ ID NO: 2910

sulfotransferase 7

CHST8
Carbohydrate (N-acetylgalactosamine 4-O)
SEQ ID NOS: 2911-

sulfotransferase 8
2914

CHSY1
Chondroitin sulfate synthase 1
SEQ ID NOS: 2915-

2916

CHSY3
Chondroitin sulfate synthase 3
SEQ ID NO: 2917

CHTF8
Chromosome transmission fidelity factor 8
SEQ ID NOS: 2918-

2928

CILP
Cartilage intermediate layer protein,
SEQ ID NO: 2929

nucleotide pyrophosphohydrolase

CILP2
Cartilage intermediate layer protein 2
SEQ ID NOS: 2930-

2931

CIRH1A
Cirrhosis, autosomal recessive 1A (cirhin)
SEQ ID NOS: 13974-

13983

CKLF
Chemokine-like factor
SEQ ID NOS: 2932-

2937

CKMT1A
Creatine kinase, mitochondrial 1A
SEQ ID NOS: 2938-

2943

CKMT1B
Creatine kinase, mitochondrial 1B
SEQ ID NOS: 2944-

2953

CLCA1
Chloride channel accessory 1
SEQ ID NOS: 2954-

2955

CLCF1
Cardiotrophin-like cytokine factor 1
SEQ ID NOS: 2956-

2957

CLDN15
Claudin 15
SEQ ID NOS: 2958-

2963

CLDN7
Claudin 7
SEQ ID NOS: 2964-

2970

CLDND1
Claudin domain containing 1
SEQ ID NOS: 2971-

2996

CLEC11A
C-type lectin domain family 11, member A
SEQ ID NOS: 2997-

2999

CLEC16A
C-type lectin domain family 16, member A
SEQ ID NOS: 3000-

3005

CLEC18A
C-type lectin domain family 18, member A
SEQ ID NOS: 3006-

3011

CLEC18B
C-type lectin domain family 18, member B
SEQ ID NOS: 3012-

3015

CLEC18C
C-type lectin domain family 18, member C
SEQ ID NOS: 3016-

3022

CLEC19A
C-type lectin domain family 19, member A
SEQ ID NOS: 3023-

3026

CLEC2B
C-type lectin domain family 2, member B
SEQ ID NOS: 3027-

3028

CLEC3A
C-type lectin domain family 3, member A
SEQ ID NOS: 3029-

3030

CLEC3B
C-type lectin domain family 3, member B
SEQ ID NOS: 3031-

3032

CLGN
Calmegin
SEQ ID NOS: 3033-

3035

CLN5
Ceroid-lipofuscinosis, neuronal 5
SEQ ID NOS: 3036-

3047

CLPS
Colipase, pancreatic
SEQ ID NOS: 3048-

3050

CLPSL1
Colipase-like 1
SEQ ID NOS: 3051-

3052

CLPSL2
Colipase-like 2
SEQ ID NOS: 3053-

3054

CLPX
Caseinolytic mitochondrial matrix peptidase
SEQ ID NOS: 3055-

chaperone subunit
3057

CLSTN3
Calsyntenin 3
SEQ ID NOS: 3058-

3064

CLU
Clusterin
SEQ ID NOS: 3065-

3078

CLUL1
Clusterin-like 1 (retinal)
SEQ ID NOS: 3079-

3086

CMA1
Chymase 1, mast cell
SEQ ID NOS: 3087-

3088

CMPK1
Cytidine monophosphate (UMP-CMP)
SEQ ID NOS: 3089-

kinase 1, cytosolic
3092

CNBD1
Cyclic nucleotide binding domain
SEQ ID NOS: 3093-

containing 1
3096

CNDP1
Carnosine dipeptidase 1 (metallopeptidase
SEQ ID NOS: 3097-

M20 family)
3099

CNPY2
Canopy FGF signaling regulator 2
SEQ ID NOS: 3107-

3111

CNPY3
Canopy FGF signaling regulator 3
SEQ ID NOS: 3112-

3113

CNPY4
Canopy FGF signaling regulator 4
SEQ ID NOS: 3114-

3116

CNTFR
Ciliary neurotrophic factor receptor
SEQ ID NOS: 3117-

3120

CNTN1
Contactin 1
SEQ ID NOS: 3121-

3130

CNTN2
Contactin 2 (axonal)
SEQ ID NOS: 3131-

3142

CNTN3
Contactin 3 (plasmacytoma associated)
SEQ ID NO: 3143

CNTN4
Contactin 4
SEQ ID NOS: 3144-

3152

CNTN5
Contactin 5
SEQ ID NOS: 3153-

3158

CNTNAP2
Contactin associated protein-like 2
SEQ ID NOS: 3159-

3162

CNTNAP3
Contactin associated protein-like 3
SEQ ID NOS: 3163-

3167

CNTNAP3B
Contactin associated protein-like 3B
SEQ ID NOS: 3168-

3176

COASY
CoA synthase
SEQ ID NOS: 3177-

3186

COCH
Cochlin
SEQ ID NOS: 3187-

3198

COG3
Component of oligomeric golgi complex 3
SEQ ID NOS: 3199-

3202

COL10A1
Collagen, type X, alpha 1
SEQ ID NOS: 3203-

3206

COL11A1
Collagen, type XI, alpha 1
SEQ ID NOS: 3207-

3217

COL11A2
Collagen, type XI, alpha 2
SEQ ID NOS: 3218-

3222

COL12A1
Collagen, type XII, alpha 1
SEQ ID NOS: 3223-

3230

COL14A1
Collagen, type XIV, alpha 1
SEQ ID NOS: 3231-

3238

COL15A1
Collagen, type XV, alpha 1
SEQ ID NOS: 3239-

3240

COL16A1
Collagen, type XVI, alpha 1
SEQ ID NOS: 3241-

3245

COL18A1
Collagen, type XVIII, alpha 1
SEQ ID NOS: 3246-

3250

COL19A1
Collagen, type XIX, alpha 1
SEQ ID NOS: 3251-

3253

COL1A1
Collagen, type I, alpha 1
SEQ ID NOS: 3254-

3255

COL1A2
Collagen, type I, alpha 2
SEQ ID NOS: 3256-

3257

COL20A1
Collagen, type XX, alpha 1
SEQ ID NOS: 3258-

3261

COL21A1
Collagen, type XXI, alpha 1
SEQ ID NOS: 3262-

3267

COL22A1
Collagen, type XXII, alpha 1
SEQ ID NOS: 3268-

3270

COL24A1
Collagen, type XXIV, alpha 1
SEQ ID NOS: 3271-

3274

COL26A1
Collagen, type XXVI, alpha 1
SEQ ID NOS: 3275-

3276

COL27A1
Collagen, type XXVII, alpha 1
SEQ ID NOS: 3277-

3279

COL28A1
Collagen, type XXVIII, alpha 1
SEQ ID NOS: 3280-

3284

COL2A1
Collagen, type II, alpha 1
SEQ ID NOS: 3285-

3286

COL3A1
Collagen, type III, alpha 1
SEQ ID NOS: 3287-

3289

COL4A1
Collagen, type IV, alpha 1
SEQ ID NOS: 3290-

3292

COL4A2
Collagen, type IV, alpha 2
SEQ ID NOS: 3293-

3295

COL4A3
Collagen, type IV, alpha 3 (Goodpasture
SEQ ID NOS: 3296-

antigen)
3299

COL4A4
Collagen, type IV, alpha 4
SEQ ID NOS: 3300-

3301

COL4A5
Collagen, type IV, alpha 5
SEQ ID NOS: 3302-

3308

COL4A6
Collagen, type IV, alpha 6
SEQ ID NOS: 3309-

3314

COL5A1
Collagen, type V, alpha 1
SEQ ID NOS: 3315-

3317

COL5A2
Collagen, type V, alpha 2
SEQ ID NOS: 3318-

3319

COL5A3
Collagen, type V, alpha 3
SEQ ID NO: 3320

COL6A1
Collagen, type VI, alpha 1
SEQ ID NOS: 3321-

3322

COL6A2
Collagen, type VI, alpha 2
SEQ ID NOS: 3323-

3328

COL6A3
Collagen, type VI, alpha 3
SEQ ID NOS: 3329-

3337

COL6A5
Collagen, type VI, alpha 5
SEQ ID NOS: 3338-

3342

COL6A6
Collagen, type VI, alpha 6
SEQ ID NOS: 3343-

3345

COL7A1
Collagen, type VII, alpha 1
SEQ ID NOS: 3346-

3347

COL8A1
Collagen, type VIII, alpha 1
SEQ ID NOS: 3348-

3351

COL8A2
Collagen, type VIII, alpha 2
SEQ ID NOS: 3352-

3354

COL9A1
Collagen, type IX, alpha 1
SEQ ID NOS: 3355-

3358

COL9A2
Collagen, type IX, alpha 2
SEQ ID NOS: 3359-

3362

COL9A3
Collagen, type IX, alpha 3
SEQ ID NOS: 3363-

3364

COLEC10
Collectin sub-family member 10 (C-type
SEQ ID NO: 3365

lectin)

COLEC11
Collectin sub-family member 11
SEQ ID NOS: 3366-

3375

COLGALT1
Collagen beta(1-O)galactosyltransferase 1
SEQ ID NOS: 3376-

3378

COLGALT2
Collagen beta(1-O)galactosyltransferase 2
SEQ ID NOS: 3379-

3381

COLQ
Collagen-like tail subunit (single strand of
SEQ ID NOS: 3382-

homotrimer) of asymmetric
3386

acetylcholinesterase

COMP
Cartilage oligomeric matrix protein
SEQ ID NOS: 3387-

3389

C0PS6
COP9 signalosome subunit 6
SEQ ID NOS: 3390-

3393

COQ6
Coenzyme Q6 monooxygenase
SEQ ID NOS: 3394-

3401

CORT
Cortistatin
SEQ ID NO: 3402

CP
Ceruloplasmin (ferroxidase)
SEQ ID NOS: 3403-

3407

CPA1
Carboxypeptidase A1 (pancreatic)
SEQ ID NOS: 3408-

3412

CPA2
Carboxypeptidase A2 (pancreatic)
SEQ ID NOS: 3413-

3414

CPA3
Carboxypeptidase A3 (mast cell)
SEQ ID NO: 3415

CPA4
Carboxypeptidase A4
SEQ ID NOS: 3416-

3421

CPA6
Carboxypeptidase A6
SEQ ID NOS: 3422-

3424

CPAMD8
C3 and PZP-like, alpha-2-macroglobulin
SEQ ID NOS: 3425-

domain containing 8
3430

CPB1
Carboxypeptidase B1 (tissue)
SEQ ID NOS: 3431-

3435

CPB2
Carboxypeptidase B2 (plasma)
SEQ ID NOS: 3436-

3438

CPE
Carboxypeptidase E
SEQ ID NOS: 3439-

3443

CPM
Carboxypeptidase M
SEQ ID NOS: 3444-

3453

CPN1
Carboxypeptidase N, polypeptide 1
SEQ ID NOS: 3454-

3455

CPN2
Carboxypeptidase N, polypeptide 2
SEQ ID NOS: 3456-

3457

CPO
Carboxypeptidase O
SEQ ID NO: 3458

CPQ
Carboxvpeptidase Q
SEQ ID NOS: 3459-

3464

CPVL
Carboxypeptidase, vitellogenic-like
SEQ ID NOS: 3465-

3475

CPXM1
Carboxypeptidase X (M14 family), member
SEQ ID NO: 3476

1

CPXM2
Carboxypeptidase X (M14 family), member
SEQ ID NOS: 3477-

2
3478

CPZ
Carboxypeptidase Z
SEQ ID NOS: 3479-

3482

CR1L
Complement component (3b/4b) receptor 1-
SEQ ID NOS: 3483-

like
3484

CRB2
Crumbs family member 2
SEQ ID NOS: 3485-

3487

CREG1
Cellular repressor of E1A-stimulated genes
SEQ ID NO: 3488

1

CREG2
Cellular repressor of E1A-stimulated genes
SEQ ID NO: 3489

2

CRELD1
Cysteine-rich with EGF-like domains 1
SEQ ID NOS: 3490-

3495

CRELD2
Cysteine-rich with EGF-like domains 2
SEQ ID NOS: 3496-

3500

CRH
Corticotropin releasing hormone
SEQ ID NO: 3501

CRHBP
Corticotropin releasing hormone binding
SEQ ID NOS: 3502-

protein
3503

CRHR1
Corticotropin releasing hormone receptor 1
SEQ ID NOS: 3504-

3515

CRHR2
Corticotropin releasing hormone receptor 2
SEQ ID NOS: 3516-

3522

CRISP1
Cysteine-rich secretory protein 1
SEQ ID NOS: 3523-

3526

CRISP2
Cysteine-rich secretory protein 2
SEQ ID NOS: 3527-

3529

CRISP3
Cysteine-rich secretory protein 3
SEQ ID NOS: 3530-

3533

CRISPLD2
Cysteine-rich secretory protein LCCL
SEQ ID NOS: 3534-

domain containing 2
3541

CRLF1
Cytokine receptor-like factor 1
SEQ ID NOS: 3542-

3543

CRP
C-reactive protein, pentraxin-related
SEQ ID NOS: 3544-

3548

CRTAC1
Cartilage acidic protein 1
SEQ ID NOS: 3549-

3553

CRTAP
Cartilage associated protein
SEQ ID NOS: 3554-

3555

CRY2
Cryptochrome circadian clock 2
SEQ ID NOS: 3556-

3559

CSAD
Cysteine sulfinic acid decarboxylase
SEQ ID NOS: 3560-

3572

CSF1
Colony stimulating factor 1 (macrophage)
SEQ ID NOS: 3573-

3580

CSF1R
Colony stimulating factor 1 receptor
SEQ ID NOS: 3581-

3585

CSF2
Colony stimulating factor 2 (granulocyte-
SEQ ID NO: 3586

macrophage)

CSF2RA
Colony stimulating factor 2 receptor, alpha,
SEQ ID NOS: 3587-

low-affinity (granulocyte-macrophage)
3598

CSF3
Colony stimulating factor 3 (granulocyte)
SEQ ID NOS: 3599-

3605

CSGALNACT1
Chondroitin sulfate N-
SEQ ID NOS: 3606-

acetylgalactosaminyltransferase 1
3614

CSH1
Chorionic somatomammotropin hormone 1
SEQ ID NOS: 3615-

(placental lactogen)
3618

CSH2
Chorionic somatomammotropin hormone 2
SEQ ID NOS: 3619-

3623

CSHL1
Chorionic somatomammotropin hormone-
SEQ ID NOS: 3624-

like 1
3630

CSN1S1
Casein alpha s1
SEQ ID NOS: 3631-

3636

CSN2
Casein beta
SEQ ID NO: 3637

CSN3
Casein kappa
SEQ ID NO: 3638

CST1
Cystatin SN
SEQ ID NOS: 3639-

3640

CST11
Cystatin 11
SEQ ID NOS: 3641-

3642

CST2
Cystatin SA
SEQ ID NO: 3643

CST3
Cystatin C
SEQ ID NOS: 3644-

3646

CST4
Cystatin S
SEQ ID NO: 3647

CST5
Cystatin D
SEQ ID NO: 3648

CST6
Cystatin E/M
SEQ ID NO: 3649

CST7
Cystatin F (leukocystatin)
SEQ ID NO: 3650

CST8
Cystatin 8 (cystatin-related epididymal
SEQ ID NOS: 3651-

specific)
3652

CST9
Cystatin 9 (testatin)
SEQ ID NO: 3653

CST9L
Cystatin 9-like
SEQ ID NO: 3654

CSTL1
Cy statin-like 1
SEQ ID NOS: 3655-

3657

CT55
Cancer/testis antigen 55
SEQ ID NOS: 3658-

3659

CTB-60B18.6

SEQ ID NOS: 74-75

CTBS
Chitobiase, di-N-acetyl-
SEQ ID NOS: 3660-

3662

CTD-

SEQ ID NO: 4160

2313N18.7

CTD-

SEQ ID NOS: 81-84

2370N5.3

CTGF
Connective tissue growth factor
SEQ ID NO: 3663

CTHRC1
Collagen triple helix repeat containing 1
SEQ ID NOS: 3664-

3667

CTLA4
Cytotoxic T-lymphocyte-associated protein
SEQ ID NOS: 3668-

4
3671

CTNS
Cystinosin, lysosomal cystine transporter
SEQ ID NOS: 3672-

3679

CTRB1
Chymotrypsinogen B1
SEQ ID NOS: 3680-

3682

CTRB2
Chymotrypsinogen B2
SEQ ID NOS: 3683-

3686

CTRC
Chymotrypsin C (caldecrin)
SEQ ID NOS: 3687-

3688

CTRL
Chymotrypsin-like
SEQ ID NOS: 3689-

3691

CTSA
Cathepsin A
SEQ ID NOS: 3692-

3700

CTSB
Cathepsin B
SEQ ID NOS: 3701-

3725

CTSC
Cathepsin C
SEQ ID NOS: 3726-

3730

CTSD
Cathepsin D
SEQ ID NOS: 3731-

3741

CTSE
Cathepsin E
SEQ ID NOS: 3742-

3743

CTSF
Cathepsin F
SEQ ID NOS: 3744-

3747

CTSG
Cathepsin G
SEQ ID NO: 3748

CTSH
Cathepsin H
SEQ ID NOS: 3749-

3754

CTSK
Cathepsin K
SEQ ID NOS: 3755-

3756

CTSL
Cathepsin L
SEQ ID NOS: 3757-

3759

CTSO
Cathepsin O
SEQ ID NO: 3760

CTSS
Cathepsin S
SEQ ID NOS: 3761-

3765

CTSV
Cathepsin V
SEQ ID NOS: 3766-

3767

CTSW
Cathepsin W
SEQ ID NOS: 3768-

3770

CTSZ
Cathepsin Z
SEQ ID NO: 3771

CUBN
Cubilin (intrinsic factor-cobalamin receptor)
SEQ ID NOS: 3772-

3775

CUTA
CutA divalent cation tolerance homolog
SEQ ID NOS: 3776-

(E. coli)
3785

CX3CL1
Chemokine (C-X3-C motif) ligand 1
SEQ ID NOS: 3786-

3789

CXADR
Coxsackie virus and adenovirus receptor
SEQ ID NOS: 3790-

3794

CXCL1
Chemokine (C-X-C motif) ligand 1
SEQ ID NO: 3795

(melanoma growth stimulating activity,

alpha)

CXCL10
Chemokine (C-X-C motif) ligand 10
SEQ ID NO: 3796

CXCL11
Chemokine (C-X-C motif) ligand 11
SEQ ID NOS: 3797-

3798

CXCL12
Chemokine (C-X-C motif) ligand 12
SEQ ID NOS: 3799-

3804

CXCL13
Chemokine (C-X-C motif) ligand 13
SEQ ID NO: 3805

CXCL14
Chemokine (C-X-C motif) ligand 14
SEQ ID NOS: 3806-

3807

CXCL17
Chemokine (C-X-C motif) ligand 17
SEQ ID NOS: 3808-

3809

CXCL2
Chemokine (C-X-C motif) ligand 2
SEQ ID NO: 3810

CXCL3
Chemokine (C-X-C motif) ligand 3
SEQ ID NO: 3811

CXCL5
Chemokine (C-X-C motif) ligand 5
SEQ ID NO: 3812

CXCL6
Chemokine (C-X-C motif) ligand 6
SEQ ID NOS: 3813-

3814

CXCL8
Chemokine (C-X-C motif) ligand 8
SEQ ID NOS: 3815-

3816

CXCL9
Chemokine (C-X-C motif) ligand 9
SEQ ID NO: 3817

CXorf36
Chromosome X open reading frame 36
SEQ ID NOS: 3818-

3819

CYB5D2
Cytochrome b5 domain containing 2
SEQ ID NOS: 3820-

3823

CYHR1
Cysteine/histidine-rich 1
SEQ ID NOS: 3824-

3831

CYP17A1
Cytochrome P450, family 17, subfamily A,
SEQ ID NOS: 3832-

polypeptide 1
3836

CYP20A1
Cytochrome P450, family 20, subfamily A,
SEQ ID NOS: 3837-

polypeptide 1
3843

CYP21A2
Cytochrome P450, family 21, subfamily A,
SEQ ID NOS: 3844-

polypeptide 2
3851

CYP26B1
Cytochrome P450, family 26, subfamily B,
SEQ ID NOS: 3852-

polypeptide 1
3856

CYP2A6
Cytochrome P450, family 2, subfamily A,
SEQ ID NOS: 3857-

polypeptide 6
3858

CYP2A7
Cytochrome P450, family 2, subfamily A,
SEQ ID NOS: 3859-

polypeptide 7
3861

CYP2B6
Cytochrome P450, family 2, subfamily B,
SEQ ID NOS: 3862-

polypeptide 6
3865

CYP2C18
Cytochrome P450, family 2, subfamily C,
SEQ ID NOS: 3866-

polypeptide 18
3867

CYP2C19
Cytochrome P450, family 2, subfamily C,
SEQ ID NOS: 3868-

polypeptide 19
3869

CYP2C8
Cytochrome P450, family 2, subfamily C,
SEQ ID NOS: 3870-

polypeptide 8
3877

CYP2C9
Cytochrome P450, family 2, subfamily C,
SEQ ID NOS: 3878-

polypeptide 9
3880

CYP2E1
Cytochrome P450, family 2, subfamily E,
SEQ ID NOS: 3881-

polypeptide 1
3886

CYP2F1
Cytochrome P450, family 2, subfamily F,
SEQ ID NOS: 3887-

polypeptide 1
3890

CYP2J2
Cytochrome P450, family 2, subfamily J,
SEQ ID NO: 3891

polypeptide 2

CYP2R1
Cytochrome P450, family 2, subfamily R,
SEQ ID NOS: 3892-

polypeptide 1
3897

CYP2S1
Cytochrome P450, family 2, subfamily S,
SEQ ID NOS: 3898-

polypeptide 1
3903

CYP2W1
Cytochrome P450, family 2, subfamily W,
SEQ ID NOS: 3904-

polypeptide 1
3906

CYP46A1
Cytochrome P450, family 46, subfamily A,
SEQ ID NOS: 3907-

polypeptide 1
3911

CYP4F11
Cytochrome P450, family 4, subfamily F,
SEQ ID NOS: 3912-

polypeptide 11
3916

CYP4F2
Cytochrome P450, family 4, subfamily F,
SEQ ID NOS: 3917-

polypeptide 2
3921

CYR61
Cysteine-rich, angiogenic inducer, 61
SEQ ID NO: 3922

CYTL1
Cytokine-like 1
SEQ ID NOS: 3923-

3925

D2HGDH
D-2-hydroxvglutarate dehydrogenase
SEQ ID NOS: 3926-

3934

DAG1
Dystroglycan 1 (dystrophin-associated
SEQ ID NOS: 3935-

glycoprotein 1)
3949

DAND5
DAN domain family member 5, BMP
SEQ ID NOS: 3950-

antagonist
3951

DAO
D-amino-acid oxidase
SEQ ID NOS: 3952-

3957

DAZAP2
DAZ associated protein 2
SEQ ID NOS: 3958-

3966

DBH
Dopamine beta-hydroxylase (dopamine
SEQ ID NOS: 3967-

beta-monooxygenase)
3968

DBNL
Drebrin-like
SEQ ID NOS: 3969-

3986

DCD
Dermcidin
SEQ ID NOS: 3987-

3989

DCN
Decorin
SEQ ID NOS: 3990-

4008

DDIAS
DNA damage-induced apoptosis suppressor
SEQ ID NOS: 4009-

4018

DDOST
Dolichyl-diphosphooligosaccharide--protein
SEQ ID NOS: 4019-

glycosyltransferase subunit (non-catalytic)
4022

DDR1
Discoidin domain receptor tyrosine kinase 1
SEQ ID NOS: 4023-

4068

DDR2
Discoidin domain receptor tyrosine kinase 2
SEQ ID NOS: 4069-

4074

DDT
D-dopachrome tautomerase
SEQ ID NOS: 4075-

4080

DDX17
DEAD (Asp-Glu-Ala-Asp) box helicase 17
SEQ ID NOS: 4081-

4085

DDX20
DEAD (Asp-Glu-Ala-Asp) box polypeptide
SEQ ID NOS: 4086-

20
4088

DDX25
DEAD (Asp-Glu-Ala-Asp) box helicase 25
SEQ ID NOS: 4089-

4095

DDX28
DEAD (Asp-Glu-Ala-Asp) box polypeptide
SEQ ID NO: 4096

28

DEAF1
DEAF1 transcription factor
SEQ ID NOS: 4097-

4099

DEF8
Differentially expressed in FDCP 8
SEQ ID NOS: 4100-

homolog (mouse)
4119

DEFA1
Defensin, alpha 1
SEQ ID NOS: 4120-

4121

DEFA1B
Defensin, alpha 1B
SEQ ID NO: 4122

DEFA3
Defensin, alpha 3, neutrophil-specific
SEQ ID NO: 4123

DEFA4
Defensin, alpha 4, corticostatin
SEQ ID NO: 4124

DEFA5
Defensin, alpha 5, Paneth cell-specific
SEQ ID NO: 4125

DEFA6
Defensin, alpha 6, Paneth cell-specific
SEQ ID NO: 4126

DEFB1
Defensin, beta 1
SEQ ID NO: 4127

DEFB103A
Defensin, beta 103A
SEQ ID NO: 4128

DEFB103B
Defensin, beta 103B
SEQ ID NO: 4129

DEFB104A
Defensin, beta 104A
SEQ ID NO: 4130

DEFB104B
Defensin, beta 104B
SEQ ID NO: 4131

DEFB105A
Defensin, beta 105A
SEQ ID NO: 4132

DEFB105B
Defensin, beta 105B
SEQ ID NO: 4133

DEFB106A
Defensin, beta 106A
SEQ ID NO: 4134

DEFB106B
Defensin, beta 106B
SEQ ID NO: 4135

DEFB107A
Defensin, beta 107A
SEQ ID NO: 4136

DEFB107B
Defensin, beta 107B
SEQ ID NO: 4137

DEFB108B
Defensin, beta 108B
SEQ ID NO: 4138

DEFB110
Defensin, beta 110
SEQ ID NOS: 4139-

4140

DEFB113
Defensin, beta 113
SEQ ID NO: 4141

DEFB114
Defensin, beta 114
SEQ ID NO: 4142

DEFB115
Defensin, beta 115
SEQ ID NO: 4143

DEFB116
Defensin, beta 116
SEQ ID NO: 4144

DEFB118
Defensin, beta 118
SEQ ID NO: 4145

DEFB119
Defensin, beta 119
SEQ ID NOS: 4146-

4148

DEFB121
Defensin, beta 121
SEQ ID NO: 4149

DEFB123
Defensin, beta 123
SEQ ID NO: 4150

DEFB124
Defensin, beta 124
SEQ ID NO: 4151

DEFB125
Defensin, beta 125
SEQ ID NO: 4152

DEFB126
Defensin, beta 126
SEQ ID NO: 4153

DEFB127
Defensin, beta 127
SEQ ID NO: 4154

DEFB128
Defensin, beta 128
SEQ ID NO: 4155

DEFB129
Defensin, beta 129
SEQ ID NO: 4156

DEFB130
Defensin, beta 130
SEQ ID NO: 4157

DEFB131
Defensin, beta 131
SEQ ID NO: 4159

DEFB132
Defensin, beta 132
SEQ ID NO: 4161

DEFB133
Defensin, beta 133
SEQ ID NO: 4162

DEFB134
Defensin, beta 134
SEQ ID NOS: 4163-

4164

DEFB135
Defensin, beta 135
SEQ ID NO: 4165

DEFB136
Defensin, beta 136
SEQ ID NO: 4166

DEFB4A
Defensin, beta 4A
SEQ ID NO: 4167

DEFB4B
Defensin, beta 4B
SEQ ID NO: 4168

DFNA5
Deafness, autosomal dominant 5
SEQ ID NOS: 6271-

6279

DFNB31
Deafness, autosomal recessive 31
SEQ ID NOS: 14251-

14254

DGCR2
DiGeorge syndrome critical region gene 2
SEQ ID NOS: 4171-

4174

DHH
Desert hedgehog
SEQ ID NO: 4175

DHRS4
Dehydrogenase/reductase (SDR family)
SEQ ID NOS: 4176-

member 4
4183

DHRS4L2
Dehydrogenase/reductase (SDR family)
SEQ ID NOS: 4184-

member 4 like 2
4193

DHRS7
Dehydrogenase/reductase (SDR family)
SEQ ID NOS: 4194-

member 7
4201

DHRS7C
Dehydrogenase/reductase (SDR family)
SEQ ID NOS: 4202-

member 7C
4204

DHRS9
Dehydrogenase/reductase (SDR family)
SEQ ID NOS: 4205-

member 9
4212

DHRSX
Dehydrogenase/reductase (SDR family) X-
SEQ ID NOS: 4213-

linked
4217

DHX29
DEAH (Asp-Glu-Ala-His) box polypeptide
SEQ ID NOS: 4218-

29
4220

DHX30
DEAH (Asp-Glu-Ala-His) box helicase 30
SEQ ID NOS: 4221-

4228

DHX8
DEAH (Asp-Glu-Ala-His) box polypeptide
SEQ ID NOS: 4229-

8
4233

DIO2
Deiodinase, iodothyronine, type II
SEQ ID NOS: 4234-

4243

DIXDC1
DIX domain containing 1
SEQ ID NOS: 4244-

4247

DKK1
Dickkopf WNT signaling pathway inhibitor
SEQ ID NO: 4248

1

DKK2
Dickkopf WNT signaling pathway inhibitor
SEQ ID NOS: 4249-

2
4251

DKK3
Dickkopf WNT signaling pathway inhibitor
SEQ ID NOS: 4252-

3
4257

DKK4
Dickkopf WNT signaling pathway inhibitor
SEQ ID NO: 4258

4

DKKL1
Dickkopf-like 1
SEQ ID NOS: 4259-

4264

DLG4
Discs, large homolog 4 (Drosophila)
SEQ ID NOS: 4265-

4273

DLK1
Delta-like 1 homolog (Drosophila)
SEQ ID NOS: 4274-

4277

DLL1
Delta-like 1 (Drosophila)
SEQ ID NOS: 4278-

4279

DLL3
Delta-like 3 (Drosophila)
SEQ ID NOS: 4280-

4282

DMBT1
Deleted in malignant brain tumors 1
SEQ ID NOS: 4283-

4289

DMKN
Dermokine
SEQ ID NOS: 4290-

4336

DMP1
Dentin matrix acidic phosphoprotein 1
SEQ ID NOS: 4337-

4338

DMRTA2
DMRT-like family A2
SEQ ID NOS: 4339-

4340

DNAAF5
Dynein, axonemal, assembly factor 5
SEQ ID NOS: 4341-

4344

DNAH14
Dynein, axonemal, heavy chain 14
SEQ ID NOS: 4345-

4359

DNAJB11
DnaJ (Hsp40) homolog, subfamily B,
SEQ ID NOS: 4360-

member 11
4361

DNAJB9
DnaJ (Hsp40) homolog, subfamily B,
SEQ ID NO: 4362

member 9

DNAJC25-
DNAJC25-GNG10 readthrough
SEQ ID NO: 4363

GNG10

DNAJC3
DnaJ (Hsp40) homolog, subfamily C,
SEQ ID NOS: 4364-

member 3
4365

DNASE1
Deoxyribonuclease I
SEQ ID NOS: 4366-

4376

DNASE1L1
Deoxyribonuclease I-like 1
SEQ ID NOS: 4377-

4387

DNASE1L2
Deoxyribonuclease I-like 2
SEQ ID NOS: 4388-

4393

DNASE1L3
Deoxyribonuclease I-like 3
SEQ ID NOS: 4394-

4399

DNASE2
Deoxyribonuclease II, lysosomal
SEQ ID NOS: 4400-

4401

DNASE2B
Deoxyribonuclease II beta
SEQ ID NOS: 4402-

4403

DPEP1
Dipeptidase 1 (renal)
SEQ ID NOS: 4404-

4408

DPEP2
Dipeptidase 2
SEQ ID NOS: 4409-

4415

DPEP3
Dipeptidase 3
SEQ ID NO: 4416

DPF3
D4, zinc and double PHD fingers, family 3
SEQ ID NOS: 4417-

4423

DPP4
Dipeptidyl-peptidase 4
SEQ ID NOS: 4424-

4428

DPP7
Dipeptidyl-peptidase 7
SEQ ID NOS: 4429-

4434

DPT
Dermatopontin
SEQ ID NO: 4435

DRAXIN
Dorsal inhibitory axon guidance protein
SEQ ID NO: 4436

DSE
Dermatan sulfate epimerase
SEQ ID NOS: 4437-

4445

DSG2
Desmoglein 2
SEQ ID NOS: 4446-

4447

DSPP
Dentin sialophosphoprotein
SEQ ID NOS: 4448-

4449

DST
Dystonin
SEQ ID NOS: 4450-

4468

DUOX1
Dual oxidase 1
SEQ ID NOS: 4469-

4473

DYNLT3
Dynein, light chain, Tctex-type 3
SEQ ID NOS: 4474-

4476

E2F5
E2F transcription factor 5, p130-binding
SEQ ID NOS: 4477-

4483

EBAG9
Estrogen receptor binding site associated,
SEQ ID NOS: 4484-

antigen, 9
4492

EBI3
Epstein-Barr virus induced 3
SEQ ID NO: 4493

ECHDC1
Ethylmalonyl-CoA decarboxylase 1
SEQ ID NOS: 4494-

4512

ECM1
Extracellular matrix protein 1
SEQ ID NOS: 4513-

4515

ECM2
Extracellular matrix protein 2, female organ
SEQ ID NOS: 4516-

and adipocyte specific
4519

ECSIT
ECSIT signalling integrator
SEQ ID NOS: 4520-

4531

EDDM3A
Epididymal protein 3A
SEQ ID NO: 4532

EDDM3B
Epididymal protein 3B
SEQ ID NO: 4533

EDEM2
ER degradation enhancer, mannosidase
SEQ ID NOS: 4534-

alpha-like 2
4535

EDEM3
ER degradation enhancer, mannosidase
SEQ ID NOS: 4536-

alpha-like 3
4538

EDIL3
EGF-like repeats and discoidin I-like
SEQ ID NOS: 4539-

domains 3
4540

EDN1
Endothelin 1
SEQ ID NO: 4541

EDN2
Endothelin 2
SEQ ID NO: 4542

EDN3
Endothelin 3
SEQ ID NOS: 4543-

4548

EDNRB
Endothelin receptor type B
SEQ ID NOS: 4549-

4557

EFEMP1
EGF containing fibulin-like extracellular
SEQ ID NOS: 4558-

matrix protein 1
4568

EFEMP2
EGF containing fibulin-like extracellular
SEQ ID NOS: 4569-

matrix protein 2
4580

EFNA1
Ephrin-A1
SEQ ID NOS: 4581-

4582

EFNA2
Ephrin-A2
SEQ ID NO: 4583

EFNA4
Ephrin-A4
SEQ ID NOS: 4584-

4586

EGFL6
EGF-like-domain, multiple 6
SEQ ID NOS: 4587-

4588

EGFL7
EGF-like-domain, multiple 7
SEQ ID NOS: 4589-

4593

EGFL8
EGF-like-domain, multiple 8
SEQ ID NOS: 4594-

4596

EGFLAM
EGF-like, fibronectin type III and laminin G
SEQ ID NOS: 4597-

domains
4605

EGFR
Epidermal growth factor receptor
SEQ ID NOS: 4606-

4613

EHBP1
EH domain binding protein 1
SEQ ID NOS: 4614-

4625

EHF
Ets homologous factor
SEQ ID NOS: 4626-

4635

EHMT1
Euchromatic histone-lysine N-
SEQ ID NOS: 4636-

methyltransferase 1
4661

EHMT2
Euchromatic histone-lysine N-
SEQ ID NOS: 4662-

methyltransferase 2
4666

EIF2AK1
Eukaryotic translation initiation factor 2-
SEQ ID NOS: 4667-

alpha kinase 1
4670

ELANE
Elastase, neutrophil expressed
SEQ ID NOS: 4671-

4672

ELN
Elastin
SEQ ID NOS: 4673-

4695

ELP2
Elongator acetyltransferase complex subunit
SEQ ID NOS: 4696-

2
4708

ELSPBP1
Epididymal sperm binding protein 1
SEQ ID NOS: 4709-

4714

EMC1
ER membrane protein complex subunit 1
SEQ ID NOS: 4715-

4721

EMC10
ER membrane protein complex subunit 10
SEQ ID NOS: 4722-

4728

EMC9
ER membrane protein complex subunit 9
SEQ ID NOS: 4729-

4732

EMCN
Endomucin
SEQ ID NOS: 4733-

4737

EMID1
EMI domain containing 1
SEQ ID NOS: 4738-

4744

EMILIN1
Elastin microfibril interfacer 1
SEQ ID NOS: 4745-

4746

EMILIN2
Elastin microfibril interfacer 2
SEQ ID NO: 4747

EMILIN3
Elastin microfibril interfacer 3
SEQ ID NO: 4748

ENAM
Enamelin
SEQ ID NO: 4749

ENDOG
Endonuclease G
SEQ ID NO: 4750

ENDOU
Endonuclease, polyU-specific
SEQ ID NOS: 4751-

4753

ENHO
Energy homeostasis associated
SEQ ID NO: 4754

ENO4
Enolase family member 4
SEQ ID NOS: 4755-

4759

ENPP6
Ectonucleotide
SEQ ID NOS: 4760-

pyrophosphatase/phosphodiesterase 6
4761

ENPP7
Ectonucleotide
SEQ ID NOS: 4762-

pyrophosphatase/phosphodiesterase 7
4763

ENTPD5
Ectonucleoside triphosphate
SEQ ID NOS: 4764-

diphosphohydrolase 5
4768

ENTPD8
Ectonucleoside triphosphate
SEQ ID NOS: 4769-

diphosphohydrolase 8
4772

EOGT
EGF domain-specific O-linked N-
SEQ ID NOS: 4773-

acetylglucosamine (GlcNAc) transferase
4780

EPCAM
Epithelial cell adhesion molecule
SEQ ID NOS: 4781-

4784

EPDR1
Ependymin related 1
SEQ ID NOS: 4785-

4788

EPGN
Epithelial mitogen
SEQ ID NOS: 4789-

4797

EPHA10
EPH receptor A10
SEQ ID NOS: 4798-

4805

EPHA3
EPH receptor A3
SEQ ID NOS: 4806-

4808

EPHA4
EPH receptor A4
SEQ ID NOS: 4809-

4818

EPHA7
EPH receptor A7
SEQ ID NOS: 4819-

4820

EPHA8
EPH receptor A8
SEQ ID NOS: 4821-

4822

EPHB2
EPH receptor B2
SEQ ID NOS: 4823-

4827

EPHB4
EPH receptor B4
SEQ ID NOS: 4828-

4830

EPHX3
Epoxide hydrolase 3
SEQ ID NOS: 4831-

4834

EPO
Erythropoietin
SEQ ID NO: 4835

EPPIN
Epididymal peptidase inhibitor
SEQ ID NOS: 4836-

4838

EPPIN-
EPPIN-WFDC6 readthrough
SEQ ID NO: 4839

WFDC6

EPS15
Epidermal growth factor receptor pathway
SEQ ID NOS: 4840-

substrate 15
4842

EPS8L1
EPS8-like 1
SEQ ID NOS: 4843-

4848

EPX
Eosinophil peroxidase
SEQ ID NO: 4849

EPYC
Epiphycan
SEQ ID NOS: 4850-

4851

EQTN
Equatorin, sperm acrosome associated
SEQ ID NOS: 4852-

4854

ERAP1
Endoplasmic reticulum aminopeptidase 1
SEQ ID NOS: 4855-

4859

ERAP2
Endoplasmic reticulum aminopeptidase 2
SEQ ID NOS: 4860-

4867

ERBB3
Erb-b2 receptor tyrosine kinase 3
SEQ ID NOS: 4868-

4881

ERLIN1
ER lipid raft associated 1
SEQ ID NOS: 4885-

4887

ERLIN2
ER lipid raft associated 2
SEQ ID NOS: 4888-

4896

ERN1
Endoplasmic reticulum to nucleus signaling
SEQ ID NOS: 4897-

1
4898

ERN2
Endoplasmic reticulum to nucleus signaling
SEQ ID NOS: 4899-

2
4903

ERO1A
Endoplasmic reticulum oxidoreductase
SEQ ID NOS: 4904-

alpha
4910

ERO1B
Endoplasmic reticulum oxidoreductase beta
SEQ ID NOS: 4911-

4913

ERP27
Endoplasmic reticulum protein 27
SEQ ID NOS: 4914-

4915

ERP29
Endoplasmic reticulum protein 29
SEQ ID NOS: 4916-

4919

ERP44
Endoplasmic reticulum protein 44
SEQ ID NO: 4920

ERV3-1
Endogenous retrovirus group 3, member 1
SEQ ID NO: 4921

ESM1
Endothelial cell-specific molecule 1
SEQ ID NOS: 4922-

4924

ESRP1
Epithelial splicing regulatory protein 1
SEQ ID NOS: 4925-

4933

EXOG
Endo/exonuclease (5′-3′), endonuclease G-
SEQ ID NOS: 4934-

like
4947

EXTL1
Exostosin-like glycosyltransferase 1
SEQ ID NO: 4948

EXTL2
Exostosin-like glycosyltransferase 2
SEQ ID NOS: 4949-

4953

F10
Coagulation factor X
SEQ ID NOS: 4954-

4957

F11
Coagulation factor XI
SEQ ID NOS: 4958-

4962

F12
Coagulation factor XII (Hageman factor)
SEQ ID NO: 4963

F13B
Coagulation factor XIII, B polypeptide
SEQ ID NO: 4964

F2
Coagulation factor II (thrombin)
SEQ ID NOS: 4965-

4967

F2R
Coagulation factor II (thrombin) receptor
SEQ ID NOS: 4968-

4969

F2RL3
Coagulation factor II (thrombin) receptor-
SEQ ID NOS: 4970-

like 3
4971

F5
Coagulation factor V (proaccelerin, labile
SEQ ID NOS: 4972-

factor)
4973

F7
Coagulation factor VII (serum prothrombin
SEQ ID NOS: 4974-

conversion accelerator)
4977

F8
Coagulation factor VIII, procoagulant
SEQ ID NOS: 4978-

component
4983

F9
Coagulation factor IX
SEQ ID NOS: 4984-

4985

FABP6
Fatty acid binding protein 6, ileal
SEQ ID NOS: 4986-

4988

FAM107B
Family with sequence similarity 107,
SEQ ID NOS: 4989-

member B
5010

FAM131A
Family with sequence similarity 131,
SEQ ID NOS: 5011-

member A
5019

FAM132A
Family with sequence similarity 132,
SEQ ID NO: 1795

member A

FAM132B
Family with sequence similarity 132,
SEQ ID NOS: 4882-

member B
4884

FAM150A
Family with sequence similarity 150,
SEQ ID NOS: 737-738

member A

FAM150B
Family with sequence similarity 150,
SEQ ID NOS: 739-745

member B

FAM171A1
Family with sequence similarity 171,
SEQ ID NOS: 5020-

member A1
5021

FAM171B
Family with sequence similarity 171,
SEQ ID NOS: 5022-

member B
5023

FAM172A
Family with sequence similarity 172,
SEQ ID NOS: 5024-

member A
5028

FAM175A
Family with sequence similarity 175,
SEQ ID NOS: 64-71

member A

FAM177A1
Family with sequence similarity 177,
SEQ ID NOS: 5029-

member A1
5038

FAM179B
Family with sequence similarity 179,
SEQ ID NOS: 13628-

member B
13633

FAM180A
Family with sequence similarity 180,
SEQ ID NOS: 5039-

member A
5041

FAM189A1
Family with sequence similarity 189,
SEQ ID NOS: 5042-

member A1
5043

FAM198A
Family with sequence similarity 198,
SEQ ID NOS: 5044-

member A
5046

FAM19A1
Family with sequence similarity 19
SEQ ID NOS: 5047-

(chemokine (C-C motif)-like), member A1
5049

FAM19A2
Family with sequence similarity 19
SEQ ID NOS: 5050-

(chemokine (C-C motif)-like), member A2
5057

FAM19A3
Family with sequence similarity 19
SEQ ID NOS: 5058-

(chemokine (C-C motif)-like), member A3
5059

FAM19A4
Family with sequence similarity 19
SEQ ID NOS: 5060-

(chemokine (C-C motif)-like), member A4
5062

FAM19A5
Family with sequence similarity 19
SEQ ID NOS: 5063-

(chemokine (C-C motif)-like), member A5
5066

FAM20A
Family with sequence similarity 20,
SEQ ID NOS: 5067-

member A
5070

FAM20C
Family with sequence similarity 20,
SEQ ID NO: 5071

member C

FAM213A
Family with sequence similarity 213,
SEQ ID NOS: 5072-

member A
5077

FAM26D
Family with sequence similarity 26,
SEQ ID NOS: 2006-

member D
2010

FAM46B
Family with sequence similarity 46,
SEQ ID NO: 5078

member B

FAM57A
Family with sequence similarity 57,
SEQ ID NOS: 5079-

member A
5084

FAM78A
Family with sequence similarity 78,
SEQ ID NOS: 5085-

member A
5087

FAM96A
Family with sequence similarity 96,
SEQ ID NOS: 5088-

member A
5092

FAM9B
Family with sequence similarity 9, member
SEQ ID NOS: 5093-

B
5096

FAP
Fibroblast activation protein, alpha
SEQ ID NOS: 5097-

5103

FAS
Fas cell surface death receptor
SEQ ID NOS: 5104-

5113

FAT1
FAT atypical cadherin 1
SEQ ID NOS: 5114-

5120

FBLN1
Fibulin 1
SEQ ID NOS: 5121-

5133

FBLN2
Fibulin 2
SEQ ID NOS: 5134-

5139

FBLN5
Fibulin 5
SEQ ID NOS: 5140-

5145

FBLN7
Fibulin 7
SEQ ID NOS: 5146-

5151

FBN1
Fibrillin 1
SEQ ID NOS: 5152-

5155

FBN2
Fibrillin 2
SEQ ID NOS: 5156-

5161

FBN3
Fibrillin 3
SEQ ID NOS: 5162-

5166

FBXW7
F-box and WD repeat domain containing 7,
SEQ ID NOS: 5167-

E3 ubiquitin protein ligase
5177

FCAR
Fc fragment of IgA receptor
SEQ ID NOS: 5178-

5187

FCGBP
Fc fragment of IgG binding protein
SEQ ID NOS: 5188-

5190

FCGR1B
Fc fragment of IgG, high affinity Ib,
SEQ ID NOS: 5191-

receptor (CD64)
5196

FCGR3A
Fc fragment of IgG, low affinity IIIa,
SEQ ID NOS: 5197-

receptor (CD16a)
5203

FCGRT
Fc fragment of IgG, receptor, transporter,
SEQ ID NOS: 5204-

alpha
5214

FCMR
Fc fragment of IgM receptor
SEQ ID NOS: 5215-

5221

FCN1
Ficolin (collagen/fibrinogen domain
SEQ ID NOS: 5222-

containing) 1
5223

FCN2
Ficolin (collagen/fibrinogen domain
SEQ ID NOS: 5224-

containing lectin) 2
5225

FCN3
Ficolin (collagen/fibrinogen domain
SEQ ID NOS: 5226-

containing) 3
5227

FCRL1
Fc receptor-like 1
SEQ ID NOS: 5228-

5230

FCRL3
Fc receptor-like 3
SEQ ID NOS: 5231-

5236

FCRL5
Fc receptor-like 5
SEQ ID NOS: 5237-

5239

FCRLA
Fc receptor-like A
SEQ ID NOS: 5240-

5251

FCRLB
Fc receptor-like B
SEQ ID NOS: 5252-

5256

FDCSP
Follicular dendritic cell secreted protein
SEQ ID NO: 5257

FETUB
Fetuin B
SEQ ID NOS: 5258-

5264

FGA
Fibrinogen alpha chain
SEQ ID NOS: 5265-

5267

FGB
Fibrinogen beta chain
SEQ ID NOS: 5268-

5270

FGF10
Fibroblast growth factor 10
SEQ ID NOS: 5271-

5272

FGF17
Fibroblast growth factor 17
SEQ ID NOS: 5273-

5274

FGF18
Fibroblast growth factor 18
SEQ ID NO: 5275

FGF19
Fibroblast growth factor 19
SEQ ID NO: 5276

FGF21
Fibroblast growth factor 21
SEQ ID NOS: 5277-

5278

FGF22
Fibroblast growth factor 22
SEQ ID NOS: 5279-

5280

FGF23
Fibroblast growth factor 23
SEQ ID NO: 5281

FGF3
Fibroblast growth factor 3
SEQ ID NO: 5282

FGF4
Fibroblast growth factor 4
SEQ ID NO: 5283

FGF5
Fibroblast growth factor 5
SEQ ID NOS: 5284-

5286

FGF7
Fibroblast growth factor 7
SEQ ID NOS: 5287-

5291

FGF8
Fibroblast growth factor 8 (androgen-
SEQ ID NOS: 5292-

induced)
5297

FGFBP1
Fibroblast growth factor binding protein 1
SEQ ID NO: 5298

FGFBP2
Fibroblast growth factor binding protein 2
SEQ ID NO: 5299

FGFBP3
Fibroblast growth factor binding protein 3
SEQ ID NO: 5300

FGFR1
Fibroblast growth factor receptor 1
SEQ ID NOS: 5301-

5322

FGFR2
Fibroblast growth factor receptor 2
SEQ ID NOS: 5323-

5344

FGFR3
Fibroblast growth factor receptor 3
SEQ ID NOS: 5345-

5352

FGFR4
Fibroblast growth factor receptor 4
SEQ ID NOS: 5353-

5362

FGFRL1
Fibroblast growth factor receptor-like 1
SEQ ID NOS: 5363-

5368

FGG
Fibrinogen gamma chain
SEQ ID NOS: 5369-

5374

FGL1
Fibrinogen-like 1
SEQ ID NOS: 5375-

5381

FGL2
Fibrinogen-like 2
SEQ ID NOS: 5382-

5383

FHL1
Four and a half LIM domains 1
SEQ ID NOS: 5384-

5411

FHOD3
Formin homology 2 domain containing 3
SEQ ID NOS: 5412-

5418

FIBIN
Fin bud initiation factor homolog
SEQ ID NO: 5419

(zebrafish)

FICD
FIC domain containing
SEQ ID NOS: 5420-

5423

FIGF
C-fos induced growth factor (vascular
SEQ ID NO: 14054

endothelial growth factor D)

FJX1
Four jointed box 1
SEQ ID NO: 5424

FKBP10
FK506 binding protein 10, 65 kDa
SEQ ID NOS: 5425-

5430

FKBP11
FK506 binding protein 11, 19 kDa
SEQ ID NOS: 5431-

5437

FKBP14
FK506 binding protein 14, 22 kDa
SEQ ID NOS: 5438-

5440

FKBP2
FK506 binding protein 2, 13 kDa
SEQ ID NOS: 5441-

5444

FKBP7
FK506 binding protein 7
SEQ ID NOS: 5445-

5450

FKBP9
FK506 binding protein 9, 63 kDa
SEQ ID NOS: 5451-

5454

FLT1
Fms-related tyrosine kinase 1
SEQ ID NOS: 5455-

5463

FLT4
Fms-related tyrosine kinase 4
SEQ ID NOS: 5464-

5468

FMO1
Flavin containing monooxygenase 1
SEQ ID NOS: 5469-

5473

FMO2
Flavin containing monooxygenase 2 (non-
SEQ ID NOS: 5474-

functional)
5476

FMO3
Flavin containing monooxygenase 3
SEQ ID NOS: 5477-

5479

FMO5
Flavin containing monooxygenase 5
SEQ ID NOS: 5480-

5486

FMOD
Fibromodulin
SEQ ID NO: 5487

FN1
Fibronectin 1
SEQ ID NOS: 5488-

5500

FNDC1
Fibronectin type III domain containing 1
SEQ ID NOS: 5501-

5502

FNDC7
Fibronectin type III domain containing 7
SEQ ID NOS: 5503-

5504

FOCAD
Focadhesin
SEQ ID NOS: 5505-

5511

FOLR2
Folate receptor 2 (fetal)
SEQ ID NOS: 5512-

5521

FOLR3
Folate receptor 3 (gamma)
SEQ ID NOS: 5522-

5526

FOXRED2
FAD-dependent oxidoreductase domain
SEQ ID NOS: 5527-

containing 2
5530

FP325331.1
Uncharacterized protein
SEQ ID NO: 5531

UNQ6126/PRO20091

FPGS
Folylpolyglutamate synthase
SEQ ID NOS: 5539-

5545

FRAS1
Fraser extracellular matrix complex subunit
SEQ ID NOS: 5546-

1
5551

FREM1
FRAS1 related extracellular matrix 1
SEQ ID NOS: 5552-

5556

FREM3
FRAS1 related extracellular matrix 3
SEQ ID NO: 5557

FRMPD2
FERM and PDZ domain containing 2
SEQ ID NOS: 5558-

5561

FRZB
Frizzled-related protein
SEQ ID NO: 5562

FSHB
Follicle stimulating hormone, beta
SEQ ID NOS: 5563-

polypeptide
5565

FSHR
Follicle stimulating hormone receptor
SEQ ID NOS: 5566-

5569

FST
Follistatin
SEQ ID NOS: 5570-

5573

FSTL1
Follistatin-like 1
SEQ ID NOS: 5574-

5577

FSTL3
Follistatin-like 3 (secreted glycoprotein)
SEQ ID NOS: 5578-

5583

FSTL4
Follistatin-like 4
SEQ ID NOS: 5584-

5586

FSTL5
Follistatin-like 5
SEQ ID NOS: 5587-

5589

FTCDNL1
Formiminotransferase cyclodeaminase N-
SEQ ID NOS: 5590-

terminal like
5593

FUCA1
Fucosidase, alpha-L- 1, tissue
SEQ ID NO: 5594

FUCA2
Fucosidase, alpha-L- 2, plasma
SEQ ID NOS: 5595-

5596

FURIN
Furin (paired basic amino acid cleaving
SEQ ID NOS: 5597-

enzyme)
5603

FUT10
Fucosyltransferase 10 (alpha (1,3)
SEQ ID NOS: 5604-

fucosyltransferase)
5606

FUT11
Fucosyltransferase 11 (alpha (1,3)
SEQ ID NOS: 5607-

fucosyltransferase)
5608

FXN
Frataxin
SEQ ID NOS: 5609-

5616

FXR1
Fragile X mental retardation, autosomal
SEQ ID NOS: 5617-

homolog 1
5629

FXYD3
FXYD domain containing ion transport
SEQ ID NOS: 5630-

regulator 3
5642

GABBR1
Gamma-aminobutyric acid (GABA) B
SEQ ID NOS: 5643-

receptor, 1
5654

GABRA1
Gamma-aminobutyric acid (GABA) A
SEQ ID NOS: 5655-

receptor, alpha 1
5670

GABRA2
Gamma-aminobutyric acid (GABA) A
SEQ ID NOS: 5671-

receptor, alpha 2
5685

GABRA5
Gamma-aminobutyric acid (GABA) A
SEQ ID NOS: 5686-

receptor, alpha 5
5694

GABRG3
Gamma-aminobutyric acid (GABA) A
SEQ ID NOS: 5695-

receptor, gamma 3
5700

GABRP
Gamma-aminobutyric acid (GABA) A
SEQ ID NOS: 5701-

receptor, pi
5709

GAL
Galanin/GMAP prepropeptide
SEQ ID NO: 5710

GAL3ST1
Galactose-3-O-sulfotransferase 1
SEQ ID NOS: 5711-

5732

GAL3ST2
Galactose-3-O-sulfotransferase 2
SEQ ID NO: 5733

GAL3ST3
Galactose-3-O-sulfotransferase 3
SEQ ID NOS: 5734-

5735

GALC
Galactosylceramidase
SEQ ID NOS: 5736-

5745

GALNS
Galactosamine (N-acetyl)-6-sulfatase
SEQ ID NOS: 5746-

5751

GALNT10
Polypeptide N-
SEQ ID NOS: 5752-

acetylgalactosaminyltransferase 10
5755

GALNT12
Polypeptide N-
SEQ ID NOS: 5756-

acetylgalactosaminyltransferase 12
5757

GALNT15
Polypeptide N-
SEQ ID NOS: 5758-

acetylgalactosaminyltransferase 15
5761

GALNT2
Polypeptide N-
SEQ ID NO: 5762

acetylgalactosaminyltransferase 2

GALNT6
Polypeptide N-
SEQ ID NOS: 5763-

acetylgalactosaminyltransferase 6
5774

GALNT8
Polypeptide N-
SEQ ID NOS: 5775-

acetylgalactosaminyltransferase 8
5778

GALNTL6
Polypeptide N-
SEQ ID NOS: 5779-

acetylgalactosaminyltransferase-like 6
5782

GALP
Galanin-like peptide
SEQ ID NOS: 5783-

5785

GANAB
Glucosidase, alpha; neutral AB
SEQ ID NOS: 5786-

5794

GARS
Glycyl-tRNA synthetase
SEQ ID NOS: 5795-

5798

GAS1
Growth arrest-specific 1
SEQ ID NO: 5799

GAS6
Growth arrest-specific 6
SEQ ID NO: 5800

GAST
Gastrin
SEQ ID NO: 5801

GBA
Glucosidase, beta, acid
SEQ ID NOS: 5811-

5814

GBGT1
Globoside alpha-1,3-N-
SEQ ID NOS: 5815-

acetylgalactosaminyltransferase 1
5823

GC
Group-specific component (vitamin D
SEQ ID NOS: 5824-

binding protein)
5828

GCG
Glucagon
SEQ ID NOS: 5829-

5830

GCGR
Glucagon receptor
SEQ ID NOS: 5831-

5833

GCNT7
Glucosaminyl (N-acetyl) transferase family
SEQ ID NOS: 5834-

member 7
5835

GCSH
Glycine cleavage system protein H
SEQ ID NOS: 5836-

(aminomethyl carrier)
5844

GDF1
Growth differentiation factor 1
SEQ ID NO: 5845

GDF10
Growth differentiation factor 10
SEQ ID NO: 5846

GDF11
Growth differentiation factor 11
SEQ ID NOS: 5847-

5848

GDF15
Growth differentiation factor 15
SEQ ID NOS: 5849-

5851

GDF2
Growth differentiation factor 2
SEQ ID NO: 5852

GDF3
Growth differentiation factor 3
SEQ ID NO: 5853

GDF5
Growth differentiation factor 5
SEQ ID NOS: 5854-

5855

GDF6
Growth differentiation factor 6
SEQ ID NOS: 5856-

5858

GDF7
Growth differentiation factor 7
SEQ ID NO: 5859

GDF9
Growth differentiation factor 9
SEQ ID NOS: 5860-

5864

GDNF
Glial cell derived neurotrophic factor
SEQ ID NOS: 5865-

5872

GFOD2
Glucose-fructose oxidoreductase domain
SEQ ID NOS: 5873-

containing 2
5878

GFPT2
Glutamine-fructose-6-phosphate
SEQ ID NOS: 5879-

transaminase 2
5881

GFRA2
GDNF family receptor alpha 2
SEQ ID NOS: 5882-

5888

GFRA4
GDNF family receptor alpha 4
SEQ ID NOS: 5889-

5891

GGA2
Golgi-associated, gamma adaptin ear
SEQ ID NOS: 5892-

containing, ARF binding protein 2
5900

GGH
Gamma-glutamyl hydrolase (conjugase,
SEQ ID NO: 5901

folylpolygammaglutamyl hydrolase)

GGT1
Gamma-glutamyltransferase 1
SEQ ID NOS: 5902-

5924

GGT5
Gamma-glutamyltransferase 5
SEQ ID NOS: 5925-

5929

GH1
Growth hormone 1
SEQ ID NOS: 5930-

5934

GH2
Growth hormone 2
SEQ ID NOS: 5935-

5939

GHDC
GH3 domain containing
SEQ ID NOS: 5940-

5947

GHRH
Growth hormone releasing hormone
SEQ ID NOS: 5948-

5950

GHRHR
Growth hormone releasing hormone
SEQ ID NOS: 5951-

receptor
5956

GHRL
Ghrelin/obestatin prepropeptide
SEQ ID NOS: 5957-

5967

GIF
Gastric intrinsic factor (vitamin B synthesis)
SEQ ID NOS: 5968-

5969

GIP
Gastric inhibitory polypeptide
SEQ ID NO: 5970

GKN1
Gastrokine 1
SEQ ID NO: 5971

GKN2
Gastrokine 2
SEQ ID NOS: 5972-

5973

GLA
Galactosidase, alpha
SEQ ID NOS: 5974-

5975

GLB1
Galactosidase, beta 1
SEQ ID NOS: 5976-

5984

GLB1L
Galactosidase, beta 1-like
SEQ ID NOS: 5985-

5992

GLB1L2
Galactosidase, beta 1-like 2
SEQ ID NOS: 5993-

5994

GLCE
Glucuronic acid epimerase
SEQ ID NOS: 5995-

5996

GLG1
Golgi glycoprotein 1
SEQ ID NOS: 5997-

6004

GLIPR1
GLI pathogenesis-related 1
SEQ ID NOS: 6005-

6008

GLIPR1L1
GLI pathogenesis-related 1 like 1
SEQ ID NOS: 6009-

6012

GLIS3
GLIS family zinc finger 3
SEQ ID NOS: 6013-

6021

GLMP
Glycosylated lysosomal membrane protein
SEQ ID NOS: 6022-

6030

GLRB
Glycine receptor, beta
SEQ ID NOS: 6031-

6036

GLS
Glutaminase
SEQ ID NOS: 6037-

6044

GLT6D1
Glycosyltransferase 6 domain containing 1
SEQ ID NOS: 6045-

6046

GLTPD2
Glycolipid transfer protein domain
SEQ ID NO: 6047

containing 2

GLUD1
Glutamate dehydrogenase 1
SEQ ID NO: 6048

GM2A
GM2 ganglioside activator
SEQ ID NOS: 6049-

6051

GML
Glycosylphosphatidylinositol anchored
SEQ ID NOS: 6052-

molecule like
6053

GNAS
GNAS complex locus
SEQ ID NOS: 6054-

6075

GNLY
Granulysin
SEQ ID NOS: 6076-

6079

GNPTG
N-acetylglucosamine-1-phosphate
SEQ ID NOS: 6080-

transferase, gamma subunit
6084

GNRH1
Gonadotropin-releasing hormone 1
SEQ ID NOS: 6085-

(luteinizing-releasing hormone)
6086

GNRH2
Gonadotropin-releasing hormone 2
SEQ ID NOS: 6087-

6090

GNS
Glucosamine (N-acetyl)-6-sulfatase
SEQ ID NOS: 6091-

6096

GOLM1
Golgi membrane protein 1
SEQ ID NOS: 6097-

6101

GORAB
Golgin, RAB6-interacting
SEQ ID NOS: 6102-

6104

GOT2
Glutamic-oxaloacetic transaminase 2,
SEQ ID NOS: 6105-

mitochondrial
6107

GP2
Glycoprotein 2 (zymogen granule
SEQ ID NOS: 6108-

membrane)
6116

GP6
Glycoprotein VI (platelet)
SEQ ID NOS: 6117-

6120

GPC2
Glypican 2
SEQ ID NOS: 6121-

6122

GPC5
Glypican 5
SEQ ID NOS: 6123-

6125

GPC6
Glypican 6
SEQ ID NOS: 6126-

6127

GPD2
Glycerol-3-phosphate dehydrogenase 2
SEQ ID NOS: 6128-

(mitochondrial)
6136

GPER1
G protein-coupled estrogen receptor 1
SEQ ID NOS: 6137-

6143

GPHA2
Glycoprotein hormone alpha 2
SEQ ID NOS: 6144-

6146

GPHB5
Glycoprotein hormone beta 5
SEQ ID NOS: 6147-

6148

GPIHBP1
Glycosylphosphatidylinositol anchored high
SEQ ID NO: 6149

density lipoprotein binding protein 1

GPLD1
Glycosylphosphatidylinositol specific
SEQ ID NO: 6150

phospholipase D1

GPNMB
Glycoprotein (transmembrane) nmb
SEQ ID NOS: 6151-

6153

GPR162
G protein-coupled receptor 162
SEQ ID NOS: 6154-

6157

GPX3
Glutathione peroxidase 3
SEQ ID NOS: 6158-

6165

GPX4
Glutathione peroxidase 4
SEQ ID NOS: 6166-

6176

GPX5
Glutathione peroxidase 5
SEQ ID NOS: 6177-

6178

GPX6
Glutathione peroxidase 6
SEQ ID NOS: 6179-

6181

GPX7
Glutathione peroxidase 7
SEQ ID NO: 6182

GREM1
Gremlin 1, DAN family BMP antagonist
SEQ ID NOS: 6183-

6185

GREM2
Gremlin 2, DAN family BMP antagonist
SEQ ID NO: 6186

GRHL3
Grainyhead-like transcription factor 3
SEQ ID NOS: 6187-

6192

GRIA2
Glutamate receptor, ionotropic, AMPA 2
SEQ ID NOS: 6193-

6204

GRIA3
Glutamate receptor, ionotropic, AMPA 3
SEQ ID NOS: 6205-

6210

GRIA4
Glutamate receptor, ionotropic, AMPA 4
SEQ ID NOS: 6211-

6222

GRIK2
Glutamate receptor, ionotropic, kainate 2
SEQ ID NOS: 6223-

6231

GRIN2B
Glutamate receptor, ionotropic, N-methyl
SEQ ID NOS: 6232-

D-aspartate 2B
6235

GRM2
Glutamate receptor, metabotropic 2
SEQ ID NOS: 6236-

6239

GRM3
Glutamate receptor, metabotropic 3
SEQ ID NOS: 6240-

6244

GRM5
Glutamate receptor, metabotropic 5
SEQ ID NOS: 6245-

6249

CRN
Granulin
SEQ ID NOS: 6250-

6265

GRP
Gastrin-releasing peptide
SEQ ID NOS: 6266-

6270

GSG1
Germ cell associated 1
SEQ ID NOS: 6280-

6288

GSN
Gelsolin
SEQ ID NOS: 6289-

6297

GTDC1
Glycosyltransferase-like domain containing
SEQ ID NOS: 6298-

1
6311

GTPBP10
GTP-binding protein 10 (putative)
SEQ ID NOS: 6312-

6320

GUCA2A
Guanylate cyclase activator 2A (guanylin)
SEQ ID NO: 6321

GUCA2B
Guanylate cyclase activator 2B
SEQ ID NO: 6322

(uroguanylin)

GUSB
Glucuronidase, beta
SEQ ID NOS: 6323-

6327

GVQW1
GVQW motif containing 1
SEQ ID NO: 6328

GXYLT1
Glucoside xylosyltransferase 1
SEQ ID NOS: 6329-

6330

GXYLT2
Glucoside xylosyltransferase 2
SEQ ID NOS: 6331-

6333

GYLTL1B
Glycosyltransferase-like 1B
SEQ ID NOS: 7702-

7707

GYPB
Glycophorin B (MNS blood group)
SEQ ID NOS: 6334-

6342

GZMA
Granzyme A (granzyme 1, cytotoxic T-
SEQ ID NO: 6343

lymphocyte-associated serine esterase 3)

GZMB
Granzyme B (granzyme 2, cytotoxic T-
SEQ ID NOS: 6344-

lymphocyte-associated serine esterase 1)
6352

GZMH
Granzyme H (cathepsin G-like 2, protein h-
SEQ ID NOS: 6353-

CCPX)
6355

GZMK
Granzyme K (granzyme 3; tryptase II)
SEQ ID NO: 6356

GZMM
Granzyme M (lymphocyte met-ase 1)
SEQ ID NOS: 6357-

6358

H6PD
Hexose-6-phosphate dehydrogenase
SEQ ID NOS: 6359-

(glucose 1-dehydrogenase)
6360

HABP2
Hyaluronan binding protein 2
SEQ ID NOS: 6361-

6362

HADHB
Hydroxyacyl-CoA dehydrogenase/3-
SEQ ID NOS: 6363-

ketoacyl-CoA thiolase/enoyl-CoA hydratase
6369

(trifunctional protein), beta subunit

HAMP
Hepcidin antimicrobial peptide
SEQ ID NOS: 6370-

6371

HAPLN1
Hyaluronan and proteoglycan link protein 1
SEQ ID NOS: 6372-

6378

HAPLN2
Hyaluronan and proteoglycan link protein 2
SEQ ID NOS: 6379-

6380

HAPLN3
Hyaluronan and proteoglycan link protein 3
SEQ ID NOS: 6381-

6384

HAPLN4
Hyaluronan and proteoglycan link protein 4
SEQ ID NO: 6385

HARS2
Histidyl-tRNA synthetase 2, mitochondrial
SEQ ID NOS: 6386-

6401

HAVCR1
Hepatitis A virus cellular receptor 1
SEQ ID NOS: 6402-

6406

HCCS
Holocytochrome c synthase
SEQ ID NOS: 6407-

6409

HCRT
Hypocretin (orexin) neuropeptide precursor
SEQ ID NO: 6410

HEATR5A
HEAT repeat containing 5A
SEQ ID NOS: 6414-

6420

HEPH
Hephaestin
SEQ ID NOS: 6421-

6428

HEXA
Hexosaminidase A (alpha polypeptide)
SEQ ID NOS: 6429-

6438

HEXB
Hexosaminidase B (beta polypeptide)
SEQ ID NOS: 6439-

6444

HFE2
Hemochromatosis type 2 (juvenile)
SEQ ID NOS: 6445-

6451

HGF
Hepatocyte growth factor (hepapoietin A;
SEQ ID NOS: 6452-

scatter factor)
6462

HGFAC
HGF activator
SEQ ID NOS: 6463-

6464

HHIP
Hedgehog interacting protein
SEQ ID NOS: 6465-

6466

HHIPL1
HHIP-like 1
SEQ ID NOS: 6467-

6468

HHIPL2
HHIP-like 2
SEQ ID NO: 6469

HHLA1
HERV-H LTR-associating 1
SEQ ID NOS: 6470-

6471

HHLA2
HERV-H LTR-associating 2
SEQ ID NOS: 6472-

6482

HIBADH
3-hydroxyisobutyrate dehydrogenase
SEQ ID NOS: 6483-

6485

HINT2
Histidine triad nucleotide binding protein 2
SEQ ID NO: 6486

HLA-A
Major histocompatibility complex, class I,
SEQ ID NOS: 6487-

A
6491

HLA-C
Major histocompatibility complex, class I, C
SEQ ID NOS: 6492-

6496

HLA-DOA
Major histocompatibility complex, class II,
SEQ ID NOS: 6497-

DO alpha
6498

HLA-DPA1
Major histocompatibility complex, class II,
SEQ ID NOS: 6499-

DP alpha 1
6502

HLA-DQA1
Major histocompatibility complex, class II,
SEQ ID NOS: 6503-

DQ alpha 1
6508

HLA-DQB1
Major histocompatibility complex, class II,
SEQ ID NOS: 6509-

DQ beta 1
6514

HLA-DQB2
Major histocompatibility complex, class II,
SEQ ID NOS: 6515-

DQ beta 2
6518

HMCN1
Hemicentin 1
SEQ ID NOS: 6519-

6520

HMCN2
Hemicentin 2
SEQ ID NOS: 6521-

6524

HMGCL
3-hydroxymethyl-3-methylglutaryl-CoA
SEQ ID NOS: 6525-

lyase
6528

HMHA1
Histocompatibility (minor) HA-1
SEQ ID NOS: 1034-

1042

HMSD
Histocompatibility (minor) serpin domain
SEQ ID NOS: 6529-

containing
6530

HP
Haptoglobin
SEQ ID NOS: 6531-

6544

HPR
Haptoglobin-related protein
SEQ ID NOS: 6545-

6547

HPSE
Heparanase
SEQ ID NOS: 6548-

6554

HPSE2
Heparanase 2 (inactive)
SEQ ID NOS: 6555-

6560

HPX
Hemopexin
SEQ ID NOS: 6561-

6562

HRC
Histidine rich calcium binding protein
SEQ ID NOS: 6563-

6565

HRG
Histidine-rich glycoprotein
SEQ ID NO: 6566

HRSP12
Heat-responsive protein 12
SEQ ID NOS: 11389-

11392

HS2ST1
Heparan sulfate 2-O-sulfotransferase 1
SEQ ID NOS: 6567-

6569

HS3ST1
Heparan sulfate (glucosamine) 3-O-
SEQ ID NOS: 6570-

sulfotransferase 1
6572

HS6ST1
Heparan sulfate 6-O-sulfotransferase 1
SEQ ID NO: 6573

HS6ST3
Heparan sulfate 6-O-sulfotransferase 3
SEQ ID NOS: 6574-

6575

HSD11B1L
Hydroxysteroid (11-beta) dehydrogenase 1-
SEQ ID NOS: 6576-

like
6594

HSD17B11
Hydroxysteroid (17-beta) dehydrogenase 11
SEQ ID NOS: 6595-

6596

HSD17B7
Hydroxysteroid (17-beta) dehydrogenase 7
SEQ ID NOS: 6597-

6601

HSP90B1
Heat shock protein 90 kDa beta (Grp94),
SEQ ID NOS: 6602-

member 1
6607

HSPA13
Heat shock protein 70 kDa family, member
SEQ ID NO: 6608

13

HSPA5
Heat shock 70 kDa protein 5 (glucose-
SEQ ID NO: 6609

regulated protein, 78 kDa)

HSPG2
Heparan sulfate proteoglycan 2
SEQ ID NOS: 6610-

6614

HTATIP2
HIV-1 Tat interactive protein 2, 30 kDa
SEQ ID NOS: 6615-

6622

HTN1
Histatin 1
SEQ ID NOS: 6623-

6625

HTN3
Histatin 3
SEQ ID NOS: 6626-

6628

HTRA1
HtrA serine peptidase 1
SEQ ID NOS: 6629-

6630

HTRA3
HtrA serine peptidase 3
SEQ ID NOS: 6631-

6632

HTRA4
HtrA serine peptidase 4
SEQ ID NO: 6633

HYAL1
Hyaluronoglucosaminidase 1
SEQ ID NOS: 6634-

6642

HYAL2
Hyaluronoglucosaminidase 2
SEQ ID NOS: 6643-

6651

HYAL3
Hyaluronoglucosaminidase 3
SEQ ID NOS: 6652-

6658

HYOU1
Hypoxia up-regulated 1
SEQ ID NOS: 6659-

6673

IAPP
Islet amyloid polypeptide
SEQ ID NOS: 6674-

6678

IBSP
Integrin-binding sialoprotein
SEQ ID NO: 6679

ICAM1
Intercellular adhesion molecule 1
SEQ ID NOS: 6680-

6682

ICAM2
Intercellular adhesion molecule 2
SEQ ID NOS: 6683-

6693

ICAM4
Intercellular adhesion molecule 4
SEQ ID NOS: 6694-

(Landsteiner-Wiener blood group)
6696

ID1
Inhibitor of DNA binding 1, dominant
SEQ ID NOS: 6697-

negative helix-loop-helix protein
6698

IDE
Insulin-degrading enzyme
SEQ ID NOS: 6699-

6702

IDNK
IdnK, gluconokinase homolog (E. coli)
SEQ ID NOS: 6703-

6708

IDS
Iduronate 2-sulfatase
SEQ ID NOS: 6709-

6714

IDUA
Iduronidase, alpha-L-
SEQ ID NOS: 6715-

6720

IFI27L2
Interferon, alpha-inducible protein 27-like 2
SEQ ID NOS: 6721-

6722

IFI30
Interferon, gamma-inducible protein 30
SEQ ID NOS: 6723-

6724

IFNA1
Interferon, alpha 1
SEQ ID NO: 6725

IFNA10
Interferon, alpha 10
SEQ ID NO: 6726

IFNA13
Interferon, alpha 13
SEQ ID NOS: 6727-

6728

IFNA14
Interferon, alpha 14
SEQ ID NO: 6729

IFNA16
Interferon, alpha 16
SEQ ID NO: 6730

IFNA17
Interferon, alpha 17
SEQ ID NO: 6731

IFNA2
Interferon, alpha 2
SEQ ID NO: 6732

IFNA21
Interferon, alpha 21
SEQ ID NO: 6733

IFNA4
Interferon, alpha 4
SEQ ID NO: 6734

IFNA5
Interferon, alpha 5
SEQ ID NO: 6735

IFNA6
Interferon, alpha 6
SEQ ID NOS: 6736-

6737

IFNA7
Interferon, alpha 7
SEQ ID NO: 6738

IFNA8
Interferon, alpha 8
SEQ ID NO: 6739

IFNAR1
Interferon (alpha, beta and omega) receptor
SEQ ID NOS: 6740-

1
6741

IFNB1
Interferon, beta 1, fibroblast
SEQ ID NO: 6742

IFNE
Interferon, epsilon
SEQ ID NO: 6743

IFNG
Interferon, gamma
SEQ ID NO: 6744

IFNGR1
Interferon gamma receptor 1
SEQ ID NOS: 6745-

6755

IFNL1
Interferon, lambda 1
SEQ ID NO: 6756

IFNL2
Interferon, lambda 2
SEQ ID NO: 6757

IFNL3
Interferon, lambda 3
SEQ ID NOS: 6758-

6759

IFNLR1
Interferon, lambda receptor 1
SEQ ID NOS: 6760-

6764

IFNW1
Interferon, omega 1
SEQ ID NO: 6765

IGF1
Insulin-like growth factor 1 (somatomedin
SEQ ID NOS: 6766-

C)
6771

IGF2
Insulin-like growth factor 2
SEQ ID NOS: 6772-

6779

IGFALS
Insulin-like growth factor binding protein,
SEQ ID NOS: 6780-

acid labile subunit
6782

IGFBP1
Insulin-like growth factor binding protein 1
SEQ ID NOS: 6783-

6785

IGFBP2
Insulin-like growth factor binding protein 2,
SEQ ID NOS: 6786-

36 kDa
6789

IGFBP3
Insulin-like growth factor binding protein 3
SEQ ID NOS: 6790-

6797

IGFBP4
Insulin-like growth factor binding protein 4
SEQ ID NO: 6798

IGFBP5
Insulin-like growth factor binding protein 5
SEQ ID NOS: 6799-

6800

IGFBP6
Insulin-like growth factor binding protein 6
SEQ ID NOS: 6801-

6803

IGFBP7
Insulin-like growth factor binding protein 7
SEQ ID NOS: 6804-

6805

IGFBPL1
Insulin-like growth factor binding protein-
SEQ ID NO: 6806

like 1

IGFL1
IGF-like family member 1
SEQ ID NO: 6807

IGFL2
IGF-like family member 2
SEQ ID NOS: 6808-

6810

IGFL3
IGF-like family member 3
SEQ ID NO: 6811

IGFLR1
IGF-like family receptor 1
SEQ ID NOS: 6812-

6820

IGIP
IgA-inducing protein
SEQ ID NO: 6821

IGLON5
IgLON family member 5
SEQ ID NO: 6822

IGSF1
Immunoglobulin superfamily, member 1
SEQ ID NOS: 6823-

6828

IGSF10
Immunoglobulin superfamily, member 10
SEQ ID NOS: 6829-

6830

IGSF11
Immunoglobulin superfamily, member 11
SEQ ID NOS: 6831-

6838

IGSF21
Immunoglobin superfamily, member 21
SEQ ID NO: 6839

IGSF8
Immunoglobulin superfamily, member 8
SEQ ID NOS: 6840-

6843

IGSF9
Immunoglobulin superfamily, member 9
SEQ ID NOS: 6844-

6846

IHH
Indian hedgehog
SEQ ID NO: 6847

IL10
Interleukin 10
SEQ ID NOS: 6848-

6849

IL11
Interleukin 11
SEQ ID NOS: 6850-

6853

IL11RA
Interleukin 11 receptor, alpha
SEQ ID NOS: 6854-

6864

IL12B
Interleukin 12B
SEQ ID NO: 6865

IL12RB1
Interleukin 12 receptor, beta 1
SEQ ID NOS: 6866-

6871

IL12RB2
Interleukin 12 receptor, beta 2
SEQ ID NOS: 6872-

6876

IL13
Interleukin 13
SEQ ID NOS: 6877-

6878

IL13RA1
Interleukin 13 receptor, alpha 1
SEQ ID NOS: 6879-

6880

IL15RA
Interleukin 15 receptor, alpha
SEQ ID NOS: 6881-

6898

IL17A
Interleukin 17A
SEQ ID NO: 6899

IL17B
Interleukin 17B
SEQ ID NO: 6900

IL17C
Interleukin 17C
SEQ ID NO: 6901

IL17D
Interleukin 17D
SEQ ID NOS: 6902-

6904

IL17F
Interleukin 17F
SEQ ID NO: 6905

IL17RA
Interleukin 17 receptor A
SEQ ID NOS: 6906-

6907

IL17RC
Interleukin 17 receptor C
SEQ ID NOS: 6908-

6923

IL17RE
Interleukin 17 receptor E
SEQ ID NOS: 6924-

6930

IL18BP
Interleukin 18 binding protein
SEQ ID NOS: 6931-

6941

IL18R1
Interleukin 18 receptor 1
SEQ ID NOS: 6942-

6945

IL18RAP
Interleukin 18 receptor accessory protein
SEQ ID NOS: 6946-

6948

IL19
Interleukin 19
SEQ ID NOS: 6949-

6951

IL1R1
Interleukin 1 receptor, type I
SEQ ID NOS: 6952-

6964

IL1R2
Interleukin 1 receptor, type II
SEQ ID NOS: 6965-

6968

IL1RAP
Interleukin 1 receptor accessory protein
SEQ ID NOS: 6969-

6982

IL1RL1
Interleukin 1 receptor-like 1
SEQ ID NOS: 6983-

6988

IL1RL2
Interleukin 1 receptor-like 2
SEQ ID NOS: 6989-

6991

IL1RN
Interleukin 1 receptor antagonist
SEQ ID NOS: 6992-

6996

IL2
Interleukin 2
SEQ ID NO: 6997

IL20
Interleukin 20
SEQ ID NOS: 6998-

7000

IL20RA
Interleukin 20 receptor, alpha
SEQ ID NOS: 7001-

7007

IL21
Interleukin 21
SEQ ID NOS: 7008-

7009

IL22
Interleukin 22
SEQ ID NOS: 7010-

7011

IL22RA2
Interleukin 22 receptor, alpha 2
SEQ ID NOS: 7012-

7014

IL23A
Interleukin 23, alpha subunit p19
SEQ ID NO: 7015

IL24
Interleukin 24
SEQ ID NOS: 7016-

7021

IL25
Interleukin 25
SEQ ID NOS: 7022-

7023

IL26
Interleukin 26
SEQ ID NO: 7024

IL27
Interleukin 27
SEQ ID NOS: 7025-

7026

IL2RB
Interleukin 2 receptor, beta
SEQ ID NOS: 7027-

7031

IL3
Interleukin 3
SEQ ID NO: 7032

IL31
Interleukin 31
SEQ ID NO: 7033

IL31RA
Interleukin 31 receptor A
SEQ ID NOS: 7034-

7041

IL32
Interleukin 32
SEQ ID NOS: 7042-

7071

IL34
Interleukin 34
SEQ ID NOS: 7072-

7075

IL3RA
Interleukin 3 receptor, alpha (low affinity)
SEQ ID NOS: 7076-

7078

IL4
Interleukin 4
SEQ ID NOS: 7079-

7081

IL4I1
Interleukin 4 induced 1
SEQ ID NOS: 7082-

7089

IL4R
Interleukin 4 receptor
SEQ ID NOS: 7090-

7103

IL5
Interleukin 5
SEQ ID NOS: 7104-

7105

IL5RA
Interleukin 5 receptor, alpha
SEQ ID NOS: 7106-

7115

IL6
Interleukin 6
SEQ ID NOS: 7116-

7122

IL6R
Interleukin 6 receptor
SEQ ID NOS: 7123-

7128

IL6ST
Interleukin 6 signal transducer
SEQ ID NOS: 7129-

7138

IL7
Interleukin 7
SEQ ID NOS: 7139-

7146

IL7R
Interleukin 7 receptor
SEQ ID NOS: 7147-

7153

IL9
Interleukin 9
SEQ ID NO: 7154

ILDR1
Immunoglobulin-like domain containing
SEQ ID NOS: 7155-

receptor 1
7159

ILDR2
Immunoglobulin-like domain containing
SEQ ID NOS: 7160-

receptor 2
7166

IMP4
IMP4, U3 small nucleolar ribonucleoprotein
SEQ ID NOS: 7167-

7172

IMPG1
Interphotoreceptor matrix proteoglycan 1
SEQ ID NOS: 7173-

7176

INHA
Inhibin, alpha
SEQ ID NO: 7177

INHBA
Inhibin, beta A
SEQ ID NOS: 7178-

7180

INHBB
Inhibin, beta B
SEQ ID NO: 7181

INHBC
Inhibin, beta C
SEQ ID NO: 7182

INHBE
Inhibin, beta E
SEQ ID NOS: 7183-

7184

INPP5A
Inositol polyphosphate-5-phosphatase A
SEQ ID NOS: 7185-

7189

INS
Insulin
SEQ ID NOS: 7190-

7194

INS-IGF2
INS-IGF2 readthrough
SEQ ID NOS: 7195-

7196

INSL3
Insulin-like 3 (Leydig cell)
SEQ ID NOS: 7197-

7199

INSL4
Insulin-like 4 (placenta)
SEQ ID NO: 7200

INSL5
Insulin-like 5
SEQ ID NO: 7201

INSL6
Insulin-like 6
SEQ ID NO: 7202

INTS3
Integrator complex subunit 3
SEQ ID NOS: 7203-

7208

IPO11
Importin 11
SEQ ID NOS: 7209-

7217

IPO9
Importin 9
SEQ ID NOS: 7218-

7219

IQCF6
IQ motif containing F6
SEQ ID NOS: 7220-

7221

IRAK3
Interleukin-1 receptor-associated kinase 3
SEQ ID NOS: 7222-

7224

IRS4
Insulin receptor substrate 4
SEQ ID NO: 7225

ISLR
Immunoglobulin superfamily containing
SEQ ID NOS: 7226-

leucine-rich repeat
7229

ISLR2
Immunoglobulin superfamily containing
SEQ ID NOS: 7230-

leucine-rich repeat 2
7239

ISM1
Isthmin 1, angiogenesis inhibitor
SEQ ID NO: 7240

ISM2
Isthmin 2
SEQ ID NOS: 7241-

7246

ITGA4
Integrin, alpha 4 (antigen CD49D, alpha 4
SEQ ID NOS: 7247-

subunit of VLA-4 receptor)
7249

ITGA9
Integrin, alpha 9
SEQ ID NOS: 7250-

7252

ITGAL
Integrin, alpha L (antigen CD11A (p180),
SEQ ID NOS: 7253-

lymphocyte function-associated antigen 1;
7262

alpha polypeptide)

ITGAX
Integrin, alpha X (complement component 3
SEQ ID NOS: 7263-

receptor 4 subunit)
7265

ITGB1
Integrin, beta 1 (fibronectin receptor, beta
SEQ ID NOS: 7266-

polypeptide, antigen CD29 includes MDF2,
7281

MSK12)

ITGB2
Integrin, beta 2 (complement component 3
SEQ ID NOS: 7282-

receptor 3 and 4 subunit)
7298

ITGB3
Integrin, beta 3 (platelet glycoprotein IIIa,
SEQ ID NOS: 7299-

antigen CD61)
7301

ITGB7
Integrin, beta 7
SEQ ID NOS: 7302-

7309

ITGBL1
Integrin, beta-like 1 (with EGF-like repeat
SEQ ID NOS: 7310-

domains)
7315

ITIH1
Inter-alpha-trypsin inhibitor heavy chain 1
SEQ ID NOS: 7316-

7321

ITIH2
Inter-alpha-trypsin inhibitor heavy chain 2
SEQ ID NOS: 7322-

7324

ITIH3
Inter-alpha-trypsin inhibitor heavy chain 3
SEQ ID NOS: 7325-

7327

ITIH4
Inter-alpha-trypsin inhibitor heavy chain
SEQ ID NOS: 7328-

family, member 4
7331

ITIH5
Inter-alpha-trypsin inhibitor heavy chain
SEQ ID NOS: 7332-

family, member 5
7335

ITIH6
Inter-alpha-trypsin inhibitor heavy chain
SEQ ID NO: 7336

family, member 6

ITLN1
Intelectin 1 (galactofuranose binding)
SEQ ID NO: 7337

ITLN2
Intelectin 2
SEQ ID NO: 7338

IZUMO1R
IZUMO1 receptor, JUNO
SEQ ID NOS: 7339-

7340

IZUMO4
IZUMO family member 4
SEQ ID NOS: 7341-

7347

JCHAIN
Joining chain of multimeric IgA and IgM
SEQ ID NOS: 7357-

7362

JMJD8
Jumonji domain containing 8
SEQ ID NOS: 7363-

7367

JSRP1
Junctional sarcoplasmic reticulum protein 1
SEQ ID NO: 7368

KANSL2
KAT8 regulatory NSL complex subunit 2
SEQ ID NOS: 7369-

7379

KAZALD1
Kazal-type serine peptidase inhibitor
SEQ ID NO: 7380

domain 1

KCNIP3
Kv channel interacting protein 3, calsenilin
SEQ ID NOS: 7381-

7383

KCNK7
Potassium channel, two pore domain
SEQ ID NOS: 7384-

subfamily K, member 7
7389

KCNN4
Potassium channel, calcium activated
SEQ ID NOS: 7390-

intermediate/small conductance subfamily
7395

N alpha, member 4

KCNU1
Potassium channel, subfamily U, member 1
SEQ ID NOS: 7396-

7400

KCP
Kielin/chordin-like protein
SEQ ID NOS: 7401-

7404

KDELC1
KDEL (Lys-Asp-Glu-Leu) containing 1
SEQ ID NO: 7405

KDELC2
KDEL (Lys-Asp-Glu-Leu) containing 2
SEQ ID NOS: 7406-

7409

KDM1A
Lysine (K)-specific demethylase 1A
SEQ ID NOS: 7410-

7413

KDM3B
Lysine (K)-specific demethylase 3B
SEQ ID NOS: 7414-

7417

KDM6A
Lysine (K)-specific demethylase 6A
SEQ ID NOS: 7418-

7427

KDM7A
Lysine (K)-specific demethylase 7A
SEQ ID NOS: 7428-

7429

KDSR
3-ketodihydrosphingosine reductase
SEQ ID NOS: 7430-

7436

KERA
Keratocan
SEQ ID NO: 7437

KIAA0100
KJAA0100
SEQ ID NOS: 7438-

7443

KIAA0319
KJAA0319
SEQ ID NOS: 7444-

7449

KIAA1324
KIAA1324
SEQ ID NOS: 7450-

7458

KIFC2
Kinesin family member C2
SEQ ID NOS: 7459-

7461

KIR2DL4
Killer cell immunoglobulin-like receptor,
SEQ ID NOS: 7462-

two domains, long cytoplasmic tail, 4
7468

KIR3DX1
Killer cell immunoglobulin-like receptor,
SEQ ID NOS: 7469-

three domains, X1
7473

KIRREL2
Kin of IRRE like 2 (Drosophila)
SEQ ID NOS: 7474-

7478

KISS1
KiSS-1 metastasis-suppressor
SEQ ID NOS: 7479-

7480

KLHL11
Kelch-like family member 11
SEQ ID NO: 7481

KLHL22
Kelch-like family member 22
SEQ ID NOS: 7482-

7488

KLK1
Kallikrein 1
SEQ ID NOS: 7489-

7490

KLK10
Kallikrein-related peptidase 10
SEQ ID NOS: 7491-

7495

KLK11
Kallikrein-related peptidase 11
SEQ ID NOS: 7496-

7504

KLK12
Kallikrein-related peptidase 12
SEQ ID NOS: 7505-

7511

KLK13
Kallikrein-related peptidase 13
SEQ ID NOS: 7512-

7520

KLK14
Kallikrein-related peptidase 14
SEQ ID NOS: 7521-

7522

KLK15
Kallikrein-related peptidase 15
SEQ ID NOS: 7523-

7527

KLK2
Kallikrein-related peptidase 2
SEQ ID NOS: 7528-

7540

KLK3
Kallikrein-related peptidase 3
SEQ ID NOS: 7541-

7552

KLK4
Kallikrein-related peptidase 4
SEQ ID NOS: 7553-

7557

KLK5
Kallikrein-related peptidase 5
SEQ ID NOS: 7558-

7561

KLK6
Kallikrein-related peptidase 6
SEQ ID NOS: 7562-

7568

KLK7
Kallikrein-related peptidase 7
SEQ ID NOS: 7569-

7573

KLK8
Kallikrein-related peptidase 8
SEQ ID NOS: 7574-

7581

KLK9
Kallikrein-related peptidase 9
SEQ ID NOS: 7582-

7583

KLKB1
Kallikrein B, plasma (Fletcher factor) 1
SEQ ID NOS: 7584-

7588

KNDC1
Kinase non-catalytic C-lobe domain
SEQ ID NOS: 7593-

(KIND) containing 1
7594

KNG1
Kininogen 1
SEQ ID NOS: 7595-

7599

KRBA2
KRAB-A domain containing 2
SEQ ID NOS: 7600-

7603

KREMEN2
Kringle containing transmembrane protein 2
SEQ ID NOS: 7604-

7609

KRTDAP
Keratinocyte differentiation-associated
SEQ ID NOS: 7610-

protein
7611

L1CAM
L1 cell adhesion molecule
SEQ ID NOS: 7612-

7621

L3MBTL2
L(3)mbt-like 2 (Drosophila)
SEQ ID NOS: 7622-

7626

LA16c-

SEQ ID NO: 72

380H5.3

LACE1
Lactation elevated 1
SEQ ID NOS: 580-583

LACRT
Lacritin
SEQ ID NOS: 7627-

7629

LACTB
Lactamase, beta
SEQ ID NOS: 7630-

7632

LAG3
Lymphocyte-activation gene 3
SEQ ID NOS: 7633-

7634

LAIR2
Leukocyte-associated immunoglobulin-like
SEQ ID NOS: 7635-

receptor 2
7638

LALBA
Lactalbumin, alpha-
SEQ ID NOS: 7639-

7640

LAMA1
Laminin, alpha 1
SEQ ID NOS: 7641-

7642

LAMA2
Laminin, alpha 2
SEQ ID NOS: 7643-

7646

LAMA3
Laminin, alpha 3
SEQ ID NOS: 7647-

7656

LAMA4
Laminin, alpha 4
SEQ ID NOS: 7657-

7671

LAMA5
Laminin, alpha 5
SEQ ID NOS: 7672-

7674

LAMB1
Laminin, beta 1
SEQ ID NOS: 7675-

7679

LAMB2
Laminin, beta 2 (laminin S)
SEQ ID NOS: 7680-

7682

LAMB3
Laminin, beta 3
SEQ ID NOS: 7683-

7687

LAMB4
Laminin, beta 4
SEQ ID NOS: 7688-

7691

LAMC1
Laminin, gamma 1 (formerly LAMB2)
SEQ ID NOS: 7692-

7693

LAMC2
Laminin, gamma 2
SEQ ID NOS: 7694-

7695

LAMC3
Laminin, gamma 3
SEQ ID NOS: 7696-

7697

LAMP3
Lysosomal-associated membrane protein 3
SEQ ID NOS: 7698-

7701

LAT
Linker for activation of T cells
SEQ ID NOS: 7708-

7717

LAT2
Linker for activation of T cells family,
SEQ ID NOS: 7718-

member 2
7726

LBP
Lipopolysaccharide binding protein
SEQ ID NO: 7727

LCAT
Lecithin-cholesterol acyltransferase
SEQ ID NOS: 7728-

7734

LCN1
Lipocalin 1
SEQ ID NOS: 7735-

7736

LCN10
Lipocalin 10
SEQ ID NOS: 7737-

7742

LCN12
Lipocalin 12
SEQ ID NOS: 7743-

7745

LCN15
Lipocalin 15
SEQ ID NO: 7746

LCN2
Lipocalin 2
SEQ ID NOS: 7747-

7749

LCN6
Lipocalin 6
SEQ ID NOS: 7750-

7751

LCN8
Lipocalin 8
SEQ ID NOS: 7752-

7753

LCN9
Lipocalin 9
SEQ ID NOS: 7754-

7755

LCORL
Ligand dependent nuclear receptor
SEQ ID NOS: 7756-

corepressor-like
7761

LDLR
Low density lipoprotein receptor
SEQ ID NOS: 7762-

7770

LDLRAD2
Low density lipoprotein receptor class A
SEQ ID NOS: 7771-

domain containing 2
7772

LEAP2
Liver expressed antimicrobial peptide 2
SEQ ID NO: 7773

LECT2
Leukocyte cell-derived chemotaxin 2
SEQ ID NOS: 7774-

7777

LEFTY1
Left-right determination factor 1
SEQ ID NOS: 7778-

7779

LEFTY2
Left-right determination factor 2
SEQ ID NOS: 7780-

7781

LEP
Leptin
SEQ ID NO: 7782

LFNG
LFNG O-fucosylpeptide 3-beta-N-
SEQ ID NOS: 7783-

acetylglucosaminyltransferase
7788

LGALS3BP
Lectin, galactoside-binding, soluble, 3
SEQ ID NOS: 7789-

binding protein
7803

LGI1
Leucine-rich, glioma inactivated 1
SEQ ID NOS: 7804-

7822

LGI2
Leucine-rich repeat LGI family, member 2
SEQ ID NOS: 7823-

7824

LGI3
Leucine-rich repeat LGI family, member 3
SEQ ID NOS: 7825-

7828

LGI4
Leucine-rich repeat LGI family, member 4
SEQ ID NOS: 7829-

7832

LGMN
Legumain
SEQ ID NOS: 7833-

7846

LGR4
Leucine-rich repeat containing G protein-
SEQ ID NOS: 7847-

coupled receptor 4
7849

LHB
Luteinizing hormone beta polypeptide
SEQ ID NO: 7850

LHCGR
Luteinizing hormone/choriogonadotropin
SEQ ID NOS: 7851-

receptor
7855

LIF
Leukemia inhibitory factor
SEQ ID NOS: 7856-

7857

LIFR
Leukemia inhibitory factor receptor alpha
SEQ ID NOS: 7858-

7862

LILRA1
Leukocyte immunoglobulin-like receptor,
SEQ ID NOS: 7863-

subfamily A (with TM domain), member 1
7864

LILRA2
Leukocyte immunoglobulin-like receptor,
SEQ ID NOS: 7865-

subfamily A (with TM domain), member 2
7871

LILRB3
Leukocyte immunoglobulin-like receptor,
SEQ ID NOS: 7872-

subfamily B (with TM and ITIM domains),
7876

member 3

LIME1
Lek interacting transmembrane adaptor 1
SEQ ID NOS: 7877-

7882

LINGO1
Leucine rich repeat and Ig domain
SEQ ID NOS: 7883-

containing 1
7893

LIPA
Lipase A, lysosomal acid, cholesterol
SEQ ID NOS: 7894-

esterase
7898

LIPC
Lipase, hepatic
SEQ ID NOS: 7899-

7902

LIPF
Lipase, gastric
SEQ ID NOS: 7903-

7906

LIPG
Lipase, endothelial
SEQ ID NOS: 7907-

7912

LIPH
Lipase, member H
SEQ ID NOS: 7913-

7917

LIPK
Lipase, family member K
SEQ ID NO: 7918

LIPM
Lipase, family member M
SEQ ID NOS: 7919-

7920

LIPN
Lipase, family member N
SEQ ID NO: 7921

LMAN2
Lectin, mannose-binding 2
SEQ ID NOS: 7922-

7926

LMNTD1
Lamin tail domain containing 1
SEQ ID NOS: 7927-

7937

LNX1
Ligand of numb-protein X 1, E3 ubiquitin
SEQ ID NOS: 7938-

protein ligase
7944

LOX
Lysyl oxidase
SEQ ID NOS: 7945-

7947

LOXL1
Lysyl oxidase-like 1
SEQ ID NOS: 7948-

7949

LOXL2
Lysyl oxidase-like 2
SEQ ID NOS: 7950-

7958

LOXL3
Lysyl oxidase-like 3
SEQ ID NOS: 7959-

7965

LOXL4
Lysyl oxidase-like 4
SEQ ID NO: 7966

LPA
Lipoprotein, Lp(a)
SEQ ID NOS: 7967-

7969

LPL
Lipoprotein lipase
SEQ ID NOS: 7970-

7974

LPO
Lactoperoxidase
SEQ ID NOS: 7975-

7981

LRAT
Lecithin retinol acyltransferase
SEQ ID NOS: 7982-

(phosphatidylcholine--retinol O-
7984

acyltransferase)

LRCH3
Leucine-rich repeats and calponin
SEQ ID NOS: 7985-

homology (CH) domain containing 3
7993

LRCOL1
Leucine rich colipase-like 1
SEQ ID NOS: 7994-

7997

LRFN4
Leucine rich repeat and fibronectin type III
SEQ ID NOS: 7998-

domain containing 4
7999

LRFN5
Leucine rich repeat and fibronectin type III
SEQ ID NOS: 8000-

domain containing 5
8002

LRG1
Leucine-rich alpha-2-glycoprotein 1
SEQ ID NO: 8003

LRP1
Low density lipoprotein receptor-related
SEQ ID NOS: 8004-

protein 1
8009

LRP11
Low density lipoprotein receptor-related
SEQ ID NOS: 8010-

protein 11
8011

LRP1B
Low density lipoprotein receptor-related
SEQ ID NOS: 8012-

protein 1B
8015

LRP2
Low density lipoprotein receptor-related
SEQ ID NOS: 8016-

protein 2
8017

LRP4
Low density lipoprotein receptor-related
SEQ ID NOS: 8018-

protein 4
8019

LRPAP1
Low density lipoprotein receptor-related
SEQ ID NOS: 8020-

protein associated protein 1
8021

LRRC17
Leucine rich repeat containing 17
SEQ ID NOS: 8022-

8024

LRRC32
Leucine rich repeat containing 32
SEQ ID NOS: 8025-

8028

LRRC3B
Leucine rich repeat containing 3B
SEQ ID NOS: 8029-

8033

LRRC4B
Leucine rich repeat containing 4B
SEQ ID NOS: 8034-

8036

LRRC70
Leucine rich repeat containing 70
SEQ ID NOS: 8037-

8038

LRRN3
Leucine rich repeat neuronal 3
SEQ ID NOS: 8039-

8042

LRRTM1
Leucine rich repeat transmembrane
SEQ ID NOS: 8043-

neuronal 1
8049

LRRTM2
Leucine rich repeat transmembrane
SEQ ID NOS: 8050-

neuronal 2
8052

LRRTM4
Leucine rich repeat transmembrane
SEQ ID NOS: 8053-

neuronal 4
8058

LRTM2
Leucine-rich repeats and transmembrane
SEQ ID NOS: 8059-

domains 2
8063

LSR
Lipolysis stimulated lipoprotein receptor
SEQ ID NOS: 8064-

8074

LST1
Leukocyte specific transcript 1
SEQ ID NOS: 8075-

8092

LTA
Lymphotoxin alpha
SEQ ID NOS: 8093-

8094

LTBP1
Latent transforming growth factor beta
SEQ ID NOS: 8095-

binding protein 1
8104

LTBP2
Latent transforming growth factor beta
SEQ ID NOS: 8105-

binding protein 2
8108

LTBP3
Latent transforming growth factor beta
SEQ ID NOS: 8109-

binding protein 3
8121

LTBP4
Latent transforming growth factor beta
SEQ ID NOS: 8122-

binding protein 4
8137

LTBR
Lymphotoxin beta receptor (TNFR
SEQ ID NOS: 8138-

superfamily, member 3)
8143

LTF
Lactotransferrin
SEQ ID NOS: 8144-

8148

LTK
Leukocyte receptor tyrosine kinase
SEQ ID NOS: 8149-

8152

LUM
Lumican
SEQ ID NO: 8153

LUZP2
Leucine zipper protein 2
SEQ ID NOS: 8154-

8157

LVRN
Laeverin
SEQ ID NOS: 8158-

8163

LY6E
Lymphocyte antigen 6 complex, locus E
SEQ ID NOS: 8164-

8177

LY6G5B
Lymphocyte antigen 6 complex, locus G5B
SEQ ID NOS: 8178-

8179

LY6G6D
Lymphocyte antigen 6 complex, locus G6D
SEQ ID NOS: 8180-

8181

LY6G6E
Lymphocyte antigen 6 complex, locus G6E
SEQ ID NOS: 8182-

(pseudogene)
8185

LY6H
Lymphocyte antigen 6 complex, locus H
SEQ ID NOS: 8186-

8189

LY6K
Lymphocyte antigen 6 complex, locus K
SEQ ID NOS: 8190-

8193

LY86
Lymphocyte antigen 86
SEQ ID NOS: 8195-

8196

LY96
Lymphocyte antigen 96
SEQ ID NOS: 8197-

8198

LYG1
Lysozyme G-like 1
SEQ ID NOS: 8199-

8200

LYG2
Lysozyme G-like 2
SEQ ID NOS: 8201-

8206

LYNX1
Ly6/neurotoxin 1
SEQ ID NOS: 8207-

8211

LYPD1
LY6/PLAUR domain containing 1
SEQ ID NOS: 8212-

8214

LYPD2
LY6/PLAUR domain containing 2
SEQ ID NO: 8215

LYPD4
LY6/PLAUR domain containing 4
SEQ ID NOS: 8216-

8218

LYPD6
LY6/PLAUR domain containing 6
SEQ ID NOS: 8219-

8223

LYPD6B
LY6/PLAUR domain containing 6B
SEQ ID NOS: 8224-

8230

LYPD8
LY6/PLAUR domain containing 8
SEQ ID NOS: 8231-

8232

LYZ
Lysozyme
SEQ ID NOS: 8233-

8235

LYZL4
Lysozyme-like 4
SEQ ID NOS: 8236-

8237

LYZL6
Lysozyme-like 6
SEQ ID NOS: 8238-

8240

M6PR
Mannose-6-phosphate receptor (cation
SEQ ID NOS: 8241-

dependent)
8251

MAD1L1
MAD1 mitotic arrest deficient-like 1 (yeast)
SEQ ID NOS: 8252-

8264

MAG
Myelin associated glycoprotein
SEQ ID NOS: 8265-

8270

MAGT1
Magnesium transporter 1
SEQ ID NOS: 8271-

8274

MALSU1
Mitochondrial assembly of ribosomal large
SEQ ID NO: 8275

subunit 1

MAMDC2
MAM domain containing 2
SEQ ID NO: 8276

MAN2B1
Mannosidase, alpha, class 2B, member 1
SEQ ID NOS: 8277-

8282

MAN2B2
Mannosidase, alpha, class 2B, member 2
SEQ ID NOS: 8283-

8285

MANBA
Mannosidase, beta A, lysosomal
SEQ ID NOS: 8286-

8299

MANEAL
Mannosidase, endo-alpha-like
SEQ ID NOS: 8300-

8304

MANF
Mesencephalic astrocyte-derived
SEQ ID NOS: 8305-

neurotrophic factor
8306

MANSC1
MANSC domain containing 1
SEQ ID NOS: 8307-

8310

MAP3K9
Mitogen-activated protein kinase 9
SEQ ID NOS: 8311-

8316

MASP1
Mannan-binding lectin serine peptidase 1
SEQ ID NOS: 8317-

(C4/C2 activating component of Ra-reactive
8324

factor)

MASP2
Mannan-binding lectin serine peptidase 2
SEQ ID NOS: 8325-

8326

MATN1
Matrilin 1, cartilage matrix protein
SEQ ID NO: 8327

MATN2
Matrilin 2
SEQ ID NOS: 8328-

8340

MATN3
Matrilin 3
SEQ ID NOS: 8341-

8342

MATN4
Matrilin 4
SEQ ID NOS: 8343-

8347

MATR3
Matrin 3
SEQ ID NOS: 8348-

8375

MAU2
MAU2 sister chromatid cohesion factor
SEQ ID NOS: 8376-

8378

MAZ
MYC-associated zinc finger protein (purine-
SEQ ID NOS: 8379-

binding transcription factor)
8393

MBD6
Methyl-CpG binding domain protein 6
SEQ ID NOS: 8394-

8405

MBL2
Mannose-binding lectin (protein C) 2,
SEQ ID NO: 8406

soluble

MBNL1
Muscleblind-like splicing regulator 1
SEQ ID NOS: 8407-

8425

MCCC1
Methylcrotonoyl-CoA carboxylase 1 (alpha)
SEQ ID NOS: 8426-

8437

MCCD1
Mitochondrial coiled-coil domain 1
SEQ ID NO: 8438

MCEE
Methylmalonyl CoA epimerase
SEQ ID NOS: 8439-

8442

MCF2L
MCF.2 cell line derived transforming
SEQ ID NOS: 8443-

sequence-like
8464

MCFD2
Multiple coagulation factor deficiency 2
SEQ ID NOS: 8465-

8476

MDFIC
MyoD family inhibitor domain containing
SEQ ID NOS: 8477-

8484

MDGA1
MAM domain containing
SEQ ID NOS: 8485-

glycosylphosphatidylinositol anchor 1
8490

MDK
Midkine (neurite growth-promoting factor
SEQ ID NOS: 8491-

2)
8500

MED20
Mediator complex subunit 20
SEQ ID NOS: 8501-

8505

MEGF10
Multiple EGF-like-domains 10
SEQ ID NOS: 8506-

8509

MEGF6
Multiple EGF-like-domains 6
SEQ ID NOS: 8510-

8513

MEI1
Meiotic double-stranded break formation
SEQ ID NOS: 8514-

protein 1
8517

MEI4
Meiotic double-stranded break formation
SEQ ID NO: 8518

protein 4

MEIS1
Meis homeobox 1
SEQ ID NOS: 8519-

8524

MEIS3
Meis homeobox 3
SEQ ID NOS: 8525-

8534

MEPE
Matrix extracellular phosphoglycoprotein
SEQ ID NOS: 8538-

8544

MESDC2
Mesoderm development candidate 2
SEQ ID NOS: 8545-

8549

MEST
Mesoderm specific transcript
SEQ ID NOS: 8550-

8563

MET
MET proto-oncogene, receptor tyrosine
SEQ ID NOS: 8564-

kinase
8569

METRN
Meteorin, glial cell differentiation regulator
SEQ ID NOS: 8570-

8574

METRNL
Meteorin, glial cell differentiation regulator-
SEQ ID NOS: 8575-

like
8578

METTL17
Methyltransferase like 17
SEQ ID NOS: 8579-

8589

METTL24
Methyltransferase like 24
SEQ ID NO: 8590

METTL7B
Methyltransferase like 7B
SEQ ID NOS: 8591-

8592

METTL9
Methyltransferase like 9
SEQ ID NOS: 8593-

8601

MEX3C
Mex-3 RNA binding family member C
SEQ ID NOS: 8602-

8604

MFAP2
Microfibrillar-associated protein 2
SEQ ID NOS: 8605-

8606

MFAP3
Microfibrillar-associated protein 3
SEQ ID NOS: 8607-

8611

MFAP3L
Microfibrillar-associated protein 3-like
SEQ ID NOS: 8612-

8621

MFAP4
Microfibrillar-associated protein 4
SEQ ID NOS: 8622-

8624

MFAP5
Microfibrillar associated protein 5
SEQ ID NOS: 8625-

8635

MFGE8
Milk fat globule-EGF factor 8 protein
SEQ ID NOS: 8636-

8642

MFI2
Antigen p97 (melanoma associated)
SEQ ID NOS: 8535-

identified by monoclonal antibodies 133.2
8537

and 96.5

MFNG
MFNG O-fucosylpeptide 3-beta-N-
SEQ ID NOS: 8643-

acetylglucosaminyltransferase
8650

MGA
MGA, MAX dimerization protein
SEQ ID NOS: 8651-

8659

MGAT2
Mannosyl (alpha-1,6-)-glycoprotein beta-
SEQ ID NO: 8660

1,2-N-acetylglucosaminyltransferase

MGAT3
Mannosyl (beta-1,4-)-glycoprotein beta-1,4-
SEQ ID NOS: 8661-

N-acetylglucosaminyltransferase
8663

MGAT4A
Mannosyl (alpha-1,3-)-glycoprotein beta-
SEQ ID NOS: 8664-

1,4-N-acetylglucosaminyltransferase,
8668

isozyme A

MGAT4B
Mannosyl (alpha-1,3-)-glycoprotein beta-
SEQ ID NOS: 8669-

1,4-N-acetylglucosaminyltransferase,
8679

isozyme B

MGAT4D
MGAT4 family, member D
SEQ ID NOS: 8680-

8685

MGLL
Monoglyceride lipase
SEQ ID NOS: 8686-

8695

MGP
Matrix Gla protein
SEQ ID NOS: 8696-

8698

MGST2
Microsomal glutathione S-transferase 2
SEQ ID NOS: 8699-

8702

MIA
Melanoma inhibitory activity
SEQ ID NOS: 8703-

8708

MIA2
Melanoma inhibitory activity 2
SEQ ID NO: 8709

MIA3
Melanoma inhibitory activity family,
SEQ ID NOS: 8710-

member 3
8714

MICU1
Mitochondrial calcium uptake 1
SEQ ID NOS: 8715-

8724

MIER1
Mesoderm induction early response 1,
SEQ ID NOS: 8725-

transcriptional regulator
8733

MINOS1-
MINOS1-NBL1 readthrough
SEQ ID NOS: 8734-

NBL1

8736

MINPP1
Multiple inositol-polyphosphate
SEQ ID NOS: 8737-

phosphatase 1
8739

MLEC
Malectin
SEQ ID NOS: 8740-

8743

MLN
Motilin
SEQ ID NOS: 8744-

8746

MLXIP
MLX interacting protein
SEQ ID NOS: 8747-

8752

MLXIPL
MLX interacting protein-like
SEQ ID NOS: 8753-

8760

MMP1
Matrix metallopeptidase 1
SEQ ID NO: 8761

MMP10
Matrix metallopeptidase 10
SEQ ID NOS: 8762-

8763

MMP11
Matrix metallopeptidase 11
SEQ ID NOS: 8764-

8767

MMP12
Matrix metallopeptidase 12
SEQ ID NO: 8768

MMP13
Matrix metallopeptidase 13
SEQ ID NOS: 8769-

8771

MMP14
Matrix metallopeptidase 14 (membrane-
SEQ ID NOS: 8772-

inserted)
8774

MMP17
Matrix metallopeptidase 17 (membrane-
SEQ ID NOS: 8775-

inserted)
8782

MMP19
Matrix metallopeptidase 19
SEQ ID NOS: 8783-

8788

MMP2
Matrix metallopeptidase 2
SEQ ID NOS: 8789-

8796

MMP20
Matrix metallopeptidase 20
SEQ ID NO: 8797

MMP21
Matrix metallopeptidase 21
SEQ ID NO: 8798

MMP25
Matrix metallopeptidase 25
SEQ ID NOS: 8799-

8800

MMP26
Matrix metallopeptidase 26
SEQ ID NOS: 8801-

8802

MMP27
Matrix metallopeptidase 27
SEQ ID NO: 8803

MMP28
Matrix metallopeptidase 28
SEQ ID NOS: 8804-

8809

MMP3
Matrix metallopeptidase 3
SEQ ID NOS: 8810-

8812

MMP7
Matrix metallopeptidase 7
SEQ ID NO: 8813

MMP8
Matrix metallopeptidase 8
SEQ ID NOS: 8814-

8819

MMP9
Matrix metallopeptidase 9
SEQ ID NO: 8820

MMRN1
Multimerin 1
SEQ ID NOS: 8821-

8823

MMRN2
Multimerin 2
SEQ ID NOS: 8824-

8828

MOXD1
Monooxygenase, DBH-like 1
SEQ ID NOS: 8829-

8831

MPO
Myeloperoxidase
SEQ ID NOS: 8840-

8841

MPPED1
Metallophosphoesterase domain containing
SEQ ID NOS: 8842-

1
8845

MPZL1
Myelin protein zero-like 1
SEQ ID NOS: 8846-

8850

MR1
Major histocompatibility complex, class I-
SEQ ID NOS: 8851-

related
8856

MRPL2
Mitochondrial ribosomal protein L2
SEQ ID NOS: 8857-

8861

MRPL21
Mitochondrial ribosomal protein L21
SEQ ID NOS: 8862-

8868

MRPL22
Mitochondrial ribosomal protein L22
SEQ ID NOS: 8869-

8873

MRPL24
Mitochondrial ribosomal protein L24
SEQ ID NOS: 8874-

8878

MRPL27
Mitochondrial ribosomal protein L27
SEQ ID NOS: 8879-

8884

MRPL32
Mitochondrial ribosomal protein L32
SEQ ID NOS: 8885-

8887

MRPL34
Mitochondrial ribosomal protein L34
SEQ ID NOS: 8888-

8892

MRPL35
Mitochondrial ribosomal protein L35
SEQ ID NOS: 8893-

8896

MRPL52
Mitochondrial ribosomal protein L52
SEQ ID NOS: 8897-

8907

MRPL55
Mitochondrial ribosomal protein L55
SEQ ID NOS: 8908-

8933

MRPS14
Mitochondrial ribosomal protein S14
SEQ ID NOS: 8934-

8935

MRPS22
Mitochondrial ribosomal protein S22
SEQ ID NOS: 8936-

8944

MRPS28
Mitochondrial ribosomal protein S28
SEQ ID NOS: 8945-

8952

MS4A14
Membrane-spanning 4-domains, subfamily
SEQ ID NOS: 8953-

A, member 14
8963

MS4A3
Membrane-spanning 4-domains, subfamily
SEQ ID NOS: 8964-

A, member 3 (hematopoietic cell-specific)
8968

MSH3
MutS homolog 3
SEQ ID NO: 8969

MSH5
MutS homolog 5
SEQ ID NOS: 8970-

8981

MSLN
Mesothelin
SEQ ID NOS: 8982-

8989

MSMB
Microseminoprotein, beta-
SEQ ID NOS: 8990-

8991

MSRA
Methionine sulfoxide reductase A
SEQ ID NOS: 8992-

8999

MSRB2
Methionine sulfoxide reductase B2
SEQ ID NOS: 9000-

9001

MSRB3
Methionine sulfoxide reductase B3
SEQ ID NOS: 9002-

9015

MST1
Macrophage stimulating 1
SEQ ID NOS: 9016-

9017

MSTN
Myostatin
SEQ ID NO: 9018

MT1G
Metallothionein 1G
SEQ ID NOS: 9019-

9022

MTHFD2
Methylenetetrahy drofolate dehydrogenase
SEQ ID NOS: 9023-

(NADP+ dependent) 2,
9027

methenyltetrahydrofolate cyclohydrolase

MTMR14
Myotubularin related protein 14
SEQ ID NOS: 9028-

9038

MTRNR2L11
MT-RNR2-like 11 (pseudogene)
SEQ ID NO: 9039

MTRR
5-methyltetrahydrofolate-homocysteine
SEQ ID NOS: 9040-

methyltransferase reductase
9052

MTTP
Microsomal triglyceride transfer protein
SEQ ID NOS: 9053-

9063

MTX2
Metaxin 2
SEQ ID NOS: 9064-

9068

MUC1
Mucin 1, cell surface associated
SEQ ID NOS: 9069-

9094

MUC13
Mucin 13, cell surface associated
SEQ ID NOS: 9095-

9096

MUC20
Mucin 20, cell surface associated
SEQ ID NOS: 9097-

9101

MUC3A
Mucin 3A, cell surface associated
SEQ ID NOS: 9102-

9104

MUC5AC
Mucin 5AC, oligomeric mucus/gel-forming
SEQ ID NO: 9105

MUC5B
Mucin 5B, oligomeric mucus/gel-forming
SEQ ID NOS: 9106-

9107

MUC6
Mucin 6, oligomeric mucus/gel-forming
SEQ ID NOS: 9108-

9111

MUC7
Mucin 7, secreted
SEQ ID NOS: 9112-

9115

MUCL1
Mucin-like 1
SEQ ID NOS: 9116-

9118

MXRA5
Matrix-remodelling associated 5
SEQ ID NO: 9119

MXRA7
Matrix-remodelling associated 7
SEQ ID NOS: 9120-

9126

MYDGF
Myeloid-derived growth factor
SEQ ID NOS: 9127-

9129

MYL1
Myosin, light chain 1, alkali; skeletal, fast
SEQ ID NOS: 9130-

9131

MYOC
Myocilin, trabecular meshwork inducible
SEQ ID NOS: 9132-

glucocorticoid response
9133

MYRFL
Myelin regulatory factor-like
SEQ ID NOS: 9134-

9138

MZB1
Marginal zone B and B1 cell-specific
SEQ ID NOS: 9139-

protein
9143

N4BP2L2
NEDD4 binding protein 2-like 2
SEQ ID NOS: 9144-

9149

NAA38
N(alpha)-acetyltransferase 38, NatC
SEQ ID NOS: 9150-

auxiliary subunit
9155

NAAA
N-acylethanolamine acid amidase
SEQ ID NOS: 9156-

9161

NAGA
N-acetylgalactosaminidase, alpha-
SEQ ID NOS: 9162-

9164

NAGLU
N-acetylglucosaminidase, alpha
SEQ ID NOS: 9165-

9169

NAGS
N-acetylglutamate synthase
SEQ ID NOS: 9170-

9171

NAPSA
Napsin A aspartic peptidase
SEQ ID NOS: 9172-

9174

NBL1
Neuroblastoma 1, DAN family BMP
SEQ ID NOS: 9180-

antagonist
9193

NCAM1
Neural cell adhesion molecule 1
SEQ ID NOS: 9194-

9213

NCAN
Neurocan
SEQ ID NOS: 9214-

9215

NCBP2-AS2
NCBP2 antisense RNA 2 (head to head)
SEQ ID NO: 9216

NCSTN
Nicastrin
SEQ ID NOS: 9217-

9226

NDNF
Neuron-derived neurotrophic factor
SEQ ID NOS: 9227-

9229

NDP
Norrie disease (pseudoglioma)
SEQ ID NOS: 9230-

9232

NDUFA10
NADH dehydrogenase (ubiquinone) 1 alpha
SEQ ID NOS: 9233-

subcomplex, 10, 42 kDa
9242

NDUFB5
NADH dehydrogenase (ubiquinone) 1 beta
SEQ ID NOS: 9243-

subcomplex, 5, 16 kDa
9251

NDUFS8
NADH dehydrogenase (ubiquinone) Fe—S
SEQ ID NOS: 9252-

protein 8, 23 kDa (NADH-coenzyme Q
9261

reductase)

NDUFV1
NADH dehydrogenase (ubiquinone)
SEQ ID NOS: 9262-

flavoprotein 1, 51 kDa
9275

NECAB3
N-terminal EF-hand calcium binding
SEQ ID NOS: 9276-

protein 3
9285

NELL1
Neural EGFL like 1
SEQ ID NOS: 9289-

9292

NELL2
Neural EGFL like 2
SEQ ID NOS: 9293-

9307

NENF
Neudesin neurotrophic factor
SEQ ID NO: 9308

NETO1
Neuropilin (NRP) and tolloid (TLL)-like 1
SEQ ID NOS: 9309-

9312

NFASC
Neurofascin
SEQ ID NOS: 9313-

9327

NFE2L1
Nuclear factor, erythroid 2-like 1
SEQ ID NOS: 9328-

9346

NFE2L3
Nuclear factor, erythroid 2-like 3
SEQ ID NOS: 9347-

9348

NGEF
Neuronal guanine nucleotide exchange
SEQ ID NOS: 9349-

factor
9354

NGF
Nerve growth factor (beta polypeptide)
SEQ ID NO: 9355

NGLY1
N-glycanase 1
SEQ ID NOS: 9356-

9362

NGRN
Neugrin, neurite outgrowth associated
SEQ ID NOS: 9363-

9364

NHLRC3
NHL repeat containing 3
SEQ ID NOS: 9365-

9367

NIDI
Nidogen 1
SEQ ID NOS: 9368-

9369

NID2
Nidogen 2 (osteonidogen)
SEQ ID NOS: 9370-

9372

NKG7
Natural killer cell granule protein 7
SEQ ID NOS: 9373-

9377

NLGN3
Neuroligin 3
SEQ ID NOS: 9378-

9382

NLGN4Y
Neuroligin 4, Y-linked
SEQ ID NOS: 9383-

9389

NLRP5
NLR family, pyrin domain containing 5
SEQ ID NOS: 9390-

9392

NMB
Neuromedin B
SEQ ID NOS: 9393-

9394

NME1
NME/NM23 nucleoside diphosphate kinase
SEQ ID NOS: 9395-

1
9401

NME1-NME2
NME1-NME2 readthrough
SEQ ID NOS: 9402-

9404

NME3
NME/NM23 nucleoside diphosphate kinase
SEQ ID NOS: 9405-

3
9409

NMS
Neuromedin S
SEQ ID NO: 9410

NMU
Neuromedin U
SEQ ID NOS: 9411-

9414

NOA1
Nitric oxide associated 1
SEQ ID NO: 9415

NODAL
Nodal growth differentiation factor
SEQ ID NOS: 9416-

9417

NOG
Noggin
SEQ ID NO: 9418

NOMO3
NODAL modulator 3
SEQ ID NOS: 9419-

9425

NOS1AP
Nitric oxide synthase 1 (neuronal) adaptor
SEQ ID NOS: 9426-

protein
9430

NOTCH3
Notch 3
SEQ ID NOS: 9431-

9434

NOTUM
Notum pectinacetylesterase homolog
SEQ ID NOS: 9435-

(Drosophila)
9437

NOV
Nephroblastoma overexpressed
SEQ ID NO: 9438

NPB
Neuropeptide B
SEQ ID NOS: 9439-

9440

NPC2
Niemann-Pick disease, type C2
SEQ ID NOS: 9441-

9449

NPFF
Neuropeptide FF-amide peptide precursor
SEQ ID NO: 9450

NPFFR2
Neuropeptide FF receptor 2
SEQ ID NOS: 9451-

9454

NPHS1
Nephrosis 1, congenital, Finnish type
SEQ ID NOS: 9455-

(nephrin)
9456

NPNT
Nephronectin
SEQ ID NOS: 9457-

9467

NPPA
Natriuretic peptide A
SEQ ID NOS: 9468-

9470

NPPB
Natriuretic peptide B
SEQ ID NO: 9471

NPPC
Natriuretic peptide C
SEQ ID NOS: 9472-

9473

NPS
Neuropeptide S
SEQ ID NO: 9474

NPTX1
Neuronal pentraxin I
SEQ ID NO: 9475

NPTX2
Neuronal pentraxin II
SEQ ID NO: 9476

NPTXR
Neuronal pentraxin receptor
SEQ ID NOS: 9477-

9478

NPVF
Neuropeptide VF precursor
SEQ ID NO: 9479

NPW
Neuropeptide W
SEQ ID NOS: 9480-

9482

NPY
Neuropeptide Y
SEQ ID NOS: 9483-

9485

NQO2
NAD(P)H dehydrogenase, quinone 2
SEQ ID NOS: 9486-

9494

NRCAM
Neuronal cell adhesion molecule
SEQ ID NOS: 9495-

9507

NRG1
Neuregulin 1
SEQ ID NOS: 9508-

9525

NRN1L
Neuritin 1-like
SEQ ID NOS: 9526-

9528

NRP1
Neuropilin 1
SEQ ID NOS: 9529-

9542

NRP2
Neuropilin 2
SEQ ID NOS: 9543-

9549

NRTN
Neurturin
SEQ ID NO: 9550

NRXN1
Neurexin 1
SEQ ID NOS: 9551-

9581

NRXN2
Neurexin 2
SEQ ID NOS: 9582-

9590

NT5C3A
5′-nucleotidase, cytosolic IIIA
SEQ ID NOS: 9591-

9601

NT5DC3
5′-nucleotidase domain containing 3
SEQ ID NOS: 9602-

9604

NT5E
5′-nucleotidase, ecto (CD73)
SEQ ID NOS: 9605-

9609

NTF3
Neurotrophin 3
SEQ ID NOS: 9610-

9611

NTF4
Neurotrophin 4
SEQ ID NOS: 9612-

9613

NTM
Neurotrimin
SEQ ID NOS: 9614-

9623

NTN1
Netrin 1
SEQ ID NOS: 9624-

9625

NTN3
Netrin 3
SEQ ID NO: 9626

NTN4
Netrin 4
SEQ ID NOS: 9627-

9631

NTN5
Netrin 5
SEQ ID NOS: 9632-

9633

NTNG1
Netrin G1
SEQ ID NOS: 9634-

9640

NTNG2
Netrin G2
SEQ ID NOS: 9641-

9642

NTS
Neurotensin
SEQ ID NOS: 9643-

9644

NUBPL
Nucleotide binding proteindike
SEQ ID NOS: 9645-

9651

NUCB1
Nucleobindin 1
SEQ ID NOS: 9652-

9658

NUCB2
Nucleobindin 2
SEQ ID NOS: 9659-

9674

NUDT19
Nudix (nucleoside diphosphate linked
SEQ ID NO: 9675

moiety X)-type motif 19

NUDT9
Nudix (nucleoside diphosphate linked
SEQ ID NOS: 9676-

moiety X)-type motif 9
9680

NUP155
Nucleoporin 155 kDa
SEQ ID NOS: 9681-

9684

NUP214
Nucleoporin 214 kDa
SEQ ID NOS: 9685-

9696

NUP85
Nucleoporin 85 kDa
SEQ ID NOS: 9697-

9711

NXPE3
Neurexophilin and PC-esterase domain
SEQ ID NOS: 9712-

family, member 3
9716

NXPE4
Neurexophilin and PC-esterase domain
SEQ ID NOS: 9717-

family, member 4
9718

NXPH1
Neurexophilin 1
SEQ ID NOS: 9719-

9722

NXPH2
Neurexophilin 2
SEQ ID NO: 9723

NXPH3
Neurexophilin 3
SEQ ID NOS: 9724-

9725

NXPH4
Neurexophilin 4
SEQ ID NOS: 9726-

9727

NYX
Nyctalopin
SEQ ID NOS: 9728-

9729

OAF
Out at first homolog
SEQ ID NOS: 9730-

9731

OBP2A
Odorant binding protein 2A
SEQ ID NOS: 9732-

9738

OBP2B
Odorant binding protein 2B
SEQ ID NOS: 9739-

9742

OC90
Otoconin 90
SEQ ID NO: 9743

OCLN
Occludin
SEQ ID NOS: 9744-

9746

ODAM
Odontogenic, ameloblast asssociated
SEQ ID NOS: 9747-

9750

OGG1
8-oxoguanine DNA glvcosylase
SEQ ID NOS: 9755-

9768

OGN
Osteoglycin
SEQ ID NOS: 9769-

9771

OIT3
Oncoprotein induced transcript 3
SEQ ID NOS: 9772-

9773

OLFM1
Olfactomedin 1
SEQ ID NOS: 9774-

9784

OLFM2
Olfactomedin 2
SEQ ID NOS: 9785-

9788

OLFM3
Olfactomedin 3
SEQ ID NOS: 9789-

9791

OLFM4
Olfactomedin 4
SEQ ID NO: 9792

OLFML1
Olfactomedin-like 1
SEQ ID NOS: 9793-

9796

OLFML2A
Olfactomedin-like 2A
SEQ ID NOS: 9797-

9799

OLFML2B
Olfactomedin-like 2B
SEQ ID NOS: 9800-

9804

OLFML3
Olfactomedin-like 3
SEQ ID NOS: 9805-

9807

OMD
Osteomodulin
SEQ ID NO: 9808

OMG
Oligodendrocyte myelin glycoprotein
SEQ ID NO: 9809

OOSP2
Oocyte secreted protein 2
SEQ ID NOS: 9810-

9811

OPCML
Opioid binding protein/cell adhesion
SEQ ID NOS: 9812-

molecule-like
9816

OPTC
Opticin
SEQ ID NOS: 9818-

9819

ORAI1
ORAI calcium release-activated calcium
SEQ ID NO: 9820

modulator 1

ORM1
Orosomucoid 1
SEQ ID NO: 9821

ORM2
Orosomucoid 2
SEQ ID NO: 9822

ORMDL2
ORMDL sphingolipid biosynthesis
SEQ ID NOS: 9823-

regulator 2
9826

OS9
Osteosarcoma amplified 9, endoplasmic
SEQ ID NOS: 9827-

reticulum lectin
9841

OSCAR
Osteoclast associated, immunoglobulin-like
SEQ ID NOS: 9842-

receptor
9852

OSM
Oncostatin M
SEQ ID NOS: 9853-

9855

OSMR
Oncostatin M receptor
SEQ ID NOS: 9856-

9860

OSTN
Osteocrin
SEQ ID NOS: 9861-

9862

OTOA
Otoancorin
SEQ ID NOS: 9863-

9868

OTOG
Otogelin
SEQ ID NOS: 9869-

9871

OTOGL
Otogelin-like
SEQ ID NOS: 9872-

9878

OTOL1
Otolin 1
SEQ ID NO: 9879

OTOR
Otoraplin
SEQ ID NO: 9880

OTOS
Otospiralin
SEQ ID NOS: 9881-

9882

OVCH1
Ovochymase 1
SEQ ID NOS: 9883-

9885

OVCH2
Ovochymase 2 (gene/pseudogene)
SEQ ID NOS: 9886-

9887

OVGP1
Oviductal glycoprotein 1, 120 kDa
SEQ ID NO: 9888

OXCT1
3-oxoacid CoA transferase 1
SEQ ID NOS: 9889-

9892

OXCT2
3-oxoacid CoA transferase 2
SEQ ID NO: 9893

OXNAD1
Oxidoreductase NAD-binding domain
SEQ ID NOS: 9894-

containing 1
9900

OXT
Oxytocin/neurophysin I prepropeptide
SEQ ID NO: 9901

P3H1
Prolyl 3-hydroxylase 1
SEQ ID NOS: 9902-

9906

P3H2
Prolyl 3-hydroxylase 2
SEQ ID NOS: 9907-

9910

P3H3
Prolyl 3-hydroxylase 3
SEQ ID NO: 9911

P3H4
Prolyl 3-hydroxylase family member 4
SEQ ID NOS: 9912-

(non-enzymatic)
9916

P4HA1
Prolyl 4-hydroxylase, alpha polypeptide I
SEQ ID NOS: 9917-

9921

P4HA2
Prolyl 4-hydroxylase, alpha polypeptide II
SEQ ID NOS: 9922-

9936

P4HA3
Prolyl 4-hydroxylase, alpha polypeptide III
SEQ ID NOS: 9937-

9941

P4HB
Prolyl 4-hydroxylase, beta polypeptide
SEQ ID NOS: 9942-

9953

PAEP
Progestagen-associated endometrial protein
SEQ ID NOS: 9954-

9962

PAM
Peptidylglycine alpha-amidating
SEQ ID NOS: 9963-

monooxygenase
9976

PAMR1
Peptidase domain containing associated
SEQ ID NOS: 9977-

with muscle regeneration 1
9983

PAPL
Iron/zinc purple acid phosphatase-like
SEQ ID NOS: 159-162

protein

PAPLN
Papilin, proteoglycan-like sulfated
SEQ ID NOS: 9984-

glycoprotein
9991

PAPPA
Pregnancy-associated plasma protein A,
SEQ ID NO: 9992

pappalysin 1

PAPPA2
Pappalysin 2
SEQ ID NOS: 9993-

9994

PARP15
Poly (ADP-ribose) polymerase family,
SEQ ID NOS: 9995-

member 15
9998

PARVB
Parvin, beta
SEQ ID NOS: 9999-

10003

PATE1
Prostate and testis expressed 1
SEQ ID NOS: 10004-

10005

PATE2
Prostate and testis expressed 2
SEQ ID NOS: 10006-

10007

PATE3
Prostate and testis expressed 3
SEQ ID NO: 10008

PATE4
Prostate and testis expressed 4
SEQ ID NOS: 10009-

10010

PATL2
Protein associated with topoisomerase II
SEQ ID NOS: 10011-

homolog 2 (yeast)
10016

PAX2
Paired box 2
SEQ ID NOS: 10017-

10022

PAX4
Paired box 4
SEQ ID NOS: 10023-

10029

PCCB
Propionyl CoA carboxylase, beta
SEQ ID NOS: 10030-

polypeptide
10044

PCDH1
Protocadherin 1
SEQ ID NOS: 10045-

10050

PCDH12
Protocadherin 12
SEQ ID NOS: 10051-

10052

PCDH15
Protocadherin-related 15
SEQ ID NOS: 10053-

10086

PCDHA1
Protocadherin alpha 1
SEQ ID NOS: 10087-

10089

PCDHA10
Protocadherin alpha 10
SEQ ID NOS: 10090-

10092

PCDHA11
Protocadherin alpha 11
SEQ ID NOS: 10093-

10095

PCDHA6
Protocadherin alpha 6
SEQ ID NOS: 10096-

10098

PCDHB12
Protocadherin beta 12
SEQ ID NOS: 10099-

10101

PCDHGA11
Protocadherin gamma subfamily A, 11
SEQ ID NOS: 10102-

10104

PCF11
PCF11 cleavage and polyadenylation factor
SEQ ID NOS: 10105-

subunit
10109

PCOLCE
Procollagen C-endopeptidase enhancer
SEQ ID NO: 10110

PCOLCE2
Procollagen C-endopeptidase enhancer 2
SEQ ID NOS: 10111-

10114

PCSK1
Proprotein convertase subtilisin/kexin type
SEQ ID NOS: 10115-

1
10117

PCSK1N
Proprotein convertase subtilisin/kexin type
SEQ ID NO: 10118

1 inhibitor

PCSK2
Proprotein convertase subtilisin/kexin type
SEQ ID NOS: 10119-

2
10121

PCSK4
Proprotein convertase subtilisin/kexin type
SEQ ID NOS: 10122-

4
10124

PCSK5
Proprotein convertase subtilisin/kexin type
SEQ ID NOS: 10125-

5
10129

PCSK9
Proprotein convertase subtilisin/kexin type
SEQ ID NO: 10130

9

PCYOX1
Prenylcysteine oxidase 1
SEQ ID NOS: 10131-

10135

PCYOX1L
Prenylcysteine oxidase 1 like
SEQ ID NOS: 10136-

10140

PDDC1
Parkinson disease 7 domain containing 1
SEQ ID NOS: 5802-

5810

PDE11A
Phosphodiesterase 11A
SEQ ID NOS: 10141-

10146

PDE2A
Phosphodiesterase 2A, cGMP-stimulated
SEQ ID NOS: 10147-

10168

PDE7A
Phosphodiesterase 7A
SEQ ID NOS: 10169-

10172

PDF
Peptide deformylase (mitochondrial)
SEQ ID NO: 10173

PDGFA
Platelet-derived growth factor alpha
SEQ ID NOS: 10174-

polypeptide
10177

PDGFB
Platelet-derived growth factor beta
SEQ ID NOS: 10178-

polypeptide
10181

PDGFC
Platelet derived growth factor C
SEQ ID NOS: 10182-

10185

PDGFD
Platelet derived growth factor D
SEQ ID NOS: 10186-

10188

PDGFRA
Platelet-derived growth factor receptor,
SEQ ID NOS: 10189-

alpha polypeptide
10195

PDGFRB
Platelet-derived growth factor receptor, beta
SEQ ID NOS: 10196-

polypeptide
10199

PDGFRL
Platelet-derived growth factor receptor-like
SEQ ID NOS: 10200-

10201

PDHA1
Pyruvate dehydrogenase (lipoamide) alpha
SEQ ID NOS: 10202-

1
10210

PDIA2
Protein disulfide isomerase family A,
SEQ ID NOS: 10211-

member 2
10214

PDIA3
Protein disulfide isomerase family A,
SEQ ID NOS: 10215-

member 3
10218

PDIA4
Protein disulfide isomerase family A,
SEQ ID NOS: 10219-

member 4
10220

PDIA5
Protein disulfide isomerase family A,
SEQ ID NOS: 10221-

member 5
10224

PDIA6
Protein disulfide isomerase family A,
SEQ ID NOS: 10225-

member 6
10231

PDILT
Protein disulfide isomerase-like, testis
SEQ ID NOS: 10232-

expressed
10233

PDYN
Prodynorphin
SEQ ID NOS: 10234-

10236

PDZD8
PDZ domain containing 8
SEQ ID NO: 10237

PDZRN4
PDZ domain containing ring finger 4
SEQ ID NOS: 10238-

10240

PEAR1
Platelet endothelial aggregation receptor 1
SEQ ID NOS: 10241-

10244

PEBP4
Phosphatidylethanolamine-binding protein 4
SEQ ID NOS: 10245-

10246

PECAM1
Platelet/endothelial cell adhesion molecule
SEQ ID NOS: 10247-

1
10250

PENK
Proenkephalin
SEQ ID NOS: 10251-

10256

PET117
PET117 homolog
SEQ ID NO: 10257

PF4
Platelet factor 4
SEQ ID NO: 10258

PF4V1
Platelet factor 4 variant 1
SEQ ID NO: 10259

PFKP
Phosphofructokinase, platelet
SEQ ID NOS: 10260-

10268

PFN1
Profilin 1
SEQ ID NOS: 10269-

10271

PGA3
Pepsinogen 3, group I (pepsinogen A)
SEQ ID NOS: 10272-

10275

PGA4
Pepsinogen 4, group I (pepsinogen A)
SEQ ID NOS: 10276-

10278

PGA5
Pepsinogen 5, group I (pepsinogen A)
SEQ ID NOS: 10279-

10281

PGAM5
PGAM family member 5, serine/threonine
SEQ ID NOS: 10282-

protein phosphatase, mitochondrial
10285

PGAP3
Post-GPI attachment to proteins 3
SEQ ID NOS: 10286-

10293

PGC
Progastricsin (pepsinogen C)
SEQ ID NOS: 10294-

10297

PGF
Placental growth factor
SEQ ID NOS: 10298-

10301

PGLYRP1
Peptidoglycan recognition protein 1
SEQ ID NO: 10302

PGLYRP2
Peptidoglycan recognition protein 2
SEQ ID NOS: 10303-

10306

PGLYRP3
Peptidoglycan recognition protein 3
SEQ ID NO: 10307

PGLYRP4
Peptidoglycan recognition protein 4
SEQ ID NOS: 10308-

10309

PHACTR1
Phosphatase and actin regulator 1
SEQ ID NOS: 10310-

10316

PHB
Prohibitin
SEQ ID NOS: 10317-

10325

PI15
Peptidase inhibitor 15
SEQ ID NOS: 10326-

10327

PI3
Peptidase inhibitor 3, skin-derived
SEQ ID NO: 10328

PIANP
PILR alpha associated neural protein
SEQ ID NOS: 10329-

10334

PIGK
Phosphatidylinositol glycan anchor
SEQ ID NOS: 10335-

biosynthesis, class K
10338

PIGL
Phosphatidylinositol glycan anchor
SEQ ID NOS: 10339-

biosynthesis, class L
10346

PIGT
Phosphatidylinositol glycan anchor
SEQ ID NOS: 10347-

biosynthesis, class T
10400

PIGZ
Phosphatidylinositol glycan anchor
SEQ ID NOS: 10401-

biosynthesis, class Z
10403

PIK3AP1
Phosphoinositide-3-kinase adaptor protein 1
SEQ ID NOS: 10404-

10406

PIK3IP1
Phosphoinositide-3-kinase interacting
SEQ ID NOS: 10407-

protein 1
10410

PILRA
Paired immunoglobin-like type 2 receptor
SEQ ID NOS: 10411-

alpha
10415

PILRB
Paired immunoglobin-like type 2 receptor
SEQ ID NOS: 10416-

beta
10427

PINLYP
Phospholipase A2 inhibitor and
SEQ ID NOS: 10428-

LY6/PLAUR domain containing
10432

PIP
Prolactin-induced protein
SEQ ID NO: 10433

PIWIL4
Piwi-like RNA-mediated gene silencing 4
SEQ ID NOS: 10434-

10438

PKDCC
Protein kinase domain containing,
SEQ ID NOS: 10439-

cytoplasmic
10440

PKHD1
Polycystic kidney and hepatic disease 1
SEQ ID NOS: 10441-

(autosomal recessive)
10442

PLA1A
Phospholipase A1 member A
SEQ ID NOS: 10443-

10447

PLA2G10
Phospholipase A2, group X
SEQ ID NOS: 10448-

10449

PLA2G12A
Phospholipase A2, group XIIA
SEQ ID NOS: 10450-

10452

PLA2G12B
Phospholipase A2, group XIIB
SEQ ID NO: 10453

PLA2G15
Phospholipase A2, group XV
SEQ ID NOS: 10454-

10461

PLA2G1B
Phospholipase A2, group IB (pancreas)
SEQ ID NOS: 10462-

10464

PLA2G2A
Phospholipase A2, group IIA (platelets,
SEQ ID NOS: 10465-

synovial fluid)
10466

PLA2G2C
Phospholipase A2, group IIC
SEQ ID NOS: 10467-

10468

PLA2G2D
Phospholipase A2, group IID
SEQ ID NOS: 10469-

10470

PLA2G2E
Phospholipase A2, group IIE
SEQ ID NO: 10471

PLA2G3
Phospholipase A2, group III
SEQ ID NO: 10472

PLA2G5
Phospholipase A2, group V
SEQ ID NO: 10473

PLA2G7
Phospholipase A2, group VII (platelet-
SEQ ID NOS: 10474-

activating factor acetylhydrolase, plasma)
10475

PLA2R1
Phospholipase A2 receptor 1, 180 kDa
SEQ ID NOS: 10476-

10477

PLAC1
Placenta-specific 1
SEQ ID NO: 10478

PLAC9
Placenta-specific 9
SEQ ID NOS: 10479-

10481

PLAT
Plasminogen activator, tissue
SEQ ID NOS: 10482-

10490

PLAU
Plasminogen activator, urokinase
SEQ ID NOS: 10491-

10493

PLAUR
Plasminogen activator, urokinase receptor
SEQ ID NOS: 10494-

10505

PLBD1
Phospholipase B domain containing 1
SEQ ID NOS: 10506-

10508

PLBD2
Phospholipase B domain containing 2
SEQ ID NOS: 10509-

10511

PLG
Plasminogen
SEQ ID NOS: 10512-

10514

PLGLB1
Plasminogen-like B1
SEQ ID NOS: 10515-

10518

PLGLB2
Plasminogen-like B2
SEQ ID NOS: 10519-

10520

PLOD1
Procollagen-lysine, 2-oxoglutarate 5-
SEQ ID NOS: 10521-

dioxygenase 1
10523

PLOD2
Procollagen-lysine, 2-oxoglutarate 5-
SEQ ID NOS: 10524-

dioxygenase 2
10529

PLOD3
Procollagen-lysine, 2-oxoglutarate 5-
SEQ ID NOS: 10530-

dioxygenase 3
10536

PLTP
Phospholipid transfer protein
SEQ ID NOS: 10537-

10541

PLXNA4
Plexin A4
SEQ ID NOS: 10542-

10545

PLXNB2
Plexin B2
SEQ ID NOS: 10546-

10554

PM20D1
Peptidase M20 domain containing 1
SEQ ID NO: 10555

PMCH
Pro-melanin-concentrating hormone
SEQ ID NO: 10556

PMEL
Premelanosome protein
SEQ ID NOS: 10557-

10568

PMEPA1
Prostate transmembrane protein, androgen
SEQ ID NOS: 10569-

induced 1
10575

PNLIP
Pancreatic lipase
SEQ ID NO: 10576

PNLIPRP1
Pancreatic lipase-related protein 1
SEQ ID NOS: 10577-

10585

PNLIPRP3
Pancreatic lipase-related protein 3
SEQ ID NO: 10586

PNOC
Prepronociceptin
SEQ ID NOS: 10587-

10589

PNP
Purine nucleoside phosphorylase
SEQ ID NOS: 10590-

10593

PNPLA4
Patatin-like phospholipase domain
SEQ ID NOS: 10594-

containing 4
10597

PODNL1
Podocan-like 1
SEQ ID NOS: 10598-

10609

POFUT1
Protein O-fucosyltransferase 1
SEQ ID NOS: 10610-

10611

POFUT2
Protein O-fucosyltransferase 2
SEQ ID NOS: 10612-

10617

POGLUT1
Protein O-glucosyltransferase 1
SEQ ID NOS: 10618-

10622

POLL
Polymerase (DNA directed), lambda
SEQ ID NOS: 10623-

10635

POMC
Proopiomelanocortin
SEQ ID NOS: 10636-

10640

POMGNT2
Protein O-linked mannose N-
SEQ ID NOS: 10641-

acetylglucosaminyltransferase 2 (beta 1,4-)
10642

PON1
Paraoxonase 1
SEQ ID NOS: 10643-

10644

PON2
Paraoxonase 2
SEQ ID NOS: 10645-

10657

PON3
Paraoxonase 3
SEQ ID NOS: 10658-

10663

POSTN
Periostin, osteoblast specific factor
SEQ ID NOS: 10664-

10669

PPBP
Pro-platelet basic protein (chemokine (C-X-
SEQ ID NO: 10670

C motif) ligand 7)

PPIB
Peptidylprolyl isomerase B (cyclophilin B)
SEQ ID NO: 10671

PPIC
Peptidylprolyl isomerase C (cyclophilin C)
SEQ ID NO: 10672

PPOX
Protoporphyrinogen oxidase
SEQ ID NOS: 10673-

10683

PPP1CA
Protein phosphatase 1, catalytic subunit,
SEQ ID NOS: 10684-

alpha isozyme
10689

PPT1
Palmitoyl-protein thioesterase 1
SEQ ID NOS: 10690-

10706

PPT2
Palmitoyl-protein thioesterase 2
SEQ ID NOS: 10707-

10714

PPY
Pancreatic polypeptide
SEQ ID NOS: 10715-

10719

PRAC2
Prostate cancer susceptibility candidate 2
SEQ ID NOS: 10720-

10721

PRADC1
Protease-associated domain containing 1
SEQ ID NO: 10722

PRAP1
Proline-rich acidic protein 1
SEQ ID NOS: 10723-

10724

PRB1
Proline-rich protein BstNI subfamily 1
SEQ ID NOS: 10725-

10728

PRB2
Proline-rich protein BstNI subfamily 2
SEQ ID NOS: 10729-

10730

PRB3
Proline-rich protein BstNI subfamily 3
SEQ ID NOS: 10731-

10732

PRB4
Proline-rich protein BstNI subfamily 4
SEQ ID NOS: 10733-

10736

PRCD
Progressive rod-cone degeneration
SEQ ID NOS: 10737-

10738

PRCP
Prolylcarboxypeptidase (angiotensinase C)
SEQ ID NOS: 10739-

10750

PRDM12
PR domain containing 12
SEQ ID NO: 10751

PRDX4
Peroxiredoxin 4
SEQ ID NOS: 10752-

10755

PRELP
Proline/arginine-rich end leucine-rich repeat
SEQ ID NO: 10756

protein

PRF1
Perforin 1 (pore forming protein)
SEQ ID NOS: 10757-

10759

PRG2
Proteoglycan 2, bone marrow (natural killer
SEQ ID NOS: 10760-

cell activator, eosinophil granule major
10762

basic protein)

PRG3
Proteoglycan 3
SEQ ID NO: 10763

PRG4
Proteoglycan 4
SEQ ID NOS: 10764-

10769

PRH1
Proline-rich protein HaeIII subfamily 1
SEQ ID NOS: 10770-

10772

PRH2
Proline-rich protein HaeIII subfamily 2
SEQ ID NOS: 10773-

10774

PRKAG1
Protein kinase, AMP-activated, gamma 1
SEQ ID NOS: 10775-

non-catalytic subunit
10789

PRKCSH
Protein kinase C substrate 80K-H
SEQ ID NOS: 10790-

10799

PRKD1
Protein kinase D1
SEQ ID NOS: 10800-

10805

PRL
Prolactin
SEQ ID NOS: 10806-

10808

PRLH
Prolactin releasing hormone
SEQ ID NO: 10809

PRLR
Prolactin receptor
SEQ ID NOS: 10810-

10828

PRNP
Prion protein
SEQ ID NOS: 10829-

10832

PRNT
Prion protein (testis specific)
SEQ ID NO: 10833

PROC
Protein C (inactivator of coagulation factors
SEQ ID NOS: 10834-

Va and VIIIa)
10841

PROK1
Prokineticin 1
SEQ ID NO: 10842

PROK2
Prokineticin 2
SEQ ID NOS: 10843-

10844

PROL1
Proline rich, lacrimal 1
SEQ ID NO: 9817

PROM1
Prominin 1
SEQ ID NOS: 10845-

10856

PROS1
Protein S (alpha)
SEQ ID NOS: 10857-

10860

PROZ
Protein Z, vitamin K-dependent plasma
SEQ ID NOS: 10861-

glycoprotein
10862

PRR27
Proline rich 27
SEQ ID NOS: 10863-

10866

PRR4
Proline rich 4 (lacrimal)
SEQ ID NOS: 10867-

10869

PRRG2
Proline rich Gla (G-carboxyglutamic acid) 2
SEQ ID NOS: 10870-

10872

PRRT3
Proline-rich transmembrane protein 3
SEQ ID NOS: 10873-

10875

PRRT4
Proline-rich transmembrane protein 4
SEQ ID NOS: 10876-

10882

PRSS1
Protease, serine, 1 (trypsin 1)
SEQ ID NOS: 10883-

10886

PRSS12
Protease, serine, 12 (neurotrypsin,
SEQ ID NO: 10887

motopsin)

PRSS16
Protease, serine, 16 (thymus)
SEQ ID NOS: 10888-

10895

PRSS2
Protease, serine, 2 (trypsin 2)
SEQ ID NOS: 10896-

10899

PRSS21
Protease, serine, 21 (testisin)
SEQ ID NOS: 10900-

10905

PRSS22
Protease, serine, 22
SEQ ID NOS: 10906-

10908

PRSS23
Protease, serine, 23
SEQ ID NOS: 10909-

10912

PRSS27
Protease, serine 27
SEQ ID NOS: 10913-

10915

PRSS3
Protease, serine, 3
SEQ ID NOS: 10916-

10920

PRSS33
Protease, serine, 33
SEQ ID NOS: 10921-

10924

PRSS35
Protease, serine, 35
SEQ ID NO: 10925

PRSS36
Protease, serine, 36
SEQ ID NOS: 10926-

10929

PRSS37
Protease, serine, 37
SEQ ID NOS: 10930-

10933

PRSS38
Protease, serine, 38
SEQ ID NO: 10934

PRSS42
Protease, serine, 42
SEQ ID NOS: 10935-

10936

PRSS48
Protease, serine, 48
SEQ ID NOS: 10937-

10938

PRSS50
Protease, serine, 50
SEQ ID NO: 10939

PRSS53
Protease, serine, 53
SEQ ID NO: 10940

PRSS54
Protease, serine, 54
SEQ ID NOS: 10941-

10945

PRSS55
Protease, serine, 55
SEQ ID NOS: 10946-

10948

PRSS56
Protease, serine, 56
SEQ ID NOS: 10949-

10950

PRSS57
Protease, serine, 57
SEQ ID NOS: 10951-

10952

PRSS58
Protease, serine, 58
SEQ ID NOS: 10953-

10954

PRSS8
Protease, serine, 8
SEQ ID NOS: 10955-

10958

PRTG
Protogenin
SEQ ID NOS: 10959-

10962

PRTN3
Proteinase 3
SEQ ID NOS: 10963-

10964

PSAP
Prosaposin
SEQ ID NOS: 10965-

10968

PSAPL1
Prosaposin-like 1 (gene/pseudogene)
SEQ ID NO: 10969

PSG1
Pregnancy specific beta-1-glycoprotein 1
SEQ ID NOS: 10970-

10977

PSG11
Pregnancy specific beta-1-glycoprotein 11
SEQ ID NOS: 10978-

10982

PSG2
Pregnancy specific beta-1-glycoprotein 2
SEQ ID NOS: 10983-

10984

PSG3
Pregnancy specific beta-1-glycoprotein 3
SEQ ID NOS: 10985-

10988

PSG4
Pregnancy specific beta-1-glycoprotein 4
SEQ ID NOS: 10989-

11000

PSG5
Pregnancy specific beta-1-glycoprotein 5
SEQ ID NOS: 11001-

11006

PSG6
Pregnancy specific beta-1-glycoprotein 6
SEQ ID NOS: 11007-

11012

PSG7
Pregnancy specific beta-1-glycoprotein 7
SEQ ID NOS: 11013-

(gene/pseudogene)
11015

PSG8
Pregnancy specific beta-1-glycoprotein 8
SEQ ID NOS: 11016-

11020

PSG9
Pregnancy specific beta-1-glycoprotein 9
SEQ ID NOS: 11021-

11028

PSMD1
Proteasome 26S subunit, non-ATPase 1
SEQ ID NOS: 11029-

11036

PSORS1C2
Psoriasis susceptibility 1 candidate 2
SEQ ID NO: 11037

PSPN
Persephin
SEQ ID NOS: 11038-

11039

PTGDS
Prostaglandin D2 synthase 21 kDa (brain)
SEQ ID NOS: 11040-

11044

PTGIR
Prostaglandin I2 (prostacyclin) receptor (IP)
SEQ ID NOS: 11045-

11049

PTGS1
Prostaglandin-endoperoxide synthase 1
SEQ ID NOS: 11050-

(prostaglandin G/H synthase and
11058

cyclooxygenase)

PTGS2
Prostaglandin-endoperoxide synthase 2
SEQ ID NOS: 11059-

(prostaglandin G/H synthase and
11060

cyclooxygenase)

PTH
Parathyroid hormone
SEQ ID NOS: 11061-

11062

PTH2
Parathyroid hormone 2
SEQ ID NO: 11063

PTHLH
Parathyroid hormone-like hormone
SEQ ID NOS: 11064-

11072

PTK7
Protein tyrosine kinase 7 (inactive)
SEQ ID NOS: 11073-

11088

PTN
Pleiotrophin
SEQ ID NOS: 11089-

11090

PTPRA
Protein tyrosine phosphatase, receptor type,
SEQ ID NOS: 11091-

A
11098

PTPRB
Protein tyrosine phosphatase, receptor type,
SEQ ID NOS: 11099-

B
11106

PTPRC
Protein tyrosine phosphatase, receptor type,
SEQ ID NOS: 11107-

C
11117

PTPRCAP
Protein tyrosine phosphatase, receptor type,
SEQ ID NO: 11118

C-associated protein

PTPRD
Protein tyrosine phosphatase, receptor type,
SEQ ID NOS: 11119-

D
11130

PTPRF
Protein tyrosine phosphatase, receptor type,
SEQ ID NOS: 11131-

F
11138

PTPRJ
Protein tyrosine phosphatase, receptor type,
SEQ ID NOS: 11139-

J
11144

PTPRO
Protein tyrosine phosphatase, receptor type,
SEQ ID NOS: 11145-

O
11153

PTPRS
Protein tyrosine phosphatase, receptor type,
SEQ ID NOS: 11154-

S
11161

PTTG1IP
Pituitary tumor-transforming 1 interacting
SEQ ID NOS: 11162-

protein
11165

PTX3
Pentraxin 3, long
SEQ ID NO: 11166

PTX4
Pentraxin 4, long
SEQ ID NOS: 11167-

11169

PVR
Poliovirus receptor
SEQ ID NOS: 11170-

11175

PVRL1
Poliovirus receptor-related 1 (herpesvirus
SEQ ID NOS: 9286-

entry mediator C)
9288

PXDN
Peroxidasin
SEQ ID NOS: 11176-

11180

PXDNL
Peroxidasin-like
SEQ ID NOS: 11181-

11183

PXYLP1
2-phosphoxylose phosphatase 1
SEQ ID NOS: 11184-

11196

PYY
Peptide YY
SEQ ID NOS: 11197-

11198

PZP
Pregnancy-zone protein
SEQ ID NOS: 11199-

11200

QPCT
Glutaminyl-peptide cyclotransferase
SEQ ID NOS: 11201-

11203

QPRT
Quinolinate phosphoribosyltransferase
SEQ ID NOS: 11204-

11205

QRFP
Pyroglutamylated RFamide peptide
SEQ ID NOS: 11206-

11207

QSOX1
Quiescin Q6 sulfhydryl oxidase 1
SEQ ID NOS: 11208-

11211

R3HDML
R3H domain containing-like
SEQ ID NO: 11212

RAB26
RAB26, member RAS oncogene family
SEQ ID NOS: 11213-

11216

RAB36
RAB36, member RAS oncogene family
SEQ ID NOS: 11217-

11219

RAB9B
RAB9B, member RAS oncogene family
SEQ ID NO: 11220

RAET1E
Retinoic acid early transcript 1E
SEQ ID NOS: 11221-

11226

RAET1G
Retinoic acid early transcript 1G
SEQ ID NOS: 11227-

11229

RAMP2
Receptor (G protein-coupled) activity
SEQ ID NOS: 11230-

modifying protein 2
11234

RAPGEF5
Rap guanine nucleotide exchange factor
SEQ ID NOS: 11235-

(GEF) 5
11241

RARRES1
Retinoic acid receptor responder (tazarotene
SEQ ID NOS: 11242-

induced) 1
11243

RARRES2
Retinoic acid receptor responder (tazarotene
SEQ ID NOS: 11244-

induced) 2
11247

RASA2
RAS p21 protein activator 2
SEQ ID NOS: 11248-

11250

RBM3
RNA binding motif (RNP1, RRM) protein 3
SEQ ID NOS: 11251-

11253

RBP3
Retinol binding protein 3, interstitial
SEQ ID NO: 11254

RBP4
Retinol binding protein 4, plasma
SEQ ID NOS: 11255-

11258

RCN1
Reticulocalbin 1, EF-hand calcium binding
SEQ ID NOS: 11259-

domain
11262

RCN2
Reticulocalbin 2, EF-hand calcium binding
SEQ ID NOS: 11263-

domain
11266

RCN3
Reticulocalbin 3, EF-hand calcium binding
SEQ ID NOS: 11267-

domain
11270

RCOR1
REST corepressor 1
SEQ ID NOS: 11271-

11272

RDH11
Retinol dehydrogenase 11 (all-trans/9-
SEQ ID NOS: 11273-

cis/11-cis)
11280

RDH12
Retinol dehydrogenase 12 (all-trans/9-
SEQ ID NOS: 11281-

cis/11-cis)
11282

RDH13
Retinol dehydrogenase 13 (all-trans/9-cis)
SEQ ID NOS: 11283-

11291

RDH5
Retinol dehydrogenase 5 (11-cis/9-cis)
SEQ ID NOS: 11292-

11296

RDH8
Retinol dehydrogenase 8 (all-trans)
SEQ ID NOS: 11297-

11298

REG1A
Regenerating islet-derived 1 alpha
SEQ ID NO: 11299

REG1B
Regenerating islet-derived 1 beta
SEQ ID NOS: 11300-

11301

REG3A
Regenerating islet-derived 3 alpha
SEQ ID NOS: 11302-

11304

REG3G
Regenerating islet-derived 3 gamma
SEQ ID NOS: 11305-

11307

REG4
Regenerating islet-derived family, member
SEQ ID NOS: 11308-

4
11311

RELN
Reelin
SEQ ID NOS: 11312-

11315

RELT
RELT tumor necrosis factor receptor
SEQ ID NOS: 11316-

11319

REN
Renin
SEQ ID NOS: 11320-

11321

REPIN1
Replication initiator 1
SEQ ID NOS: 11322-

11335

REPS2
RALBP1 associated Eps domain containing
SEQ ID NOS: 11336-

2
11337

RET
Ret proto-oncogene
SEQ ID NOS: 11338-

11343

RETN
Resistin
SEQ ID NOS: 11344-

11346

RETNLB
Resistin like beta
SEQ ID NO: 11347

RETSAT
Retinol saturase (all-trans-retinol 13,14-
SEQ ID NOS: 11348-

reductase)
11352

RFNG
RFNG O-fucosylpeptide 3-beta-N-
SEQ ID NOS: 11353-

acetylglucosaminyltransferase
11355

RGCC
Regulator of cell cycle
SEQ ID NO: 11356

RGL4
Ral guanine nucleotide dissociation
SEQ ID NOS: 11357-

stimulator-like 4
11363

RGMA
Repulsive guidance molecule family
SEQ ID NOS: 11364-

member a
11373

RGMB
Repulsive guidance molecule family
SEQ ID NOS: 11374-

member b
11375

RHOQ
Ras homolog family member Q
SEQ ID NOS: 11376-

11380

RIC3
RIC3 acety lcholine receptor chaperone
SEQ ID NOS: 11381-

11388

RIMS1
Regulating sy naptic membrane exocytosis 1
SEQ ID NOS: 11393-

11408

RIPPLY1
Ripply transcriptional repressor 1
SEQ ID NOS: 11409-

11410

RLN1
Relaxin 1
SEQ ID NO: 11411

RLN2
Relaxin 2
SEQ ID NOS: 11412-

11413

RLN3
Relaxin 3
SEQ ID NOS: 11414-

11415

RMDN1
Regulator of microtubule dynamics 1
SEQ ID NOS: 11416-

11429

RNASE1
Ribonuclease, RNase A family, 1
SEQ ID NOS: 11430-

(pancreatic)
11434

RNASE10
Ribonuclease, RNase A family, 10 (non-
SEQ ID NOS: 11435-

active)
11436

RNASE11
Ribonuclease, RNase A family, 11 (non-
SEQ ID NOS: 11437-

active)
11447

RNASE12
Ribonuclease, RNase A family, 12 (non-
SEQ ID NO: 11448

active)

RNASE13
Ribonuclease, RNase A family, 13 (non-
SEQ ID NO: 11449

active)

RNASE2
Ribonuclease, RNase A family, 2 (liver,
SEQ ID NO: 11450

eosinophil-derived neurotoxin)

RNASE3
Ribonuclease, RNase A family, 3
SEQ ID NO: 11451

RNASE4
Ribonuclease, RNase A family, 4
SEQ ID NOS: 11452-

11454

RNASE6
Ribonuclease, RNase A family, k6
SEQ ID NO: 11455

RNASE7
Ribonuclease, RNase A family, 7
SEQ ID NOS: 11456-

11457

RNASE8
Ribonuclease, RNase A family, 8
SEQ ID NO: 11458

RNASE9
Ribonuclease, RNase A family, 9 (non-
SEQ ID NOS: 11459-

active)
11469

RNASEH1
Ribonuclease H1
SEQ ID NOS: 11470-

11472

RNASET2
Ribonuclease T2
SEQ ID NOS: 11473-

11480

RNF146
Ring finger protein 146
SEQ ID NOS: 11481-

11492

RNF148
Ring finger protein 148
SEQ ID NOS: 11493-

11494

RNF150
Ring finger protein 150
SEQ ID NOS: 11495-

11499

RNF167
Ring finger protein 167
SEQ ID NOS: 11500-

11510

RNF220
Ring finger protein 220
SEQ ID NOS: 11511-

11517

RNF34
Ring finger protein 34, E3 ubiquitin protein
SEQ ID NOS: 11518-

ligase
11525

RNLS
Renalase, FAD-dependent amine oxidase
SEQ ID NOS: 11526-

11528

RNPEP
Arginyl aminopeptidase (aminopeptidase B)
SEQ ID NOS: 11529-

11534

ROR1
Receptor tyrosine kinase-like orphan
SEQ ID NOS: 11535-

receptor 1
11537

RP11-

SEQ ID NO: 4158

1236K1.1

RP11-14J7.7

SEQ ID NOS: 674-675

RP11-

SEQ ID NOS: 85-87

196G11.1

RP11-

SEQ ID NO: 683

350O14.18

RP11-

SEQ ID NO: 8194

520P18.5

RP11-

SEQ ID NO: 89

812E19.9

RP11-

SEQ ID NO: 676

903H12.5

RP11-

SEQ ID NOS: 78-80

977G19.10

RP4-576H24.4

SEQ ID NOS: 670-672

RP4-608O15.3
Complement factor H-related protein 2
SEQ ID NO: 1649

RPL3
Ribosomal protein L3
SEQ ID NOS: 11538-

11543

RPLP2
Ribosomal protein, large, P2
SEQ ID NOS: 11544-

11546

RPN2
Ribophorin II
SEQ ID NOS: 11547-

11553

RPS27L
Ribosomal protein S27-like
SEQ ID NOS: 11554-

11559

RQCD1
RCD1 required for cell differentiation1
SEQ ID NOS: 3100-

homolog (S. pombe)
3106

RS1
Retinoschisin 1
SEQ ID NO: 11560

RSF1
Remodeling and spacing factor 1
SEQ ID NOS: 11561-

11567

RSPO1
R-spondin 1
SEQ ID NOS: 11568-

11571

RSPO2
R-spondin 2
SEQ ID NOS: 11572-

11579

RSPO3
R-spondin 3
SEQ ID NOS: 11580-

11581

RSPO4
R-spondin 4
SEQ ID NOS: 11582-

11583

RSPRY1
Ring finger and SPRY domain containing 1
SEQ ID NOS: 11584-

11590

RTBDN
Retbindin
SEQ ID NOS: 11591-

11603

RTN4RL1
Reticulon 4 receptor-like 1
SEQ ID NO: 11604

RTN4RL2
Reticulon 4 receptor-like 2
SEQ ID NOS: 11605-

11607

SAA1
Serum amyloid A1
SEQ ID NOS: 11608-

11610

SAA2
Serum amyloid A2
SEQ ID NOS: 11611-

11616

SAA4
Serum amyloid A4, constitutive
SEQ ID NO: 11617

SAP30
Sin3A-associated protein, 30 kDa
SEQ ID NO: 11618

SAR1A
Secretion associated, Ras related GTPase
SEQ ID NOS: 11619-

1A
11625

SARAF
Store-operated calcium entry-associated
SEQ ID NOS: 11626-

regulatory factor
11636

SARM1
Sterile alpha and TIR motif containing 1
SEQ ID NOS: 11637-

11640

SATB1
SATB homeobox 1
SEQ ID NOS: 11641-

11653

SAXO2
Stabilizer of axonemal microtubules 2
SEQ ID NOS: 11654-

11658

SBSN
Suprabasin
SEQ ID NOS: 11659-

11661

SBSPON
Somatomedin B and thrombospondin, type
SEQ ID NO: 11662

1 domain containing

SCARF1
Scavenger receptor class F, member 1
SEQ ID NOS: 11663-

11667

SCG2
Secretogranin II
SEQ ID NOS: 11668-

11670

SCG3
Secretogranin III
SEQ ID NOS: 11671-

11673

SCG5
Secretogranin V
SEQ ID NOS: 11674-

11678

SCGB1A1
Secretoglobin, family 1A, member 1
SEQ ID NOS: 11679-

(uteroglobin)
11680

SCGB1C1
Secretoglobin, family 1C, member 1
SEQ ID NO: 11681

SCGB1C2
Secretoglobin, family 1C, member 2
SEQ ID NO: 11682

SCGB1D1
Secretoglobin, family 1D, member 1
SEQ ID NO: 11683

SCGB1D2
Secretoglobin, family 1D, member 2
SEQ ID NO: 11684

SCGB1D4
Secretoglobin, family 1D, member 4
SEQ ID NO: 11685

SCGB2A1
Secretoglobin, family 2A, member 1
SEQ ID NO: 11686

SCGB2A2
Secretoglobin, family 2A, member 2
SEQ ID NOS: 11687-

11688

SCGB2B2
Secretoglobin, family 2B, member 2
SEQ ID NOS: 11689-

11690

SCGB3A1
Secretoglobin, family 3A, member 1
SEQ ID NO: 11691

SCGB3A2
Secretoglobin, family 3A, member 2
SEQ ID NOS: 11692-

11693

SCN1B
Sodium channel, voltage gated, type I beta
SEQ ID NOS: 11694-

subunit
11699

SCN3B
Sodium channel, voltage gated, type III beta
SEQ ID NOS: 11700-

subunit
11704

SCPEP1
Serine carboxypeptidase 1
SEQ ID NOS: 11705-

11712

SCRG1
Stimulator of chondrogenesis 1
SEQ ID NOS: 11713-

11714

SCT
Secretin
SEQ ID NO: 11715

SCUBE1
Signal peptide, CUB domain, EGF-like 1
SEQ ID NOS: 11716-

11719

SCUBE2
Signal peptide, CUB domain, EGF-like 2
SEQ ID NOS: 11720-

11726

SCUBE3
Signal peptide, CUB domain, EGF-like 3
SEQ ID NO: 11727

SDC1
Syndecan 1
SEQ ID NOS: 11728-

11732

SDF2
Stromal cell-derived factor 2
SEQ ID NOS: 11733-

11735

SDF2L1
Stromal cell-derived factor 2-like 1
SEQ ID NO: 11736

SDF4
Stromal cell derived factor 4
SEQ ID NOS: 11737-

11740

SDHAF2
Succinate dehydrogenase complex assembly
SEQ ID NOS: 11741-

factor 2
11748

SDHAF4
Succinate dehydrogenase complex assembly
SEQ ID NO: 11749

factor 4

SDHB
Succinate dehydrogenase complex, subunit
SEQ ID NOS: 11750-

B, iron sulfur (Ip)
11752

SDHD
Succinate dehydrogenase complex, subunit
SEQ ID NOS: 11753-

D, integral membrane protein
11762

SEC14L3
SEC14-like lipid binding 3
SEQ ID NOS: 11763-

11769

SEC16A
SEC16 homolog A, endoplasmic reticulum
SEQ ID NOS: 11770-

export factor
11776

SEC16B
SEC16 homolog B, endoplasmic reticulum
SEQ ID NOS: 11777-

export factor
11780

SEC22C
SEC22 homolog C, vesicle trafficking
SEQ ID NOS: 11781-

protein
11793

SEC31A
SEC31 homolog A, COPII coat complex
SEQ ID NOS: 11794-

component
11823

SECISBP2
SECIS binding protein 2
SEQ ID NOS: 11824-

11828

SECTM1
Secreted and transmembrane 1
SEQ ID NOS: 11829-

11836

SEL1L
Sel-1 suppressor of lin-12-like (C. elegans)
SEQ ID NOS: 11837-

11839

SELM
Selenoprotein M
SEQ ID NOS: 11847-

11849

SELO
Selenoprotein O
SEQ ID NOS: 11854-

11855

SEMA3A
Serna domain, immunoglobulin domain
SEQ ID NOS: 11862-

(Ig), short basic domain, secreted,
11866

(semaphorin) 3A

SEMA3B
Serna domain, immunoglobulin domain
SEQ ID NOS: 11867-

(Ig), short basic domain, secreted,
11873

(semaphorin) 3B

SEMA3C
Serna domain, immunoglobulin domain
SEQ ID NOS: 11874-

(Ig), short basic domain, secreted,
11878

(semaphorin) 3C

SEMA3E
Serna domain, immunoglobulin domain
SEQ ID NOS: 11879-

(Ig), short basic domain, secreted,
11883

(semaphorin) 3E

SEMA3F
Serna domain, immunoglobulin domain
SEQ ID NOS: 11884-

(Ig), short basic domain, secreted,
11890

(semaphorin) 3F

SEMA3G
Serna domain, immunoglobulin domain
SEQ ID NOS: 11891-

(Ig), short basic domain, secreted,
11893

(semaphorin) 3G

SEMA4A
Serna domain, immunoglobulin domain
SEQ ID NOS: 11894-

(Ig), transmembrane domain (TM) and short
11902

cytoplasmic domain, (semaphorin) 4A

SEMA4B
Serna domain, immunoglobulin domain
SEQ ID NOS: 11903-

(Ig), transmembrane domain (TM) and short
11913

cytoplasmic domain, (semaphorin) 4B

SEMA4C
Serna domain, immunoglobulin domain
SEQ ID NOS: 11914-

(Ig), transmembrane domain (TM) and short
11916

cytoplasmic domain, (semaphorin) 4C

SEMA4D
Sema domain, immunoglobulin domain
SEQ ID NOS: 11917-

(Ig), transmembrane domain (TM) and short
11930

cytoplasmic domain, (semaphorin) 4D

SEMA4F
Sema domain, immunoglobulin domain
SEQ ID NOS: 11931-

(Ig), transmembrane domain (TM) and short
11939

cytoplasmic domain, (semaphorin) 4F

SEMA4G
Sema domain, immunoglobulin domain
SEQ ID NOS: 11940-

(Ig), transmembrane domain (TM) and short
11947

cytoplasmic domain, (semaphorin) 4G

SEMA5A
Sema domain, seven thrombospondin
SEQ ID NOS: 11948-

repeats (type 1 and type 1-like),
11949

transmembrane domain (TM) and short

cytoplasmic domain, (semaphorin) 5A

SEMA6A
Sema domain, transmembrane domain
SEQ ID NOS: 11950-

(TM), and cytoplasmic domain,
11957

(semaphorin) 6A

SEMA6C
Sema domain, transmembrane domain
SEQ ID NOS: 11958-

(TM), and cytoplasmic domain,
11963

(semaphorin) 6C

SEMA6D
Sema domain, transmembrane domain
SEQ ID NOS: 11964-

(TM), and cytoplasmic domain,
11977

(semaphorin) 6D

SEMG1
Semenogelin I
SEQ ID NO: 11978

SEMG2
Semenogelin II
SEQ ID NO: 11979

SEPN1
Selenoprotein N, 1
SEQ ID NOS: 11850-

11853

SEPP1
Selenoprotein P, plasma, 1
SEQ ID NOS: 11856-

11861

SEPT15
15 kDa selenoprotein
SEQ ID NOS: 11840-

11846

SEPT9
Septin 9
SEQ ID NOS: 11980-

12016

SERPINA1
Serpin peptidase inhibitor, clade A (alpha-1
SEQ ID NOS: 12017-

antiproteinase, antitrypsin), member 1
12033

SERPINA10
Serpin peptidase inhibitor, clade A (alpha-1
SEQ ID NOS: 12034-

anti proteinase, antitrypsin), member 10
12037

SERPINA11
Serpin peptidase inhibitor, clade A (alpha-1
SEQ ID NO: 12038

antiproteinase, antitrypsin), member 11

SERPINA12
Serpin peptidase inhibitor, clade A (alpha-1
SEQ ID NOS: 12039-

anti proteinase, antitrypsin), member 12
12040

SERPINA3
Serpin peptidase inhibitor, clade A (alpha-1
SEQ ID NOS: 673-

antiproteinase, antitrypsin), member 3
12047

SERPINA4
Serpin peptidase inhibitor, clade A (alpha-1
SEQ ID NOS: 12048-

antiproteinase, antitrypsin), member 4
12050

SERPINA5
Serpin peptidase inhibitor, clade A (alpha-1
SEQ ID NOS: 12051-

antiproteinase, antitrypsin), member 5
12062

SERPINA6
Serpin peptidase inhibitor, clade A (alpha-1
SEQ ID NOS: 12063-

antiproteinase, antitrypsin), member 6
12065

SERPINA7
Serpin peptidase inhibitor, clade A (alpha-1
SEQ ID NOS: 12066-

antiproteinase, antitrypsin), member 7
12067

SERPINA9
Serpin peptidase inhibitor, clade A (alpha-1
SEQ ID NOS: 12068-

antiproteinase, antitrypsin), member 9
12074

SERPINB2
Serpin peptidase inhibitor, clade B
SEQ ID NOS: 12075-

(ovalbumin), member 2
12079

SERPINC1
Serpin peptidase inhibitor, clade C
SEQ ID NOS: 12080-

(antithrombin), member 1
12081

SERPIND1
Serpin peptidase inhibitor, clade D (heparin
SEQ ID NOS: 12082-

cofactor), member 1
12083

SERPINE1
Serpin peptidase inhibitor, clade E (nexin.
SEQ ID NO: 12084

plasminogen activator inhibitor type 1),

member 1

SERPINE2
Serpin peptidase inhibitor, clade E (nexin,
SEQ ID NOS: 12085-

plasminogen activator inhibitor type 1),
12091

member 2

SERPINE3
Serpin peptidase inhibitor, clade E (nexin,
SEQ ID NOS: 12092-

plasminogen activator inhibitor type 1),
12095

member 3

SERPINF1
Serpin peptidase inhibitor, clade F (alpha-2
SEQ ID NOS: 12096-

antiplasmin, pigment epithelium derived
12104

factor), member 1

SERPINF2
Serpin peptidase inhibitor, clade F (alpha-2
SEQ ID NOS: 12105-

antiplasmin, pigment epithelium derived
12109

factor), member 2

SERPING1
Serpin peptidase inhibitor, clade G (C1
SEQ ID NOS: 12110-

inhibitor), member 1
12120

SERPINH1
Serpin peptidase inhibitor, clade H (heat
SEQ ID NOS: 12121-

shock protein 47), member 1, (collagen
12135

binding protein 1)

SERPINI1
Serpin peptidase inhibitor, clade I
SEQ ID NOS: 12136-

(neuroserpin), member 1
12140

SERPINI2
Serpin peptidase inhibitor, clade I (pancpin),
SEQ ID NOS: 12141-

member 2
12147

SETD8
SET domain containing (lysine
SEQ ID NOS: 7589-

methyltransferase) 8
7592

SEZ6L2
Seizure related 6 homolog (mouse)-like 2
SEQ ID NOS: 12148-

12154

SFRP1
Secreted frizzled-related protein 1
SEQ ID NOS: 12155-

12156

SFRP2
Secreted frizzled-related protein 2
SEQ ID NO: 12157

SFRP4
Secreted frizzled-related protein 4
SEQ ID NOS: 12158-

12159

SFRP5
Secreted frizzled-related protein 5
SEQ ID NO: 12160

SFTA2
Surfactant associated 2
SEQ ID NOS: 12161-

12162

SFTPA1
Surfactant protein A1
SEQ ID NOS: 12163-

12167

SFTPA2
Surfactant protein A2
SEQ ID NOS: 12168-

12172

SFTPB
Surfactant protein B
SEQ ID NOS: 12173-

12177

SFTPD
Surfactant protein D
SEQ ID NOS: 12178-

12179

SFXN5
Sideroflexin 5
SEQ ID NOS: 12180-

12184

SGCA
Sarcoglycan, alpha (50 kDa dystrophin-
SEQ ID NOS: 12185-

associated glycoprotein)
12192

SGSH
N-sulfoglucosamine sulfohydrolase
SEQ ID NOS: 12193-

12201

SH3RF3
SH3 domain containing ring finger 3
SEQ ID NO: 12202

SHBG
Sex hormone-binding globulin
SEQ ID NOS: 12203-

12221

SHE
Src homology 2 domain containing E
SEQ ID NOS: 12222-

12224

SHH
Sonic hedgehog
SEQ ID NOS: 12225-

12228

SHKBP1
SH3KBP1 binding protein 1
SEQ ID NOS: 12229-

12244

SIAE
Sialic acid acetylesterase
SEQ ID NOS: 12245-

12247

SIDT2
SID1 transmembrane family, member 2
SEQ ID NOS: 12248-

12257

SIGLEC10
Sialic acid binding Ig-like lectin 10
SEQ ID NOS: 12258-

12266

SIGLEC6
Sialic acid binding Ig-like lectin 6
SEQ ID NOS: 12267-

12272

SIGLEC7
Sialic acid binding Ig-like lectin 7
SEQ ID NOS: 12273-

12277

SIGLECL1
SIGLEC family like 1
SEQ ID NOS: 12278-

12283

SIGMAR1
Sigma non-opioid intracellular receptor 1
SEQ ID NOS: 12284-

12287

SIL1
SIL1 nucleotide exchange factor
SEQ ID NOS: 12288-

12296

SIRPB1
Signal-regulatory protein beta 1
SEQ ID NOS: 12297-

12309

SIRPD
Signal-regulatory protein delta
SEQ ID NOS: 12310-

12312

SLAMF1
Signaling lymphocytic activation molecule
SEQ ID NOS: 12313-

family member 1
12315

SLAMF7
SLAM family member 7
SEQ ID NOS: 12316-

12324

SLC10A3
Solute carrier family 10, member 3
SEQ ID NOS: 12325-

12329

SLC15A3
Solute carrier family 15 (oligopeptide
SEQ ID NOS: 12330-

transporter), member 3
12335

SLC25A14
Solute carrier family 25 (mitochondrial
SEQ ID NOS: 12336-

carrier, brain), member 14
12342

SLC25A25
Solute carrier family 25 (mitochondrial
SEQ ID NOS: 12343-

carrier; phosphate carrier), member 25
12349

SLC2A5
Solute carrier family 2 (facilitated
SEQ ID NOS: 12350-

glucose/fructose transporter), member 5
12358

SLC35E3
Solute carrier family 35, member E3
SEQ ID NOS: 12359-

12360

SLC39A10
Solute carrier family 39 (zinc transporter),
SEQ ID NOS: 12361-

member 10
12367

SLC39A14
Solute carrier family 39 (zinc transporter),
SEQ ID NOS: 12368-

member 14
12378

SLC39A4
Solute carrier family 39 (zinc transporter),
SEQ ID NOS: 12379-

member 4
12381

SLC39A5
Solute carrier family 39 (zinc transporter),
SEQ ID NOS: 12382-

member 5
12388

SLC3A1
Solute carrier family 3 (amino acid
SEQ ID NOS: 12389-

transporter heavy chain), member 1
12398

SLC51A
Solute carrier family 51, alpha subunit
SEQ ID NOS: 12399-

12403

SLC52A2
Solute carrier family 52 (riboflavin
SEQ ID NOS: 12404-

transporter), member 2
12414

SLC5A6
Solute carrier family 5
SEQ ID NOS: 12415-

(sodium/multivitamin and iodide
12425

cotransporter), member 6

SLC6A9
Solute carrier family 6 (neurotransmitter
SEQ ID NOS: 12426-

transporter, glycine), member 9
12433

SLC8A1
Solute carrier family 8 (sodium/calcium
SEQ ID NOS: 12434-

exchanger), member 1
12445

SLC8B1
Solute carrier family 8
SEQ ID NOS: 12446-

(sodium/lithium/calcium exchanger),
12456

member B1

SLC9A6
Solute carrier family 9, subfamily A
SEQ ID NOS: 12457-

(NHE6, cation proton antiporter 6), member
12468

6

SLCO1A2
Solute carrier organic anion transporter
SEQ ID NOS: 12469-

family, member 1A2
12481

SLIT1
Slit guidance ligand 1
SEQ ID NOS: 12482-

12485

SLIT2
Slit guidance ligand 2
SEQ ID NOS: 12486-

12494

SLIT3
Slit guidance ligand 3
SEQ ID NOS: 12495-

12497

SLITRK3
SLIT and NTRK-like family, member 3
SEQ ID NOS: 12498-

12500

SLPI
Secretory leukocyte peptidase inhibitor
SEQ ID NO: 12501

SLTM
SAFB-like, transcription modulator
SEQ ID NOS: 12502-

12515

SLURP1
Secreted LY6/PLAUR domain containing 1
SEQ ID NO: 12516

SMARCA2
SWI/SNF related, matrix associated, actin
SEQ ID NOS: 12517-

dependent regulator of chromatin, subfamily
12562

a, member 2

SMG6
SMG6 nonsense mediated mRNA decay
SEQ ID NOS: 12563-

factor
12574

SMIM7
Small integral membrane protein 7
SEQ ID NOS: 12575-

12591

SMOC1
SPARC related modular calcium binding 1
SEQ ID NOS: 12592-

12593

SMOC2
SPARC related modular calcium binding 2
SEQ ID NOS: 12594-

12598

SMPDL3A
Sphingomyelin phosphodiesterase, acid-like
SEQ ID NOS: 12599-

3A
12600

SMPDL3B
Sphingomyelin phosphodiesterase, acid-like
SEQ ID NOS: 12601-

3B
12605

SMR3A
Submaxillary gland androgen regulated
SEQ ID NO: 12606

protein 3A

SMR3B
Submaxillary gland androgen regulated
SEQ ID NOS: 12607-

protein 3B
12609

SNED1
Sushi, nidogen and EGF-like domains 1
SEQ ID NOS: 12610-

12616

SNTB1
Syntrophin, beta 1 (dystrophin-associated
SEQ ID NOS: 12617-

protein A1, 59 kDa, basic component 1)
12619

SNTB2
Syntrophin, beta 2 (dystrophin-associated
SEQ ID NOS: 12620-

protein A1, 59 kDa, basic component 2)
12624

SNX14
Sorting nexin 14
SEQ ID NOS: 12625-

12636

SOD3
Superoxide dismutase 3, extracellular
SEQ ID NOS: 12637-

12638

SOST
Sclerostin
SEQ ID NO: 12639

SOSTDC1
Sclerostin domain containing 1
SEQ ID NOS: 12640-

12641

SOWAHA
Sosondowah ankyrin repeat domain family
SEQ ID NO: 12642

member A

SPACA3
Sperm acrosome associated 3
SEQ ID NOS: 12643-

12645

SPACA4
Sperm acrosome associated 4
SEQ ID NO: 12646

SPACA5
Sperm acrosome associated 5
SEQ ID NOS: 12647-

12648

SPACA5B
Sperm acrosome associated 5B
SEQ ID NO: 12649

SPACA7
Sperm acrosome associated 7
SEQ ID NOS: 12650-

12653

SPAG11A
Sperm associated antigen 11A
SEQ ID NOS: 12654-

12662

SPAG11B
Sperm associated antigen 11B
SEQ ID NOS: 12663-

12671

SPARC
Secreted protein, acidic, cysteine-rich
SEQ ID NOS: 12672-

(osteonectin)
12676

SPARCL1
SPARC-like 1 (hevin)
SEQ ID NOS: 12677-

12686

SPATA20
Spermatogenesis associated 20
SEQ ID NOS: 12687-

12700

SPESP1
Sperm equatorial segment protein 1
SEQ ID NO: 12701

SPINK1
Serine peptidase inhibitor, Kazal type 1
SEQ ID NOS: 12702-

12703

SPINK13
Serine peptidase inhibitor, Kazal type 13
SEQ ID NOS: 12704-

(putative)
12706

SPINK14
Serine peptidase inhibitor, Kazal type 14
SEQ ID NOS: 12707-

(putative)
12708

SPINK2
Serine peptidase inhibitor, Kazal type 2
SEQ ID NOS: 12709-

(acrosin-trypsin inhibitor)
12714

SPINK4
Serine peptidase inhibitor, Kazal type 4
SEQ ID NOS: 12715-

12716

SPINK5
Serine peptidase inhibitor, Kazal type 5
SEQ ID NOS: 12717-

12722

SPINK6
Serine peptidase inhibitor, Kazal type 6
SEQ ID NOS: 12723-

12725

SPINK7
Serine peptidase inhibitor, Kazal type 7
SEQ ID NOS: 12726-

(putative)
12727

SPINK8
Serine peptidase inhibitor, Kazal type 8
SEQ ID NO: 12728

(putative)

SPINK9
Serine peptidase inhibitor, Kazal type 9
SEQ ID NOS: 12729-

12730

SPINT1
Serine peptidase inhibitor, Kunitz type 1
SEQ ID NOS: 12731-

12738

SPINT2
Serine peptidase inhibitor, Kunitz type, 2
SEQ ID NOS: 12739-

12746

SPINT3
Serine peptidase inhibitor, Kunitz type, 3
SEQ ID NO: 12747

SPINT4
Serine peptidase inhibitor, Kunitz type 4
SEQ ID NO: 12748

SPOCK1
Sparc/osteonectin, cwcv and kazal-like
SEQ ID NOS: 12749-

domains proteoglycan (testican) 1
12752

SPOCK2
Sparc/osteonectin, cwcv and kazal-like
SEQ ID NOS: 12753-

domains proteoglycan (testican) 2
12756

SPOCK3
Sparc/osteonectin, cwcv and kazal-like
SEQ ID NOS: 12757-

domains proteoglycan (testican) 3
12782

SPON1
Spondin 1, extracellular matrix protein
SEQ ID NO: 12783

SPON2
Spondin 2, extracellular matrix protein
SEQ ID NOS: 12784-

12793

SPP1
Secreted phosphoprotein 1
SEQ ID NOS: 12794-

12798

SPP2
Secreted phosphoprotein 2, 24 kDa
SEQ ID NOS: 12799-

12801

SPRN
Shadow of prion protein homolog
SEQ ID NO: 12802

(zebrafish)

SPRYD3
SPRY domain containing 3
SEQ ID NOS: 12803-

12806

SPRYD4
SPRY domain containing 4
SEQ ID NO: 12807

SPTY2D1-AS1
SPTY2D1 antisense RNA 1
SEQ ID NOS: 12808-

12813

SPX
Spexin hormone
SEQ ID NOS: 12814-

12815

SRGN
Serglycin
SEQ ID NO: 12816

SRL
Sarealumenin
SEQ ID NOS: 12817-

12819

SRP14
Signal recognition particle 14 kDa
SEQ ID NOS: 12820-

(homologous Alu RNA binding protein)
12823

SRPX
Sushi-repeat containing protein, X-linked
SEQ ID NOS: 12824-

12827

SRPX2
Sushi-repeat containing protein, X-linked 2
SEQ ID NOS: 12828-

12831

SSC4D
Scavenger receptor cysteine rich family, 4
SEQ ID NO: 12832

domains

SSC5D
Scavenger receptor cysteine rich family, 5
SEQ ID NOS: 12833-

domains
12836

SSPO
SCO-spondin
SEQ ID NO: 12837

SSR2
Signal sequence receptor, beta (translocon-
SEQ ID NOS: 12838-

associated protein beta)
12847

SST
Somatostatin
SEQ ID NO: 12848

ST3GAL1
ST3 beta-galactoside alpha-2,3-
SEQ ID NOS: 12849-

sialyltransferase 1
12856

ST3GAL4
ST3 beta-galactoside alpha-2,3-
SEQ ID NOS: 12857-

sialyltransferase 4
12872

ST6GAL1
ST6 beta-galactosamide alpha-2,6-
SEQ ID NOS: 12873-

sialyltranferase 1
12888

ST6GALNAC2
ST6 (alpha-N-acetyl-neuraminyl-2,3-beta-
SEQ ID NOS: 12889-

galactosyl-1,3)-N-acetylgalactosaminide
12893

alpha-2,6-sialyltransferase 2

ST6GALNAC5
ST6 (alpha-N-acetyl-neuraminyl-2,3-beta-
SEQ ID NOS: 12894-

galactosyl-1,3)-N-acetylgalactosaminide
12895

alpha-2,6-sialyltransferase 5

ST6GALNAC6
ST6 (alpha-N-acetyl-neuraminyl-2,3-beta-
SEQ ID NOS: 12896-

galactosyl-1,3)-N-acetylgalactosaminide
12903

alpha-2,6-sialyltransferase 6

ST8SIA2
ST8 alpha-N-acetyl-neuraminide alpha-2,8-
SEQ ID NOS: 12904-

sialyltransferase 2
12906

ST8SIA4
ST8 alpha-N-acetyl-neuraminide alpha-2,8-
SEQ ID NOS: 12907-

sialyltransferase 4
12909

ST8SIA6
ST8 alpha-N-acetyl-neuraminide alpha-2,8-
SEQ ID NOS: 12910-

sialyltransferase 6
12911

STARD7
StAR-related lipid transfer (START)
SEQ ID NOS: 12912-

domain containing 7
12913

STATH
Statherin
SEQ ID NOS: 12914-

12916

STC1
Stanniocalcin 1
SEQ ID NOS: 12917-

12918

STC2
Stanniocalcin 2
SEQ ID NOS: 12919-

12921

STMND1
Stathmin domain containing 1
SEQ ID NOS: 12922-

12923

STOML2
Stomatin (EPB72)-like 2
SEQ ID NOS: 12926-

12929

STOX1
Storkhead box 1
SEQ ID NOS: 12930-

12934

STRC
Stereocilin
SEQ ID NOS: 12935-

12940

SUCLG1
Succinate-CoA ligase, alpha subunit
SEQ ID NOS: 12941-

12942

SUDS3
SDS3 homolog, SIN3A corepressor
SEQ ID NO: 12943

complex component

SULF1
Sulfatase 1
SEQ ID NOS: 12944-

12954

SULF2
Sulfatase 2
SEQ ID NOS: 12955-

12959

SUMF1
Sulfatase modifying factor 1
SEQ ID NOS: 12960-

12964

SUMF2
Sulfatase modifying factor 2
SEQ ID NOS: 12965-

12978

SUSD1
Sushi domain containing 1
SEQ ID NOS: 12979-

12984

SUSD5
Sushi domain containing 5
SEQ ID NOS: 12985-

12986

SVEP1
Sushi, von Willebrand factor type A, EGF
SEQ ID NOS: 12987-

and pentraxin domain containing 1
12989

SWSAP1
SWIM-type zinc finger 7 associated protein
SEQ ID NO: 12990

1

SYAP1
Synapse associated protein 1
SEQ ID NO: 12991

SYCN
Syncollin
SEQ ID NO: 12992

TAC1
Tachykinin, precursor 1
SEQ ID NOS: 12993-

12995

TAC3
Tachykinin 3
SEQ ID NOS: 12996-

13005

TAC4
Tachykinin 4 (hemokinin)
SEQ ID NOS: 13006-

13011

TAGLN2
Transgelin 2
SEQ ID NOS: 13012-

13015

TAPBP
TAP binding protein (tapasin)
SEQ ID NOS: 13016-

13021

TAPBPL
TAP binding protein-like
SEQ ID NOS: 13022-

13023

TBL2
Transducin (beta)-like 2
SEQ ID NOS: 13024-

13036

TBX10
T-box 10
SEQ ID NO: 13037

TCF12
Transcription factor 12
SEQ ID NOS: 13038-

13051

TCN1
Transcobalamin I (vitamin B12 binding
SEQ ID NO: 13052

protein, R binder family)

TCN2
Transcobalamin II
SEQ ID NOS: 13053-

13056

TCTN1
Tectonic family member 1
SEQ ID NOS: 13057-

13075

TCTN3
Tectonic family member 3
SEQ ID NOS: 13076-

13080

TDP2
Tyrosyl-DNA phosphodiesterase 2
SEQ ID NOS: 13081-

13082

TEK
TEK tyrosine kinase, endothelial
SEQ ID NOS: 13097-

13101

TEPP
Testis, prostate and placenta expressed
SEQ ID NOS: 13102-

13103

TEX101
Testis expressed 101
SEQ ID NOS: 13104-

13105

TEX264
Testis expressed 264
SEQ ID NOS: 13106-

13117

TF
Transferrin
SEQ ID NOS: 13121-

13127

TFAM
Transcription factor A, mitochondrial
SEQ ID NOS: 13128-

13130

TFF1
Trefoil factor 1
SEQ ID NO: 13131

TFF2
Trefoil factor 2
SEQ ID NO: 13132

TFF3
Trefoil factor 3 (intestinal)
SEQ ID NOS: 13133-

13135

TFPI
Tissue factor pathway inhibitor (lipoprotein-
SEQ ID NOS: 13136-

associated coagulation inhibitor)
13145

TFPI2
Tissue factor pathway inhibitor 2
SEQ ID NOS: 13146-

13147

TG
Thyroglobulin
SEQ ID NOS: 13148-

13157

TGFB1
Transforming growth factor, beta 1
SEQ ID NOS: 13158-

13159

TGFB2
Transforming growth factor, beta 2
SEQ ID NOS: 13160-

13161

TGFB3
Transforming growth factor, beta 3
SEQ ID NOS: 13162-

13163

TGFBI
Transforming growth factor, beta-induced,
SEQ ID NOS: 13164-

68 kDa
13171

TGFBR1
Transforming growth factor, beta receptor 1
SEQ ID NOS: 13172-

13181

TGFBR3
Transforming growth factor, beta receptor
SEQ ID NOS: 13182-

III
13188

THBS1
Thrombospondin 1
SEQ ID NOS: 13189-

13190

THBS2
Thrombospondin 2
SEQ ID NOS: 13191-

13193

THBS3
Thrombospondin 3
SEQ ID NOS: 13194-

13198

THBS4
Thrombospondin 4
SEQ ID NOS: 13199-

13200

THOC3
THO complex 3
SEQ ID NOS: 13201-

13210

THPO
Thrombopoietin
SEQ ID NOS: 13211-

13216

THSD4
Thrombospondin, type I, domain containing
SEQ ID NOS: 13217-

4
13220

THY1
Thy-1 cell surface antigen
SEQ ID NOS: 13221-

13226

TIE1
Tyrosine kinase with immunoglobulin-like
SEQ ID NOS: 13227-

and EGF-like domains 1
13228

TIMMDC1
Translocase of inner mitochondrial
SEQ ID NOS: 13229-

membrane domain containing 1
13236

TIMP1
TIMP metallopeptidase inhibitor 1
SEQ ID NOS: 13237-

13241

TIMP2
TIMP metallopeptidase inhibitor 2
SEQ ID NOS: 13242-

13246

TIMP3
TIMP metallopeptidase inhibitor 3
SEQ ID NO: 13247

TIMP4
TIMP metallopeptidase inhibitor 4
SEQ ID NO: 13248

TINAGL1
Tubulointerstitial nephritis antigen-like 1
SEQ ID NOS: 13249-

13251

TINF2
TERF1 (TRF1)-interacting nuclear factor 2
SEQ ID NOS: 13252-

13261

TLL2
Tolloid-like 2
SEQ ID NO: 13262

TLR1
Toll-like receptor 1
SEQ ID NOS: 13263-

13268

TLR3
Toll-like receptor 3
SEQ ID NOS: 13269-

13271

TM2D2
TM2 domain containing 2
SEQ ID NOS: 13272-

13277

TM2D3
TM2 domain containing 3
SEQ ID NOS: 13278-

13285

TM7SF3
Transmembrane 7 superfamily member 3
SEQ ID NOS: 13286-

13300

TM9SF1
Transmembrane 9 superfamily member 1
SEQ ID NOS: 13301-

13311

TMCO6
Transmembrane and coiled-coil domains 6
SEQ ID NOS: 13312-

13319

TMED1
Transmembrane p24 trafficking protein 1
SEQ ID NOS: 13320-

13326

TMED2
Transmembrane p24 trafficking protein 2
SEQ ID NOS: 13327-

13329

TMED3
Transmembrane p24 trafficking protein 3
SEQ ID NOS: 13330-

13333

TMED4
Transmembrane p24 trafficking protein 4
SEQ ID NOS: 13334-

13336

TMED5
Transmembrane p24 trafficking protein 5
SEQ ID NOS: 13337-

13340

TMED7
Transmembrane p24 trafficking protein 7
SEQ ID NOS: 13341-

13342

TMED7-
TMED7-TICAM2 readthrough
SEQ ID NOS: 13343-

TICAM2

13344

TMEM108
Transmembrane protein 108
SEQ ID NOS: 13345-

13353

TMEM116
Transmembrane protein 116
SEQ ID NOS: 13354-

13365

TMEM119
Transmembrane protein 119
SEQ ID NOS: 13366-

13369

TMEM155
Transmembrane protein 155
SEQ ID NOS: 13370-

13373

TMEM168
Transmembrane protein 168
SEQ ID NOS: 13374-

13379

TMEM178A
Transmembrane protein 178A
SEQ ID NOS: 13380-

13381

TMEM179
Transmembrane protein 179
SEQ ID NOS: 13382-

13387

TMEM196
Transmembrane protein 196
SEQ ID NOS: 13388-

13392

TMEM199
Transmembrane protein 199
SEQ ID NOS: 13393-

13396

TMEM205
Transmembrane protein 205
SEQ ID NOS: 13397-

13410

TMEM213
Transmembrane protein 213
SEQ ID NOS: 13411-

13414

TMEM25
Transmembrane protein 25
SEQ ID NOS: 13415-

13431

TMEM30C
Transmembrane protein 30C
SEQ ID NO: 13432

TMEM38B
Transmembrane protein 38B
SEQ ID NOS: 13433-

13437

TMEM44
Transmembrane protein 44
SEQ ID NOS: 13438-

13447

TMEM52
Transmembrane protein 52
SEQ ID NOS: 13448-

13452

TMEM52B
Transmembrane protein 52B
SEQ ID NOS: 13453-

13455

TMEM59
Transmembrane protein 59
SEQ ID NOS: 13456-

13463

TMEM67
Transmembrane protein 67
SEQ ID NOS: 13464-

13475

TMEM70
Transmembrane protein 70
SEQ ID NOS: 13476-

13478

TMEM87A
Transmembrane protein 87A
SEQ ID NOS: 13479-

13488

TMEM94
Transmembrane protein 94
SEQ ID NOS: 13489-

13504

TMEM95
Transmembrane protein 95
SEQ ID NOS: 13505-

13507

TMIGD1
Transmembrane and immunoglobulin
SEQ ID NOS: 13508-

domain containing 1
13509

TMPRSS12
Transmembrane (C-terminal) protease,
SEQ ID NOS: 13510-

serine 12
13511

TMPRSS5
Transmembrane protease, serine 5
SEQ ID NOS: 13512-

13523

TMUB1
Transmembrane and ubiquitin-like domain
SEQ ID NOS: 13524-

containing 1
13530

TMX2
Thioredoxin-related transmembrane protein
SEQ ID NOS: 13531-

2
13538

TMX3
Thioredoxin-related transmembrane protein
SEQ ID NOS: 13539-

3
13546

TNC
Tenascin C
SEQ ID NOS: 13547-

13555

TNFAIP6
Tumor necrosis factor, alpha-induced
SEQ ID NO: 13556

protein 6

TNFRSF11A
Tumor necrosis factor receptor superfamily,
SEQ ID NOS: 13557-

member 11a, NFKB activator
13561

TNFRSF11B
Tumor necrosis factor receptor superfamily,
SEQ ID NOS: 13562-

member 11b
13563

TNFRSF12A
Tumor necrosis factor receptor superfamily,
SEQ ID NOS: 13564-

member 12A
13569

TNFRSF14
Tumor necrosis factor receptor superfamily,
SEQ ID NOS: 13570-

member 14
13576

TNFRSF18
Tumor necrosis factor receptor superfamily,
SEQ ID NOS: 13577-

member 18
13580

TNFRSF1A
Tumor necrosis factor receptor superfamily,
SEQ ID NOS: 13581-

member 1A
13589

TNFRSF1B
Tumor necrosis factor receptor superfamily,
SEQ ID NOS: 13590-

member 1B
13591

TNFRSF25
Tumor necrosis factor receptor superfamily,
SEQ ID NOS: 13592-

member 25
13603

TNFRSF6B
Tumor necrosis factor receptor superfamily,
SEQ ID NO: 13604

member 6b, decoy

TNFSF11
Tumor necrosis factor (ligand) superfamily,
SEQ ID NOS: 13605-

member 11
13609

TNFSF12
Tumor necrosis factor (ligand) superfamily,
SEQ ID NOS: 13610-

member 12
13611

TNFSF12-
TNFSF12-TNFSF13 readthrough
SEQ ID NO: 13612

TNFSF13

TNFSF15
Tumor necrosis factor (ligand) superfamily,
SEQ ID NOS: 13613-

member 15
13614

TNN
Tenascin N
SEQ ID NOS: 13615-

13617

TNR
Tenascin R
SEQ ID NOS: 13618-

13620

TNXB
Tenascin XB
SEQ ID NOS: 13621-

13627

TOMM7
Translocase of outer mitochondrial
SEQ ID NOS: 13634-

membrane 7 homolog (yeast)
13637

TOP1MT
Topoisomerase (DNA) I, mitochondrial
SEQ ID NOS: 13638-

13652

TOR1A
Torsin family 1, member A (torsin A)
SEQ ID NO: 13653

TOR1B
Torsin family 1, member B (torsin B)
SEQ ID NOS: 13654-

13655

TOR2A
Torsin family 2, member A
SEQ ID NOS: 13656-

13662

TOR3A
Torsin family 3, member A
SEQ ID NOS: 13663-

13667

TPD52
Tumor protein D52
SEQ ID NOS: 13668-

13680

TPO
Thyroid peroxidase
SEQ ID NOS: 13681-

13691

TPP1
Tripeptidyl peptidase I
SEQ ID NOS: 13692-

13709

TPSAB1
Tryptase alpha/beta 1
SEQ ID NOS: 13710-

13712

TPSB2
Tryptase beta 2 (gene/pseudogene)
SEQ ID NOS: 13713-

13715

TPSD1
Tryptase delta 1
SEQ ID NOS: 13716-

13717

TPST1
Tyrosylprotein sulfotransferase 1
SEQ ID NOS: 13718-

13720

TPST2
Tyrosylprotein sulfotransferase 2
SEQ ID NOS: 13721-

13729

TRABD2A
TraB domain containing 2A
SEQ ID NOS: 13730-

13732

TRABD2B
TraB domain containing 2B
SEQ ID NO: 13733

TREH
Trehalase (brush-border membrane
SEQ ID NOS: 13734-

glycoprotein)
13736

TREM1
Triggering receptor expressed on myeloid
SEQ ID NOS: 13737-

cells 1
13740

TREM2
Triggering receptor expressed on myeloid
SEQ ID NOS: 13741-

cells 2
13743

TRH
Thyrotropin-releasing hormone
SEQ ID NOS: 13744-

13745

TRIM24
Tripartite motif containing 24
SEQ ID NOS: 13746-

13747

TRIM28
Tripartite motif containing 28
SEQ ID NOS: 13748-

13753

TRIO
Trio Rho guanine nucleotide exchange
SEQ ID NOS: 13754-

factor
13760

TRNP1
TMF1-regulated nuclear protein 1
SEQ ID NOS: 13761-

13762

TSC22D4
TSC22 domain family, member 4
SEQ ID NOS: 13763-

13766

TSHB
Thyroid stimulating hormone, beta
SEQ ID NOS: 13767-

13768

TSHR
Thyroid stimulating hormone receptor
SEQ ID NOS: 13769-

13776

TSKU
Tsukushi, small leucine rich proteoglycan
SEQ ID NOS: 13777-

13781

TSLP
Thymic stromal lymphopoietin
SEQ ID NOS: 13782-

13784

TSPAN3
Tetraspanin 3
SEQ ID NOS: 13785-

13790

TSPAN31
Tetraspanin 31
SEQ ID NOS: 13791-

13797

TSPEAR
Thrombospondin-type laminin G domain
SEQ ID NOS: 13798-

and EAR repeats
13801

TTC13
Tetratricopeptide repeat domain 13
SEQ ID NOS: 13802-

13808

TTC19
Tetratricopeptide repeat domain 19
SEQ ID NOS: 13809-

13814

TTC9B
Tetratricopeptide repeat domain 9B
SEQ ID NO: 13815

TTLL11
Tubulin tyrosine ligase-like family member
SEQ ID NOS: 13816-

11
13820

TTR
Transthyretin
SEQ ID NOS: 13821-

13823

TWSG1
Twisted gastrulation BMP signaling
SEQ ID NOS: 13824-

modulator 1
13826

TXNDC12
Thioredoxin domain containing 12
SEQ ID NOS: 13827-

(endoplasmic reticulum)
13829

TXNDC15
Thioredoxin domain containing 15
SEQ ID NOS: 13830-

13836

TXNDC5
Thioredoxin domain containing 5
SEQ ID NOS: 13837-

(endoplasmic reticulum)
13838

TXNRD2
Thioredoxin reductase 2
SEQ ID NOS: 13839-

13851

TYRP1
Tyrosinase-related protein 1
SEQ ID NOS: 13852-

13854

UBAC2
UBA domain containing 2
SEQ ID NOS: 13855-

13859

UBALD1
UBA-like domain containing 1
SEQ ID NOS: 13860-

13868

UBAP2
Ubiquitin associated protein 2
SEQ ID NOS: 13869-

13875

UBXN8
UBX domain protein 8
SEQ ID NOS: 13876-

13882

UCMA
Upper zone of growth plate and cartilage
SEQ ID NOS: 13883-

matrix associated
13884

UCN
Urocortin
SEQ ID NO: 13885

UCN2
Urocortin 2
SEQ ID NO: 13886

UCN3
Urocortin 3
SEQ ID NO: 13887

UGGT2
UDP-glucose glycoprotein
SEQ ID NOS: 13888-

glucosyltransferase 2
13893

UGT1A10
UDP glucuronosyltransferase 1 family,
SEQ ID NOS: 13894-

polypeptide A10
13895

UGT2A1
UDP glucuronosyltransferase 2 family,
SEQ ID NOS: 13896-

polypeptide A1, complex locus
13900

UGT2B11
UDP glucuronosyltransferase 2 family,
SEQ ID NO: 13901

polypeptide B11

UGT2B28
UDP glucuronosyltransferase 2 family,
SEQ ID NOS: 13902-

polypeptide B28
13903

UGT2B4
UDP glucuronosyltransferase 2 family,
SEQ ID NOS: 13904-

polypeptide B4
13907

UGT2B7
UDP glucuronosyltransferase 2 family,
SEQ ID NOS: 13908-

polypeptide B7
13911

UGT3A1
UDP glycosyltransferase 3 family,
SEQ ID NOS: 13912-

polypeptide A1
13917

UGT3A2
UDP glycosyltransferase 3 family,
SEQ ID NOS: 13918-

polypeptide A2
13921

UGT8
UDP glycosyltransferase 8
SEQ ID NOS: 13922-

13924

ULBP3
UL16 binding protein 3
SEQ ID NOS: 13925-

13926

UMOD
Uromodulin
SEQ ID NOS: 13927-

13938

UNC5C
Unc-5 netrin receptor C
SEQ ID NOS: 13939-

13943

UPK3B
Uroplakin 3B
SEQ ID NOS: 13944-

13946

USP11
Ubiquitin specific peptidase 11
SEQ ID NOS: 13947-

13950

USP14
Ubiquitin specific peptidase 14 (tRNA-
SEQ ID NOS: 13951-

guanine transglycosylase)
13957

USP3
Ubiquitin specific peptidase 3
SEQ ID NOS: 13958-

13973

UTS2
Urotensin 2
SEQ ID NOS: 13984-

13986

UTS2B
Urotensin 2B
SEQ ID NOS: 13987-

13992

UTY
Ubiquitously transcribed tetratricopeptide
SEQ ID NOS: 13993-

repeat containing, Y-linked
14005

UXS1
UDP-glucuronate decarboxylase 1
SEQ ID NOS: 14006-

14013

VASH1
Vasohibin 1
SEQ ID NOS: 14014-

14016

VCAN
Versican
SEQ ID NOS: 14017-

14023

VEGFA
Vascular endothelial growth factor A
SEQ ID NOS: 14024-

14049

VEGFB
Vascular endothelial growth factor B
SEQ ID NOS: 14050-

14052

VEGFC
Vascular endothelial growth factor C
SEQ ID NO: 14053

VGF
VGF nerve growth factor inducible
SEQ ID NOS: 14055-

14057

VIP
Vasoactive intestinal peptide
SEQ ID NOS: 14058-

14060

VIPR2
Vasoactive intestinal peptide receptor 2
SEQ ID NOS: 14061-

14064

VIT
Vitrin
SEQ ID NOS: 14065-

14072

VKORC1
Vitamin K epoxide reductase complex,
SEQ ID NOS: 14073-

subunit 1
14080

VLDLR
Very low density lipoprotein receptor
SEQ ID NOS: 14081-

14083

VMO1
Vitelline membrane outer layer 1 homolog
SEQ ID NOS: 14084-

(chicken)
14087

VNN1
Vanin 1
SEQ ID NO: 14088

VNN2
Vanin 2
SEQ ID NOS: 14089-

14102

VNN3
Vanin 3
SEQ ID NOS: 14103-

14114

VOPP1
Vesicular, overexpressed in cancer,
SEQ ID NOS: 14115-

prosurvival protein 1
14127

VPREB1
Pre-B lymphocyte 1
SEQ ID NOS: 14128-

14129

VPREB3
Pre-B lymphocyte 3
SEQ ID NOS: 14130-

14131

VPS37B
Vacuolar protein sorting 37 homolog B
SEQ ID NOS: 14132-

(S. cerevisiae)
14134

VPS51
Vacuolar protein sorting 51 homolog
SEQ ID NOS: 14135-

(S. cerevisiae)
14146

VSIG1
V-set and immunoglobulin domain
SEQ ID NOS: 14147-

containing 1
14149

VSIG10
V-set and immunoglobulin domain
SEQ ID NOS: 14150-

containing 10
14151

VSTM1
V-set and transmembrane domain
SEQ ID NOS: 14152-

containing 1
14158

VSTM2A
V-set and transmembrane domain
SEQ ID NOS: 14159-

containing 2A
14162

VSTM2B
V-set and transmembrane domain
SEQ ID NO: 14163

containing 2B

VSTM2L
V-set and transmembrane domain
SEQ ID NOS: 14164-

containing 2 like
14166

VSTM4
V-set and transmembrane domain
SEQ ID NOS: 14167-

containing 4
14168

VTN
Vitronectin
SEQ ID NOS: 14169-

14170

VWA1
Von Willebrand factor A domain containing
SEQ ID NOS: 14171-

1
14174

VWA2
Von Willebrand factor A domain containing
SEQ ID NOS: 14175-

2
14176

VWA5B2
Von Willebrand factor A domain containing
SEQ ID NOS: 14177-

5B2
14178

VWA7
Von Willebrand factor A domain containing
SEQ ID NO: 14179

7

VWC2
Von Willebrand factor C domain containing
SEQ ID NO: 14180

2

VWC2L
Von Willebrand factor C domain containing
SEQ ID NOS: 14181-

protein 2-like
14182

VWCE
Von Willebrand factor C and EGF domains
SEQ ID NOS: 14183-

14187

VWDE
Von Willebrand factor D and EGF domains
SEQ ID NOS: 14188-

14193

VWF
Von Willebrand factor
SEQ ID NOS: 14194-

14196

WDR25
WD repeat domain 25
SEQ ID NOS: 14197-

14203

WDR81
WD repeat domain 81
SEQ ID NOS: 14204-

14213

WDR90
WD repeat domain 90
SEQ ID NOS: 14214-

14221

WFDC1
WAP four-disulfide core domain 1
SEQ ID NOS: 14222-

14224

WFDC10A
WAP four-disulfide core domain 10A
SEQ ID NO: 14225

WFDC10B
WAP four-disulfide core domain 10B
SEQ ID NOS: 14226-

14227

WFDC11
WAP four-disulfide core domain 11
SEQ ID NOS: 14228-

14230

WFDC12
WAP four-disulfide core domain 12
SEQ ID NO: 14231

WFDC13
WAP four-disulfide core domain 13
SEQ ID NO: 14232

WFDC2
WAP four-disulfide core domain 2
SEQ ID NOS: 14233-

14237

WFDC3
WAP four-disulfide core domain 3
SEQ ID NOS: 14238-

14241

WFDC5
WAP four-disulfide core domain 5
SEQ ID NOS: 14242-

14243

WFDC6
WAP four-disulfide core domain 6
SEQ ID NOS: 14244-

14245

WFDC8
WAP four-disulfide core domain 8
SEQ ID NOS: 14246-

14247

WFIKKN1
WAP, follistatin/kazal, immunoglobulin,
SEQ ID NO: 14248

kunitz and netrin domain containing 1

WFIKKN2
WAP, follistatin/kazal, immunoglobulin,
SEQ ID NOS: 14249-

kunitz and netrin domain containing 2
14250

WIF1
WNT inhibitory factor 1
SEQ ID NOS: 14255-

14257

WISP1
WNT1 inducible signaling pathway protein
SEQ ID NOS: 14258-

1
14262

WISP2
WNT1 inducible signaling pathway protein
SEQ ID NOS: 14263-

2
14265

WISP3
WNT1 inducible signaling pathway protein
SEQ ID NOS: 14266-

3
14273

WNK1
WNK lysine deficient protein kinase 1
SEQ ID NOS: 14274-

14287

WNT1
Wingless-type MMTV integration site
SEQ ID NOS: 14288-

family, member 1
14289

WNT10B
Wingless-type MMTV integration site
SEQ ID NOS: 14290-

family, member 10B
14294

WNT11
Wingless-type MMTV integration site
SEQ ID NOS: 14295-

family, member 11
14297

WNT16
Wingless-type MMTV integration site
SEQ ID NOS: 14298-

family, member 16
14299

WNT2
Wingless-type MMTV integration site
SEQ ID NOS: 14300-

family member 2
14302

WNT3
Wingless-type MMTV integration site
SEQ ID NO: 14303

family, member 3

WNT3A
Wingless-type MMTV integration site
SEQ ID NO: 14304

family, member 3A

WNT5A
Wingless-type MMTV integration site
SEQ ID NOS: 14305-

family, member 5A
14308

WNT5B
Wingless-type MMTV integration site
SEQ ID NOS: 14309-

family, member 5B
14315

WNT6
Wingless-type MMTV integration site
SEQ ID NO: 14316

family, member 6

WNT7A
Wingless-type MMTV integration site
SEQ ID NO: 14317

family, member 7A

WNT7B
Wingless-type MMTV integration site
SEQ ID NOS: 14318-

family, member 7B
14322

WNT8A
Wingless-type MMTV integration site
SEQ ID NOS: 14323-

family, member 8A
14326

WNT8B
Wingless-type MMTV integration site
SEQ ID NO: 14327

family, member 8B

WNT9A
Wingless-type MMTV integration site
SEQ ID NO: 14328

family, member 9A

WNT9B
Wingless-type MMTV integration site
SEQ ID NOS: 14329-

family, member 9B
14331

WSB1
WD repeat and SOCS box containing 1
SEQ ID NOS: 14332-

14341

WSCD1
WSC domain containing 1
SEQ ID NOS: 14342-

14351

WSCD2
WSC domain containing 2
SEQ ID NOS: 14352-

14355

XCL1
Chemokine (C motif) ligand 1
SEQ ID NO: 14356

XCL2
Chemokine (C motif) ligand 2
SEQ ID NO: 14357

XPNPEP2
X-prolyl aminopeptidase (aminopeptidase
SEQ ID NOS: 14358-

P) 2, membrane-bound
14359

XXbac-

SEQ ID NOS: 679- 680

BPG116M5.17

XXbac-

SEQ ID NO: 681

BPG181M17.5

XXbac-

SEQ ID NO: 682

BPG32J3.20

XXYLT1
Xyloside xylosyltransferase 1
SEQ ID NOS: 14360-

14365

XYLT1
Xylosyltransferase I
SEQ ID NO: 14366

XYLT2
Xylosyltransferase II
SEQ ID NOS: 14367-

14372

ZFYVE21
Zinc finger, FYVE domain containing 21
SEQ ID NOS: 14373-

14377

ZG16
Zymogen granule protein 16
SEQ ID NO: 14378

ZG16B
Zymogen granule protein 16B
SEQ ID NOS: 14379-

14382

ZIC4
Zic family member 4
SEQ ID NOS: 14383-

14391

ZNF207
Zinc finger protein 207
SEQ ID NOS: 14392-

14402

ZNF26
Zinc finger protein 26
SEQ ID NOS: 14403-

14406

ZNF34
Zinc finger protein 34
SEQ ID NOS: 14407-

14410

ZNF419
Zinc finger protein 419
SEQIDNOS: 14411-

14425

ZNF433
Zinc finger protein 433
SEQ ID NOS: 14426-

14435

ZNF449
Zinc finger protein 449
SEQ ID NOS: 14436-

14437

ZNF488
Zinc finger protein 488
SEQ ID NOS: 14438-

14439

ZNF511
Zinc finger protein 511
SEQ ID NOS: 14440-

14441

ZNF570
Zinc finger protein 570
SEQ ID NOS: 14442-

14447

ZNF691
Zinc finger protein 691
SEQ ID NOS: 14448-

14455

ZNF98
Zinc finger protein 98
SEQ ID NOS: 14456-

14459

ZPBP
Zona pellucida binding protein
SEQ ID NOS: 14460-

14463

ZPBP2
Zona pellucida binding protein 2
SEQ ID NOS: 14464-

14467

ZSCAN29
Zinc finger and SCAN domain containing
SEQ ID NOS: 14468-

29
14474

Cas-Clover

The disclosure provides a composition comprising a guide RNA and a fusion protein or a sequence encoding the fusion protein wherein the fusion protein comprises a dCas9 and a Clo051 endonuclease or a nuclease domain thereof.

Small Cas9 (SaCas9)

The disclosure provides compositions comprising a small, Cas9 (Cas9) operatively-linked to an effector. In certain embodiments, the disclosure provides a fusion protein comprising, consisting essentially of or consisting of a DNA localization component and an effector molecule, wherein the effector comprises a small, Cas9 (Cas9). In certain embodiments, a small Cas9 construct of the disclosure may comprise an effector comprising a type IIS endonuclease.

Amino acid sequence of Staphylococcus aureus Cas9 with an active catalytic site.

(SEQ ID NO: 17051)

1
mkrnyilgld igitsvgygi idyetrdvid agvrlfkean

vennegrrsk rgarrlkrrr

61
rhriqrvkkl lfdynlltdh selsginpye arvkglsgkl

seeefsaall hlakrrgvhn

121
vneveedtgn elstkeqisr nskaleekyv aelqlerlkk

dgevrgsinr fktsdvvkea

181
kgllkvqkay hqldqsfidt yidlletrrt yyegpgegsp

fgwkdikewy emlmghctyf

241
peelrsvkya ynadlynaln dlnnlvitrd enekleyyek

fqiienvfkq kkkptlkqia

301
keilvneedi kgyrvtstgk peftnlkvyh dikditarke

iienaelldq iakiltiyqs

361
sediqeeltn lnseltqeei egisnikgyt gthnlslkai

nlildelwht ndnqiaifnr

421
lklvpkkvdl sqqkeipttl vddfilspvv krsfiqsikv

inaiikkygl pndiiielar

481
eknskdaqkm inemqkrnrq tnerieeiir ttgkanakyl

iekiklhdmq egkclyslea

541
ipledllnnp fnyevdhiip rsvsfdnsfn nkvlvkqeen

skkgnrtpfq ylsssdskis

601
yetfkkhiln lakgkgrisk tkkeylleer dinrfsvqkd

finrnlvdtr yatrglmnll

661
rsyfrvnnld vkvksinggf tsflrrkwkf kkernkgykh

haedaliian adfifkewkk

721
ldkakkvmen qmfeekqaes mpeieteqey keifitphqi

khikdfkdyk yshrvdkkpn

781
relindtlys trkddkgntl ivnnlnglyd kdndklkkli

nkspekllmy hhdpqtyqkl

841
klimeqygde knplykyyee tgnyltkysk kdngpvikki

kyygnklnah lditddypns

901
rnkvvklslk pyrfdvyldn gvykfvtvkn ldvikkenyy

evnskcyeea kklkkisnqa

961
efiasfynnd likingelyr vigvnndlln rievnmidit

yreylenmnd krppriikti

1021
asktqsikky stdilgnlye vkskkhpqii kkg

Inactivated Small Cas9 (dSaCas9)

The disclosure provides compositions comprising an inactivated, small, Cas9 (dSaCas9) operatively-linked to an effector. In certain embodiments, the disclosure provides a fusion protein comprising, consisting essentially of or consisting of a DNA localization component and an effector molecule, wherein the effector comprises a small, inactivated Cas9 (dSaCas9). In certain embodiments, a small, inactivated Cas9 (dSaCas9) construct of the disclosure may comprise an effector comprising a type IIS endonuclease.

dSaCas9 Sequence: D10A and N580A mutations (bold, capitalized, and underlined) inactivate the catalytic site.

(SEQ ID NO: 17052)

1
mkrnyilglA igitsvgvgi idyetrdvid agvrlfkean

vennegrrsk rgarrlkrrr

61
rhriqrvkkl lfdynlltdh selsginpye arvkglsqkl

seeefsaall hlakrrgvhn

121
vneveedtgn elstkeqisr nskaleekyv aelqlerlkk

dgevrgsinr fktsdyvkea

181
kqllkvqkay hqldgsfidt yidlletrrt yyegpgegsp

fgwkdikewy emlmghctyf

241
peelrsvkya ynadlynaln dlnnlvitrd enekleyyek

fqiienvfkq kkkptlkgia

301
keilvneedi kgyrvtstgk peftnlkvyh dikditarke

iienaelldq iakiltiyqs

361
sediqeeltn lnseltqeei egisnlkgyt gthnlslkai

nlildelwht ndnqiaifnr

421
lklvpkkvdl sqqkeipttl vddfilspvv krsfiqsikv

inaiikkygl pndiiielar

481
eknskdaqkm inemqkrnrq tnerieeiir ttgkenakyl

iekiklhdmq egkclyslea

541
ipledllnnp fnyevdhiip rsvsfdnsfn nkvlvkqeeA

skkgnrtpfq ylsssdskis

601
yetfkkhiln lakgkgrisk tkkeylleer dinrftvqkd

finrnlvdtr yatrglmnll

661
rsyfrvnnld vkvksinggf tsflrrkwkf kkernkgykh

haedaliian adfifkewkk

721
ldkakkvmen qmfeekqaes mpeieteqey keifitphqi

khikdfkdyk yshrvdkkpn

781
relindtlys trkddkgntl ivnnlnglyd kdndklkkli

nkspekllmy hhdpqtyqkl

841
klimegygde knplykyyee tgnyltkysk kdngpvikki

kyygnklnah lditddypns

901
rnkvvklslk pyrfdvyldn gvykfvtvkn ldvikkenyy

evnskoyeea kklkkisnqa

961
efiasfynnd likingelyr vigvnndlln rievnmidit

yreylenmnd krppriikti

1021
asktqsikky stdilgnlye vkskkhpqii kkg

Inactivated Cas9 (dCas9)

The disclosure provides compositions comprising an inactivated Cas9 (dCas9) operatively-linked to an effector. In certain embodiments, the disclosure provides a fusion protein comprising, consisting essentially of or consisting of a DNA localization component and an effector molecule, wherein the effector comprises an inactivated Cas9 (dCas9). In certain embodiments, an inactivated Cas9 (dCas9) construct of the disclosure may comprise an effector comprising a type IIS endonuclease.

In certain embodiments, the dCas9 of the disclosure comprises a dCas9 isolated or derived from Staphyloccocus pyogenes. In certain embodiments, the dCas9 comprises a dCas9 with substitutions at positions 10 and 840 of the amino acid sequence of the dCas9 which inactivate the catalytic site. In certain embodiments, these substitutions are D10A and H840A. In certain embodiments, the amino acid sequence of the dCas9 comprises the sequence of:

(SEQ ID NO: 17053)

1
XDKKYSIGLA IGTNSVGWAV ITDEYKVPSK KFKVLGNTDR

HSIKKNLIGA LLFDSGETAE

61
ATRLKRTARR RYTRRKNRIC YLQEIFSNEM AKVDDSFFHR

LEESFLVEED KKHERHPIFG

121
NIVDEVAYHE KYPTIYHLRK KLVDSTDKAD LRLIYLALAH

MIKFRGHFLI EGDLNPDNSD

181
VDKLFIQLVQ TYNQLFEENP INASGVDAKA ILSARLSKSR

RLENLIAQLP GEKKNGLFGN

241
LIALSLGLTP NFKSNFDLAE DAKLQLSKDT YDDDLDNLLA

QIGDQYADLF LAAKNLSDAI

301
LLSDILRVNT EITKAPLSAS MIKRYDEHHQ DLTLLKALVR

QQLPEKYKEI FFDQSKNGYA

361
GYIDGGASQE EFYKFIKPIL EKMDGTEELL VKLNREDLLR

KQRTFDNGSI PHQILGELH

421
AILRRQEDFY PFLKDNREKI EKILTFRIPY YVGPLARGNS

RFAWMTRKSE ETITPWNFEE

481
VVDKGASAQS FIERMTNFDK NLPNEKVLPK HSLLYEYFTV

YNELTKVKYV TEGMRKPAFL

541
SGEQKKAIVD LLFKTNRKVT VKQLKEDYFK KIECFDSVEI

SGVEDRFNAS LGTYHDLLKI

601
IKDKDFLDNE ENEDILEDIV LTLTLFEDRE MIEERLKTYA

HLFDDKVMKQ LKRRRYTGWG

661
RLSRKLINGI RDKQSGKTIL DFLKSDGFAN RNFMQLIHDD

SLTFKEDIQK AQVSGQGDSL

721
HEHIANLAGS PAIKKGILQT VKVVDELVKV MGRHKPENIV

IEMARENQTT QKGQKNSRER

781
MKRIEEGIKE LGSQILKEHP VENTQLQNEK LYLYYLQNGR

DMYVDQELDI NRLSDYDVDA

841
IVPQSFLKDD SIDNKVLTRS DKNRGKSDNV PSEEVVKKMK

NYWRQLLNAK LITQRKFDNL

901
TKAERGGLSE LDKAGFIKRQ LVETRQITKH VAQILDSRMN

TKYDENDKLI REVKVITLKS

961
KLVSDERKDF QFYKVREINN YHHAHDAYLN AVVGTALIKK

YPKLESEFVY GDYKVYDVRK

1021
MIAKSEQEIG KATAKYFFYS NIMNFFKTEI TLANGEIRKR

PLIETNGETG EIVWDKGRDF

1081
ATVRKVLSMP QVNIVKKTEV QTGGESKESI LPKRNSDKLI

ARKKDWDPKK YGGFDSPTVA

1141
YSVLVVAKVE KGKSKKLKSV KELLGITIME RSSFEKNPID

FLEAKGYKEV KKDLIIKLPK

1201
YSLFELENGR KRMLASAGEL QKGNELALPS KYVNFLYLAS

HYEKLKGSPE DNEQKQLFVE

1261
QHKHYIDEII EQISEFSKRV ILADANLDKV LSAYNKHRDK

PIREQAENII HLFTLTNLGA

1321
PAAFKYFDTT IDRKRYTSTK EVLDATLIHQ SITGLYETRI

DLSQLGGD.

In certain embodiments, the amino acid sequence of the dCas9 comprises the sequence of:

(SEQ ID NO: 17054)

1
MDKKYSIGLA IGTNSVGWAV ITDEYKVPSK KFKVLGNTDR

HSIKKNLIGA LLFDSGETAE

61
ATPLKRTARR RYTRRKNPIC YLQEIFSNEM AKVDDSFFER

LEESELVEED KKHERHPIFG

121
NIVDEVAYHE KYPTIYHLRK KLVDSTDKAD LRLIYLALSH

MIKFRGHFLI EGDLNPDNSD

181
VDKLFIQLVQ TYNOLFEENP INASGVDAKA ILSARLSKSR

RLENLIAQLP GEKKNGLFGN

241
LIALSLGLTP NEKSNFDLAE DAKLQLSKDT YDDDLDNLLA

QIGDQYADLF LAAKNLSDAI

301
LLSDILRVNT EITKAPLSAS MIKRYDEHHQ DLTLLKALVR

QQLPEKYKEI FFDQSKNGYA

361
GYIDGGASQE EFYKFIKPIL EKMDGTEELL VKLNREDLLR

KQRTFDNGSI PHQIHLGELH

421
AILPPQEDFY PFLKDNREKI EKILTFRIPY YVGPLARGNS

RFAWMTRKSE ETITPWNFEE

481
YVDKGASAQS FIERMTNFDK NLPNEKVLPK HSLLYEYFTV

YNELTKVKYV TEGMRKPAFL

541
SGEQKKAIVD LLFKTNRKVT VKQLKEDYFK KIECFDSVEI

SGVEDRFNAS LGTYHDLLKI

601
IKDKDFLDNE ENEDILEDIV LTLTLFEDRE MIEEPIKTYA

HLFDDKVMKQ LKRRRYTGWG

661
RLSRKLINGI RDKQSGKTIL DFLKSDGFAN RNFMQLIHDD

SLTFKEDIQK AQVSGQGDSL

721
HEHIANLAGS PAIKKGILQT VKVVDELVKV MGRHKPENIV

IEMARENQTT QKGQKNSRER

781
MKRIEEGIKE LGSQILKEHP VENTQLQNEK LYLYYLQNGR

DMYVDQELDI NRLSDYDVDA

841
IVPQSFLKDD SIDNKVLTRS DKNRGKSDNV PSEEVVKKMK

NYWRQLLNAK LITQRKFDNL

901
TKAERGGLSE LDKAGFIKRQ LVETRQITKH VAQILDSRMN

TKYDENDKLI REVKVITLKS

961
KLVSDFRKDF QFYKVREINN YHHAHDAYLN AVVGTALIKK

YPKLESEFVY GDYKVYDVRK

1021
MIAKSEQEIG KATAKYFFYS NIMNFFKTEI TLANGEIRKR

PLIETNGETG EIVWDKGRDF

1081
ATVRKVLSMP QVNIVKKTEV QTGGFSKESI LPKRNSDKLI

ARKKDWDPKK YGGFDSPTVA

1141
YSVLVVAKVE KGKSKKLKSV KELLGITIME RSSFEKNPID

FLEAKGYKEV KKDLIIKLPK

1201
YSLFELENGR KRMLASAGEL QKGNELALPS KYVNFLYLAS

HYEKLKGSPE DNEQKQLFVE

1261
QHKHYLDEII EQISEFSKRV ILADANLDKV LSAYNKHRDK

PIREQAENII HLFTLTNLGA

1321
PAAFKYFDTT IDRKRYTSTK EVLDATLIHQ SITGLYETRI

DLSQLGGD.

Clo051 Endonuclease

An exemplary Clo051 nuclease domain may comprise, consist essentially of or consist of, the amino acid sequence of:

(SEQ ID NO: 17055)

EGIKSNISILKDELRGQISHISHEYLSLIDLAFDSKQNRLFEMKVLELLV

NEYGFKGRHLGGSRKPDGIVYSTTLEDNFGIIVDTKAYSEGYSLPISQAD

EMERYVRENSNRDEEVNPNKWWENFSEEVKKYYFVFISGSFKGKFEEQLR

RLSMTTGVNGSAVNVVNLLLGAEKIRSGEMTIEELERAMFNNSEFILKY.

Cas-Clover Fusion Protein

In certain embodiments, an exemplary dCas9-Clo051 fusion protein (embodiment 1) may comprise, consist essentially of or consist of, the amino acid sequence of (Clo051 sequence underlined, linker bold italics, dCas9 sequence (Streptoccocus pyogenes) in italics):

(SEQ ID NO: 17056)

MAPKKKRKVEGIKSNISLLKDELRGQISHISHEYLSLIDLAFDSKQNRLFE

MKVLELLVNEYGFKGRHLGGSRKPDGINYSTTLEDNFGIIVDTKAYSEGYS

LPISQADEMERYVRENSNRDEEVNPNKWWENFSEEVKKYYFVFISGSFKGK

FEEQLRRLSMTTGVNGSAVNVVNLLLGAEKIRSGEMTIEELERAMFNNSEF

ILKY
custom-character

DKKYSIGLAIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSI

KKNLIGALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVD

DSFFHRLEESFLVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDS

TDKADLRLIYLALAHMIKFRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLF

EENPINASGVDAKAILSARLSKSRRLENLIAQLPGEKKNGLFGNLIALSLG

LTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQIGDQYADLFLAAKNLSD

AILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVRQQLPEKYKE

IFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLR

KQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYY

VGPLARGNSRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNL

PNEKVLPKHSLLYEYFTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLF

KTNRKVTVKQLKEDYFKKIECFDSVEISGVEDRFNASLGTYHDLLKIIKDK

DFLDNEENEDILEDIVLTLTLFEDREMIEERLKTYAHLFDDKVMKQLKRRR

YTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFMQLIHDDSLTFKE

DIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGRHKPE

NIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQN

EKLYLYYLQNGRDMYVDQELDINRLSDYDVDAIVPQSFLKDDSIDNKVLTR

SDKNRGKSDNVPSEEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSE

LDKAGFIKRQLVETRQITKHVAQILDSRMNTKYDENDKLIREVKVITLKSK

LVSDFRKDFQFYKVREINNYHHAHDAYLNAVVGTALIKKYPKLESEFVYGD

YKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKTEITLANGEIRKRPLI

ETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSKESILPKR

NSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLG

ITIMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLAS

AGELQKGNELALPSMYVNFLYLASHYEKLKGSPEDNEQKQLPVEQHKHYLD

EIIEQSSBFSKRVILADANLDKVLSAYNKHRDKPIREQAENIIHLFTLTNL

GAPAAFKYFDTTIDRKRYTSTKEVLDATLIHQSITGLYETRIDLSQLGGDG

SPKKKRKVSS.

In certain embodiments, an exemplary dCas9-Clo051 fusion protein (embodiment 1) may comprise, consist essentially of or consist of, the nucleic acid sequence of (dCas9 sequence derived from Streptoccocus pyogenes):

(SEQ ID NO: 17057)

1
atggcaccaa agaagaaaag aaaagtggag ggcatcaagt

caaacatcag cctgctgaaa

61
gacgaactgc ggggacagat tagtcacatc agtcacgagt

acctgtcact gattgatctg

121
gccttcgaca gcaagcagaa tagactgttt gagatgaaag

tgctggaact gctggtcaac

181
gagtatggct tcaagggcag acatctgggc gggtctagga

aacctgacgg catcgtgtac

241
agtaccacac tggaagacaa cttcggaatc attgtcgata

ccaaggctta ttccgagggc

301
tactctctgc caattagtca ggcagatgag atggaaaggt

acqtgcgcga aaactcaaat

361
agggacgagg aagtcaaccc caataagtgg tgggagaatt

tcagcgagga agtgaagaaa

421
tactacttcg tctttatctc aggcagcttc aaagggaagt

ttgaggaaca gctgcggaga

481
ctgtccatga ctaccggggt gaacggatct gctgtcaacg

tggtcaatct gctgctgggc

541
gcagaaaaga tcaggtccgg ggagatgaca attgaggaac

tggaacgcgc catgttcaac

601
aattctgagt ttatcctgaa gtatggaggc gggggaagcg

ataagaaata ctccatcgga

661
ctggccattg gcaccaattc cgtgggctgg gctgtcatca

cagacgagta caaggtgcca

721
agcaagaagt tcaaggtcct ggggaacacc gatcgccaca

gtatcaagaa aaatctgatt

781
ggagccctgc tgttcgactc aggcgagact gctgaagcaa

cccgactgaa gcggactgct

841
aggcgccgat atacccggag aaaaaatcgg atctgctacc

tgcaggaaat tttcagcaac

901
gagatggcca aggtggacga tagtttcttt caccgcctgg

aggaatcatt cctggtggag

961
gaagataaga aacacgagcg gcatcccatc tttggcaaca

ttgtggacga agtcgcttat

1021
cacgagaagt accctactat ctatcatctg aggaagaaac

tggtggactc caccgataag

1081
gcagacctgc gcctgatcta tctggccctg gctcacatga

tcaagttccg ggggcatttt

1141
ctgatcgagg gagatctgaa ccctgacaat tctgatgtgg

acaagctgtt catccagctg

1201
gtccagacat acaatcagct gtttgaggaa aacccaatta

atgcctcagg cgtggacgca

1261
aaggccatcc tgagcgccag actgtccaaa tctaggcgcc

tggaaaacct gatcgctcag

1321
ctgccaggag agaagaaaaa cggcctgttt gqqaatctga

ttgcactgtc cctgggcctg

1381
acacccaact tcaagtctaa ttttgatctg gccgaggacg

ctaagctgca gctgtccaaa

1441
gacacttatg acgatgacct ggataacctg ctggctcaga

tcggcgatca gtacgcagac

1501
ctgttcctgg ccgctaagaa tctgagtgac gccatcctgc

tgtcagatat tctgcgcgtg

1561
aacacagaga ttactaaggc cccactgagt gcttcaatga

tcaaaagata tgacgagcac

1621
catcaggatc tgaccctgct gaaggctctg gtgaggcagc

agctgcccga gaaatacaag

1681
gaaatcttct ttgatcagag caagaatgga tacgccggct

atattgacgg cggggcttcc

1741
caggaggagt tctacaagtt catcaagccc attctggaaa

agatggacgg caccgaggaa

1801
ctgctggtga agctgaatcg ggaggacctg ctgagaaaac

agaggacatt tgataacgga

1861
agcatccctc accagattca tctgggcgaa ctgcacgcca

tcctgcgacg gcaggaggac

1921
ttctacccat ttctgaagga taaccgcqag aaaatcgaaa

agatcctgac cttcagaatc

1981
ccctactatg tggggcctct ggcacgggga aataqtagat

ttgcctggat gacaagaaag

2041
tcagaggaaa ctatcacccc ctggaacttc gaggaagtgg

tcgataaagg cgctagcgca

2101
cagtccttca ttgaaaggat gacaaatttt gacaagaacc

tgccaaatga gaaggtgctg

2161
cccaaacaca gcctgctgta cgaatatttc acagtgtata

acgagctgac taaagtgaag

2221
tacgtcaccg aagggatgcg caagcccgca ttcctgtccg

gagagcagaa gaaagccatc

2281
gtggacctgc tgtttaagac aaatcggaaa gtgactgtca

aacagctgaa ggaagactat

2341
ttcaagaaaa ttgagtgttt cgattcagtg gaaatcagcg

gcgtcgagga caggtttaac

2401
gcctccctgg ggacctacca cgatctgctg aagatcatca

aggataagga cttcctggac

2461
aacgaggaaa atgaggacat cctggaggac attgtgctga

cactgactct gtttgaggat

2521
cgcgaaatga tcgaggaacg actgaagact tatgcccatc

tgttcgatga caaagtgatg

2581
aagcagctga aaagaaggcg ctacaccqga tggggacgcc

tqagccgaaa actgatcaat

2641
gggattagag acaagcagag cggaaaaact atcctggact

ttctgaagtc cgatggcttc

2701
gccaacagga acttcatgca gctgattcac gatgactctc

tgaccttcaa ggaggacatc

2761
cagaaagcac aggtgtctgg ccagggggac agtctgcacg

agcatatcgc aaacctggcc

2821
ggcagccccg ccatcaagaa agggattctg cagaccgtga

aggtggtgga cgaactggtc

2881
aaggtcatgg gacgacacaa acctgagaac atcgtgattg

agatggcccg cgaaaatcag

2941
acaactcaga agggccagaa aaacagtcga gaacggatga

agagaatcga ggaaggcatc

3001
aaggagctgg ggtcacagat cctgaaggag catcctgtgg

aaaacactca gctgcagaat

3061
gagaaactgt atctgtacta totgcagaat ggacgggata

tgtacgtgga ccaggagctg

3121
gatattaaca gactgagtga ttatgacgtg gatgccatcg

tccctcagag cttcctgaag

3181
gatgactcca ttgacaacaa ggtgctgacc aggtccgaca

agaaccgcgg caaatcagat

3241
aatgtqccaa gcgaggaagt ggtcaagaaa atgaaqaact

actggaggca gctgctgaat

3301
gccaagctga tcacacagcg gaaatttgat aacctgacta

aggcagaaag aggaggcctg

3361
tctgagctgg acaaggccgg cttcatcaag cggcagctgg

tggagacaag acagatcact

3421
aagcacgtcg ctcagattct ggatagcaga atgaacacaa

agtacgatga aaacgacaag

3481
ctgatcaggg aggtgaaagt cattactctg aaatccaagc

tggtgtctga ctttagaaag

3541
gatttccagt tttataaagt cagggagatc aacaactacc

accatgctca tgacgcatac

3601
ctgaacgcag tggtcgggac cgccctgatt aagaaatacc

ccaagctgga gtccgagttc

3661
gtgtacggag actataaagt gtacgatgtc cggaagatga

tcgccaaatc tgagcaggaa

3721
attggcaagg ccaccgctaa gtatttcttt tacagtaaca

tcatgaattt ctttaagacc

3781
gaaatcacac tggcaaatgg ggagatcaga aaaaggcctc

tgattgagac caacggggag

3841
acaggagaaa tcgtgtggga caagggaagg gattttgcta

ccgtgcgcaa agtcctgtcc

3901
atgccccaag tgaatattgt caagaaaact gaagtgcaga

ccgggggatt ctctaaggag

3961
agtattctgc ctaagcgaaa ctctgataaa ctgatcgccc

ggaagaaaga ctgggacccc

4021
aagaagtatg gcgggttcga ctctccaaca gtggcttaca

gtgtcctggt ggtcgcaaag

4081
gtggaaaagg ggaagtccaa gaaactgaag tctgtcaaag

agctgctggg aatcactatt

4141
atggaacgca gctccttcga gaagaatcct atcgattttc

tggaagccaa gggctataaa

4201
gaggtgaaga aagacctgat cattaagctg ccaaaatact

cactgtttga gctggaaaac

4261
ggacgaaagc gaatgctggc aagcgccgga gaactgcaga

agggcaatga gctggccctg

4321
ccctccaaat acgtgaactt cctgtatctg gctagccact

acgagaaact gaaggggtcc

4381
cctgaggata acgaacagaa gcagctgttt gtggagcagc

acaaacatta tctggacgag

4441
atcattgaac agatttcaga gttcagcaag agagtgatcc

tggctgacgc aaatctggat

4501
aaagtcctga gcgcatacaa caagcaccga gacaaaccaa

tccgggagca ggccgaaaat

4561
atcattcatc tgttcaccct gacaaacctg ggcgcccctg

cagccttcaa gtattttgac

4621
accacaatcg atcggaagag atacacttct accaaagagg

tqctggatgc taccctgatc

4681
caccagagta ttaccggcct gtatgagaca cgcatcgacc

tgtcacagct gggaggcgat

4741
gggagcccca agaaaaagcg gaaggtgtct agttaa.

In certain embodiments, the nucleic acid sequence encoding a dCas9-Clo051 fusion protein (embodiment 1) of the disclosure may comprise a DNA. In certain embodiments, the nucleic acid sequence encoding a dCas9-Clo051 fusion protein (embodiment 1) of the disclosure may comprise an RNA.

In certain embodiments, an exemplary dCas9-Clo051 fusion protein (embodiment 2) may comprise, consist essentially of or consist of, the amino acid sequence of (Clo051 sequence underlined, linker bold italics, dCas9 sequence (Streptoccocus pyogenes) in italics):

(SEQ ID NO: 17058)

1
MPKKKRKVEG IKSNISLLKD ELRGQISHIS HEYLSLIDLA

FDSKQNRLFE MKVLELLVNE

61

YGFKGRHLGG SRKPDGIVYS TTLEDNEGII VDTKAYSEGY

SLPISQADEM ERYVRENSNR

121

DEEVNPNKWW ENFSEEVKKY YFVFISGSFK GKFEEQLRRL

SMTTGVNGSA VNVVNLLLGA

181

EKIRSGEMTI EELERAMENN SEFILKY
custom-character

DRKYSIGL

AIGTNSVGWA VITDEYKVPS

241

KKFKVLGNTD RHSIKKNLIG ALLFDSGETA EATRLKRTAR

RRYTRRRNRI CYLQEIFSNE

301

MAKVDDSFFH RLEESFLVEE DKKHERHPIF GNIVDEVAYH

EKYPTIYHLR KKLVDSTDKA

361

DLRLIYLALA HMIKERGHFL IEGDLNPDNS DVDRIFIQLV

QTYNQLFEEN PINASGVDAK

421

AILSARLSKS RRLENLIAQL PGEKKNGLFG NLIALSLGLT

PNFKSNFDLA EDAKLQLSKD

481

TYDDDLDNLL AQIGDOYADL FLAAENLSDA ILLSDILRVN

TEITKAPLSA SMIKRYDEHH

541

QDLTILKALV RQQLPEKYKE IFFDQSRNGY AGYIDGGASQ

EEFYKFIKPI LEKMDGTEEL

601

LVKLNREDLL RKQRTEDNGS IPHQIHLGEL HAILRRQEDF

YPFLKDNREK IEKILTFRIP

661

YYVGPLARGN SRFAWMTRKS EETITPWNFE EVVDKGASAQ

SFIERMTNFD KNLPNEKVLP

721

KHSLLYEYFT VYNELTKVKY VTEGMRKPAF LSGEQRRAIV

DLLFKTNRKV TVKQLKEDYF

781

KKIECFDSVE TSGVEDRFNA SLGTYRDLLK IIKDKDFLDN

EENEDILEDI VLTLTLFEDR

841

EMIEEPLKTY AHLFDDKVMK QLKRRRYTGW GRLSRKLING

IRDKQSGKTI LDFLKSDGFA

901

NRNFMQLIHD DSLTFKEDIQ KAQVSGQGDS LHEHTANLAG

SPAIKKGTLQ TVKVVDELVK

961

VMGRHKPENI VIEMARENQT TQKGQKNSRE RMKRIEEGIK

ELGSQILKEH PVENTQLQNE

1021

KLYLYYLQNG RDMYVDQELD INRLSDYDVD AIVPQSFLKD

DSIDNKVLTR SDKNRGKSDN

1081

VPSEEVVKKM KNYWRQLLNA KLITQRKFDN LTRAERGGLS

ELDKAGFIKR QLVETRQITK

1141

HVAQILDSRM NTKYDENDKL IREVRVITLK SKLVSDFRKD

FQTYKVREIN NYHHAHDAYL

1201

NAVVGIALIK KYPKLESEFV YGDYKVYDVR KMIAKSEQEI

GKATAKYFFY SNIMNFFKTE

1261

ITLANGEIRK RPLIETNGET GEIVWDKGRD FATVRKVLSM

PQVNIVKKTE VQTGGFSKES

1321

ILPKRNSDKL IARKKDWDPK KYGGEDSPTV AYSVLVVAKV

EKGKSKKLKS VKELLGITIM

1381

ERSSFEKNPI DFLEAKGYRE VKKDLIIKLP KYSLFELENG

RKRMLASAGE LQKGNELALP

1441

SKYVNFLYLA SHYEKLKGSP EDNEQKQLFV EQHKHYLDEI

IEQISEFSKR VILADANLDK

1501

VLSAYNKHRD KPIREQAENI IHLFTLTNLG APAAFKYFDT

TIDRKRYTST KEVLDATLIH

1561

QSITGLYETR IDLSQLGGDG SPKKKRKV.

In certain embodiments, an exemplary dCas9-Clo051 fusion protein (embodiment 2) may comprise, consist essentially of or consist of, the nucleic acid sequence of (dCas9 sequence derived from Streptoccocus pyogenes):

(SEQ ID NO: 17059)

1
atgcctaaga agaagcggaa ggtggaaggc atcaaaagca

acatctccct cctgaaagac

61
gaactccggg ggcagattag ccacattagt cacgaatacc

tctccctcat cgacctggct

121
ttcgatagca agcagaacag gctctttgag atgaaagtgc

tggaactgct cgtcaatgag

181
tacgggttca agggtcgaca cctcggcgga tctaggaaac

cagacggcat cgtgtatagt

241
accacactgg aagacaactt tgggatcatt gtggatacca

aggcatactc tgagggttat

301
agtctgccca tttcacaggc cgacgagatg gaacggtacg

tgcgcgagaa ctcaaataga

361
gatgaggaag tcaaccctaa caagtggtgg gagaacttct

ctgaggaagt gaagaaatac

421
tacttcgtct ttatcagcgg gtccttcaag ggtaaatttg

aggaacagct caggagactg

481
agcatgacta ccggcgtgaa tggcagcgcc gtcaacgtgg

tcaatctgct cctgggcgct

541
gaaaagattc ggagcggaga gatgaccatc gaagagctgg

agagggcaat gtttaataat

601
agcgagttta tcctgaaata cggtggcggt ggatccgata

aaaagtattc tattggttta

661
gccatcggca ctaattccgt tggatgggct gtcataaccg

atgaatacaa agtaccttca

721
aagaaattta aggtgttggg gaacacagac cgtcattcga

ttaaaaagaa tcttatcggt

781
gccctcctat tcgatagtgg cgaaacggca gaggcgactc

gcctgaaacg aaccgctcgg

841
agaaggtata cacgtcgcaa gaaccgaata tgttacttac

aagaaatttt tagcaatgag

901
atggccaaag ttgacgattc tttctttcac cgtttggaag

agtccttcct tgtcgaagag

961
gacaagaaac atgaacggca ccccatcttt ggaaacatag

tagatgaggt ggcatatcat

1021
gaaaagtacc caacgattta tcacctcaga aaaaagctag

ttgactcaac tgataaagcg

1081
gacctgaggt taatctactt ggctcttgcc catatgataa

agttccgtgg gcactttctc

1141
attgagggtg atctaaatcc ggacaactcg gatgtcgaca

aactgttcat ccagttagta

1201
caaacctata atcagttgtt tgaagagaac cctataaatg

caagtggcgt ggatgcgaag

1261
gctattctta gcgcccgcct ctctaaatcc cgacggctag

aaaacctgat cgcacaatta

1321
cccggagaga agaaaaatgg gttgttcggt aaccttatag

cgctctcact aggcctgaca

1381
ccaaatttta agtcgaactt cgacttagct gaagatgcca

aattgcagct tagtaaggac

1441
acgtacgatg acgatctcga caatctactg gcacaaattg

gagatcagta tgcggactta

1501
tttttggctg ccaaaaacct tagcgatgca atcctcctat

ctgacatact gagagttaat

1561
actgagatta ccaaggcgcc gttatccgct tcaatgatca

aaaggtacga tgaacatcac

1621
caagacttga cacttctcaa ggccctagtc cgtcagcaac

tgcctgagaa atataaggaa

1681
atattctttg atcagtcgaa aaacgggtac gcaggttata

ttgacggcgg agcgagtcaa

1741
gaggaattct acaagtttat caaacccata ttagagaaga

tggatgggac ggaagagttg

1801
cttgtaaaac tcaatcgcga agatctactg cgaaagcagc

ggactttcga caacggtagc

1861
attccacatc aaatccactt aggcgaattg catgctatac

ttagaaggca ggaggatttt

1921
tatccgttcc tcaaagacaa tcgtgaaaag attgagaaaa

tcctaacctt tcgcatacct

1981
tactatgtgg gacccctggc ccgagggaac tctcggttcg

catggatgac aagaaagtcc

2041
gaagaaacga ttactccatg gaattttgag gaagttgtcg

ataaaggtgc gtcagctcaa

2101
tcgttcatcg agaggatgac caactttgac aagaatttac

cgaacgaaaa agtattgcct

2161
aagcacagtt tactttacga gtatttcaca gtgtacaatg

aactcacgaa agttaagtat

2221
gtcactgagg gcatgcgtaa acccgccttt ctaagcggag

aacagaagaa agcaatagta

2281
gatctgttat tcaagaccaa ccgcaaagtg acagttaagc

aattgaaaga ggactacttt

2341
aagaaaattg aatgcttcga ttctgtcgag atctccgggg

tagaagatcg atttaatgcg

2401
tcacttggta cgtatcatga cctcctaaag ataattaaag

ataaggactt cctggataac

2461
gaagagaatg aagatatctt agaagatata gtgttgactc

ttaccctctt tgaagatcgg

2521
gaaatgattg aggaaagact aaaaacatac gctcacctgt

tcgacgataa ggttatgaaa

2581
cagttaaaga ggcgtcgcta tacgggctgg ggacgattgt

cgcggaaact tatcaacggg

2641
ataagagaca agcaaagtgg taaaactatt ctcgattttc

taaagagcga cggcttcgcc

2701
aataggaact ttatgcagct gatccatgat gactctttaa

ccttcaaaga ggatatacaa

2761
aaggcacagg tttccggaca aggggactca ttgcacgaac

atattgcgaa tcttgctggt

2821
tcgccagcca tcaaaaaggg catactccag acagtcaaag

tagtggatga gctagttaag

2881
gtcatgggac gtcacaaacc ggaaaacatt gtaatcgaga

tggcacgcga aaatcaaacg

2941
actcagaagg ggcaaaaaaa cagtcgagag cggatgaaga

gaatagaaga gggtattaaa

3001
gaactgggca gccagatctt aaaggagcat cctgtggaaa

atacccaatt gcagaacgag

3061
aaactttacc tctattacct acaaaatgga agggacatgt

atgttgatca ggaactggac

3121
ataaaccgtt tatctgatta cgacgtcgat gccattgtac

cccaatcctt tttgaaggac

3181
gattcaatcg acaataaagt gcttacacgc tcggataaga

accgagggaa aagtgacaat

3241
gttccaagcg aggaagtcgt aaagaaaatg aagaactatt

ggcggcagct cctaaatgcg

3301
aaactgataa cgcaaagaaa gttcgataac ttaactaaag

ctgagagggg tggcttgtct

3361
gaacttgaca aggccggatt tattaaacgt cagctcgtgg

aaacccgcca aatcacaaag

3421
catgttgcac agatactaga ttcccgaatg aatacgaaat

acgacgagaa cgataagctg

3481
attcgggaag tcaaagtaat cactttaaag tcaaaattgg

tgtcggactt cagaaaggat

3541
tttcaattct ataaagttag ggagataaat aactaccacc

atgcgcacqa cgcttatctt

3601
aatgccgtcg tagggaccgc actcattaag aaatacccga

agctagaaag tgagtttgtg

3661
tatggtgatt acaaagttta tgacgtccgt aagatgatcg

cgaaaagcqa acaggagata

3721
ggcaaggcta cagccaaata cttcttttat tctaacatta

tgaatttctt taagacggaa

3781
atcactctgg caaacggaga gatacgcaaa cgacctttaa

ttgaaaccaa tggggagaca

3841
ggtgaaatcg tatgggataa gggccgggac ttcgcgacgg

tgagaaaagt tttgtccatg

3901
ccccaagtca acatagtaaa gaaaactgag gtgcagaccg

gagggttttc aaaggaatcg

3961
attcttccaa aaaggaatag tgataagctc atcgctcgta

aaaaggactg ggacccgaaa

4021
aagtacggtg gcttcgatag ccctacagtt gcctattctg

tcctagtagt ggcaaaagtt

4081
gagaagggaa aatccaagaa actgaagtca gtcaaagaat

tattggggat aacgattatg

4141
gagcgctcgt cttttgaaaa gaaccccatc gacttccttg

aggcgaaagg ttacaaggaa

4201
gtaaaaaagg atctcataat taaactacca aagtatagtc

tgtttgagtt agaaaatggc

4261
cgaaaacgga tgttggctag cgccggagag cttcaaaagg

ggaacgaact cgcactaccg

4321
tctaaatacg tgaatttcct gtatttagcg tcccattacg

agaagttgaa aggttcacct

4381
gaagataacg aacagaagca actttttgtt gagcagcaca

aacattatct cgacgaaatc

4441
atagagcaaa tttcggaatt cagtaagaga gtcatcctag

ctgatgccaa tctggacaaa

4501
gtattaagcg catacaacaa gcacagggat aaacccatac

gtgagcaggc ggaaaatatt

4561
atccatttgt ttactcttac caacctcggc gctccagccg

cattcaagta ttttgacaca

4621
acgatagatc gcaaacgata cacttctacc aaggaggtgc

tagacgcgac actgattcac

4681
caatccatca cgggattata tgaaactcgg atagatttgt

cacagcttgg gggtgacgga

4741
tcccccaaga agaagaggaa agtctga.

In certain embodiments, the nucleic acid sequence encoding a dCas9-Clo051 fusion protein (embodiment 2) of the disclosure may comprise a DNA. In certain embodiments, the nucleic acid sequence encoding a dCas9-Clo051 fusion protein (embodiment 2) of the disclosure may comprise an RNA.

Transposition Systems

Exemplary transposon/transposase systems of the disclosure include, but are not limited to, piggyBac® transposons and transposases, Sleeping Beauty transposons and transposases, Helraiser transposons and transposases and Tol2 transposons and transposases.

The piggyBac® transposase recognizes transposon-specific inverted terminal repeat sequences (ITRs) on the ends of the transposon, and moves the contents between the ITRs into TTAA chromosomal sites. The piggyBac® transposon system has no payload limit for the genes of interest that can be included between the ITRs. In certain embodiments, and, in particular, those embodiments wherein the transposon is a piggyBac transposon, the transposase is a piggyBac® or a Super piggyBac™ (SPB) transposase. In certain embodiments, and, in particular, those embodiments wherein the transposase is a Super piggyBac™ (SPB) transposase, the sequence encoding the transposase is an mRNA sequence.

In certain embodiments of the methods of the disclosure, the transposase enzyme is a piggyBac® (PB) transposase enzyme. The piggyBac® (PB) transposase enzyme may comprise or consist of an amino acid sequence at least 75%, 80%, 85%, 90%, 95%, 99% or any percentage in between identical to:

(SEQ ID NO: 14487)

1
MGSSIDDEHI LSALLQDDE LVGEDSDSEI SDHVSEDDVQ

SDTEEAFIDE VHEVQPTSSG

61
SEILDEQNVI EQPGSSLASN RILTLPQRTI PGKNKHCWST

SKSTRRSRVS ALNIVRSQRG

121
PTRMCRNIYD PLLCFKLFFT DEIISEIVKW TNAEISLKRR

ESMTGATFRD TNEDEIYAFF

181
GILVMTAVRK DNHMSTDDLF DPSLSMVYVS VMSRDREDFL

IRCLRMDDKS IRPTLRENDV

241
FTPVRKIWDL FIHQCIQNYT PGAHLTIDEQ LLGFRGRCPF

RMYIPNKPSK YGIKILMMCD

301
SGTKYMINGM PYLGRGTQTN GVPLGEYYVK ELSKPVHGSC

RNITCDNWFT SIPLAKNLLQ

361
EPYKLTIVGT VRSNKREIPE VLKNSRSRPV GTSMFCFDGP

LTLVSYKPKP AKMVYLLSSC

421
DEDASINEST GKPQMVMYYN QTKGGVDTLD QMCSVMTCSR

KTNRWPMALL YGMINIACIN

481
SFIIYSHNVS SKGEKVQSRK KFMRNLYMSL TSSFMRKRLE

APTLKRYLRD NISNILPNEV

541
PGTSDDSTEE PVMKKRTYCT YCPSKIRRKA NASCKKCKKV

ICREHNIDMC QSCF.

In certain embodiments of the methods of the disclosure, the transposase enzyme is a piggyBac® (PB) transposase enzyme that comprises or consists of an amino acid sequence having an amino acid substitution at one or more of positions 30, 165, 282, or 538 of the sequence:

In certain embodiments, the transposase enzyme is a piggyBac® (PB) transposase enzyme that comprises or consists of an amino acid sequence having an amino acid substitution at two or more of positions 30, 165, 282, or 538 of the sequence of SEQ ID NO: 14487. In certain embodiments, the transposase enzyme is a piggyBac® (PB) transposase enzyme that comprises or consists of an amino acid sequence having an amino acid substitution at three or more of positions 30, 165, 282, or 538 of the sequence of SEQ ID NO: 14487. In certain embodiments, the transposase enzyme is a piggyBac® (PB) transposase enzyme that comprises or consists of an amino acid sequence having an amino acid substitution at each of the following positions 30, 165, 282, and 538 of the sequence of SEQ ID NO: 14487. In certain embodiments, the amino acid substitution at position 30 of the sequence of SEQ ID NO: 14487 is a substitution of a valine (V) for an isoleucine (I). In certain embodiments, the amino acid substitution at position 165 of the sequence of SEQ ID NO: 14487 is a substitution of a serine (S) for a glycine (G). In certain embodiments, the amino acid substitution at position 282 of the sequence of SEQ ID NO: 14487 is a substitution of a valine (V) for a methionine (M). In certain embodiments, the amino acid substitution at position 538 of the sequence of SEQ ID NO: 14487 is a substitution of a lysine (K) for an asparagine (N).

In certain embodiments of the methods of the disclosure, the transposase enzyme is a Super piggyBac™ (SPB) transposase enzyme. In certain embodiments, the Super piggyBac™ (SPB) transposase enzymes of the disclosure may comprise or consist of the amino acid sequence of the sequence of SEQ ID NO: 14487 wherein the amino acid substitution at position 30 is a substitution of a valine (V) for an isoleucine (I), the amino acid substitution at position 165 is a substitution of a serine (S) for a glycine (G), the amino acid substitution at position 282 is a substitution of a valine (V) for a methionine (M), and the amino acid substitution at position 538 is a substitution of a lysine (K) for an asparagine (N). In certain embodiments, the Super piggyBac™ (SPB) transposase enzyme may comprise or consist of an amino acid sequence at least 75%, 80%, 85%, 90%, 95%, 99% or any percentage in between identical to:

(SEQ ID NO: 14484)

1
MGSSIDDEHI LSALLQDDE LVGEDSDSEI SDHVSEDDVQ

SDTEEAFIDE VHEVQPTSSG

61
SEILDEQNVI EQPGSSLASN RILTLPQRTI PGKNKHCWST

SKSTRRSRVS ALNIVRSQRG

121
PTRMCRNIYD PLLCFKLFFT DEIISEIVKW TNAEISLKRR

ESMTGATFRD TNEDEIYAFF

181
GILVMTAVRK DNHMSTDDLF DPSLSMVYVS VMSRDREDFL

IRCLRMDDKS IRPTLRENDV

241
FTPVRKIWDL FIHQCIQNYT PGAHLTIDEQ LLGFRGRCPF

RMYIPNKPSK YGIKILMMCD

301
SGTKYMINGM PYLGRGTQTN GVPLGEYYVK ELSKPVHGSC

RNITCDNWFT SIPLAKNLLQ

361
EPYKLTIVGT VRSNKREIPE VLKNSRSRPV GTSMFCFDGP

LTLVSYKPKP AKMVYLLSSC

421
DEDASINEST GKPQMVMYYN QTKGGVDTLD QMCSVMTCSR

KTNRWPMALL YGMINIACIN

481
SFIIYSHNVS SKGEKVQSRK KFMRNLYMSL TSSFMRKRLE

APTLKRYLRD NISNILPNEV

541
PGTSDDSTEE PVMKKRTYCT YCPSKIRRKA NASCKKCKKV

ICREHNIDMC QSCF.

In certain embodiments of the methods of the disclosure, including those embodiments wherein the transposase comprises the above-described mutations at positions 30, 165, 282 and/or 538, the piggyBac® or Super piggyBac™ transposase enzyme may further comprise an amino acid substitution at one or more of positions 3, 46, 82, 103, 119, 125, 177, 180, 185, 187, 200, 207, 209, 226, 235, 240, 241, 243, 258, 296, 298, 311, 315, 319, 327, 328, 340, 421, 436, 456, 470, 486, 503, 552, 570 and 591 of the sequence of SEQ ID NO: 14487 or SEQ ID NO: 14484. In certain embodiments, including those embodiments wherein the transposase comprises the above-described mutations at positions 30, 165, 282 and/or 538, the piggyBac® or Super piggyBac™ transposase enzyme may further comprise an amino acid substitution at one or more of positions 46, 119, 125, 177, 180, 185, 187, 200, 207, 209, 226, 235, 240, 241, 243, 296, 298, 311, 315, 319, 327, 328, 340, 421, 436, 456, 470, 485, 503, 552 and 570. In certain embodiments, the amino acid substitution at position 3 of SEQ ID NO: 14487 or SEQ ID NO: 14484 is a substitution of an asparagine (N) for a serine (S). In certain embodiments, the amino acid substitution at position 46 of SEQ ID NO: 14487 or SEQ ID NO: 14484 is a substitution of a serine (S) for an alanine (A). In certain embodiments, the amino acid substitution at position 46 of SEQ ID NO: 14487 or SEQ ID NO: 14484 is a substitution of a threonine (T) for an alanine (A). In certain embodiments, the amino acid substitution at position 82 of SEQ ID NO: 14487 or SEQ ID NO: 14484 is a substitution of a tryptophan (W) for an isoleucine (I). In certain embodiments, the amino acid substitution at position 103 of SEQ ID NO: 14487 or SEQ ID NO: 14484 is a substitution of a proline (P) for a serine (S). In certain embodiments, the amino acid substitution at position 119 of SEQ ID NO: 14487 or SEQ ID NO: 14484 is a substitution of a proline (P) for an arginine (R). In certain embodiments, the amino acid substitution at position 125 of SEQ ID NO: 14487 or SEQ ID NO: 14484 is a substitution of an alanine (A) a cysteine (C). In certain embodiments, the amino acid substitution at position 125 of SEQ ID NO: 14487 or SEQ ID NO: 14484 is a substitution of a leucine (L) for a cysteine (C). In certain embodiments, the amino acid substitution at position 177 of SEQ ID NO: 14487 or SEQ ID NO: 14484 is a substitution of a lysine (K) for a tyrosine (Y). In certain embodiments, the amino acid substitution at position 177 of SEQ ID NO: 14487 or SEQ ID NO: 14484 is a substitution of a histidine (H) for a tyrosine (Y). In certain embodiments, the amino acid substitution at position 180 of SEQ ID NO: 14487 or SEQ ID NO: 14484 is a substitution of a leucine (L) for a phenylalanine (F). In certain embodiments, the amino acid substitution at position 180 of SEQ ID NO: 14487 or SEQ ID NO: 14484 is a substitution of an isoleucine (I) for a phenylalanine (F). In certain embodiments, the amino acid substitution at position 180 of SEQ ID NO: 14487 or SEQ ID NO: 14484 is a substitution of a valine (V) for a phenylalanine (F). In certain embodiments, the amino acid substitution at position 185 of SEQ ID NO: 14487 or SEQ ID NO: 14484 is a substitution of a leucine (L) for a methionine (M). In certain embodiments, the amino acid substitution at position 187 of SEQ ID NO: 14487 or SEQ ID NO: 14484 is a substitution of a glycine (G) for an alanine (A). In certain embodiments, the amino acid substitution at position 200 of SEQ ID NO: 14487 or SEQ ID NO: 14484 is a substitution of a tryptophan (W) for a phenylalanine (F). In certain embodiments, the amino acid substitution at position 207 of SEQ ID NO: 14487 or SEQ ID NO: 14484 is a substitution of a proline (P) for a valine (V). In certain embodiments, the amino acid substitution at position 209 of SEQ ID NO: 14487 or SEQ ID NO: 14484 is a substitution of a phenylalanine (F) for a valine (V). In certain embodiments, the amino acid substitution at position 226 of SEQ ID NO: 14487 or SEQ ID NO: 14484 is a substitution of a phenylalanine (F) for a methionine (M). In certain embodiments, the amino acid substitution at position 235 of SEQ ID NO: 14487 or SEQ ID NO: 14484 is a substitution of an arginine (R) for a leucine (L). In certain embodiments, the amino acid substitution at position 240 of SEQ ID NO: 14487 or SEQ ID NO: 14484 is a substitution of a lysine (K) for a valine (V). In certain embodiments, the amino acid substitution at position 241 of SEQ ID NO: 14487 or SEQ ID NO: 14484 is a substitution of a leucine (L) for a phenylalanine (F). In certain embodiments, the amino acid substitution at position 243 of SEQ ID NO: 14487 or SEQ ID NO: 14484 is a substitution of a lysine (K) for a proline (P). In certain embodiments, the amino acid substitution at position 258 of SEQ ID NO: 14487 or SEQ ID NO: 14484 is a substitution of a serine (S) for an asparagine (N). In certain embodiments, the amino acid substitution at position 296 of SEQ ID NO: 14487 or SEQ ID NO: 14484 is a substitution of a tryptophan (W) for a leucine (L). In certain embodiments, the amino acid substitution at position 296 of SEQ ID NO: 14487 or SEQ ID NO: 14484 is a substitution of a tyrosine (Y) for a leucine (L). In certain embodiments, the amino acid substitution at position 2% of SEQ ID NO: 14487 or SEQ ID NO: 14484 is a substitution of a phenylalanine (F) for a leucine (L). In certain embodiments, the amino acid substitution at position 298 of SEQ ID NO: 14487 or SEQ ID NO: 14484 is a substitution of a leucine (L) for a methionine (M). In certain embodiments, the amino acid substitution at position 298 of SEQ ID NO: 14487 or SEQ ID NO: 14484 is a substitution of an alanine (A) for a methionine (M). In certain embodiments, the amino acid substitution at position 298 of SEQ ID NO: 14487 or SEQ ID NO: 14484 is a substitution of a valine (V) for a methionine (M). In certain embodiments, the amino acid substitution at position 311 of SEQ ID NO: 14487 or SEQ ID NO: 14484 is a substitution of an isoleucine (I) for a proline (P). In certain embodiments, the amino acid substitution at position 311 of SEQ ID NO: 14487 or SEQ ID NO: 14484 is a substitution of a valine for a proline (P). In certain embodiments, the amino acid substitution at position 315 of SEQ ID NO: 14487 or SEQ ID NO: 14484 is a substitution of a lysine (K) for an arginine (R). In certain embodiments, the amino acid substitution at position 319 of SEQ ID NO: 14487 or SEQ ID NO: 14484 is a substitution of a glycine (G) for a threonine (T). In certain embodiments, the amino acid substitution at position 327 of SEQ ID NO: 14487 or SEQ ID NO: 14484 is a substitution of an arginine (R) for a tyrosine (Y). In certain embodiments, the amino acid substitution at position 328 of SEQ ID NO: 14487 or SEQ ID NO: 14484 is a substitution of a valine (V) for a tyrosine (Y). In certain embodiments, the amino acid substitution at position 340 of SEQ ID NO: 14487 or SEQ ID NO: 14484 is a substitution of a glycine (G) for a cysteine (C). In certain embodiments, the amino acid substitution at position 340 of SEQ ID NO: 14487 or SEQ ID NO: 14484 is a substitution of a leucine (L) for a cysteine (C). In certain embodiments, the amino acid substitution at position 421 of SEQ ID NO: 14487 or SEQ ID NO: 14484 is a substitution of a histidine (H) for the aspartic acid (D). In certain embodiments, the amino acid substitution at position 436 of SEQ ID NO: 14487 or SEQ ID NO: 14484 is a substitution of an isoleucine (I) for a valine (V). In certain embodiments, the amino acid substitution at position 456 of SEQ ID NO: 14487 or SEQ ID NO: 14484 is a substitution of a tyrosine (Y) for a methionine (M). In certain embodiments, the amino acid substitution at position 470 of SEQ ID NO: 14487 or SEQ ID NO: 14484 is a substitution of a phenylalanine (F) for a leucine (L). In certain embodiments, the amino acid substitution at position 485 of SEQ ID NO: 14487 or SEQ ID NO: 14484 is a substitution of a lysine (K) for a serine (S). In certain embodiments, the amino acid substitution at position 503 of SEQ ID NO: 14487 or SEQ ID NO: 14484 is a substitution of a leucine (L) for a methionine (M). In certain embodiments, the amino acid substitution at position 503 of SEQ ID NO: 14487 or SEQ ID NO: 14484 is a substitution of an isoleucine (I) for a methionine (M). In certain embodiments, the amino acid substitution at position 552 of SEQ ID NO: 14487 or SEQ ID NO: 14484 is a substitution of a lysine (K) for a valine (V). In certain embodiments, the amino acid substitution at position 570 of SEQ ID NO: 14487 or SEQ ID NO: 14484 is a substitution of a threonine (T) for an alanine (A). In certain embodiments, the amino acid substitution at position 591 of SEQ ID NO: 14487 or SEQ ID NO: 14484 is a substitution of a proline (P) for a glutamine (Q). In certain embodiments, the amino acid substitution at position 591 of SEQ ID NO: 14487 or SEQ ID NO: 14484 is a substitution of an arginine (R) for a glutamine (Q).

The sleeping beauty transposon is transposed into the target genome by the Sleeping Beauty transposase that recognizes ITRs, and moves the contents between the ITRs into TA chromosomal sites. In various embodiments, SB transposon-mediated gene transfer, or gene transfer using any of a number of similar transposons, may be used in the compositions and methods of the disclosure.

In certain embodiments, and, in particular, those embodiments wherein the transposon is a Sleeping Beauty transposon, the transposase is a Sleeping Beauty transposase or a hyperactive Sleeping Beauty transposase (SB100X).

In certain embodiments of the methods of the disclosure, the Sleeping Beauty transposase enzyme comprises an amino acid sequence at least 75%, 80%, 85%, 90%, 95%, 99% or any percentage in between identical to:

(SEQ ID NO: 14485)

1
MGKSKEISQD LRKKIVIDLHK SGSSLGAISK RLKVPRSSVQ

TIVRKYKHHG TTQPSYRSGR

61
RPVLSPRDER TLVRKVQINP RTTAKDLVKM LEETGTKVSI

STVKRVLYRH NLKGRSARKK

121
PLLQNRHKKP RLRFATAHGD KDRTFWRNVL WSDETKIELF

GHNDHRYVWR KKGEACKPKN

181
TIPTVKHGGG SIMLWGCFAA GGTGAIHKID GIMRKENYVD

ILKQHLKTSV RKLKLGRKWV

241
FQMDNDPKHT SKVVAKWLKD NKVKVLEWPS QSPDLNPIEN

LWAELKKRVR ARRPTNLTQL

301
HQLCQEEWAK IHPTYCGKLV EGYPKRLTQV KQFKGNATKY.

In certain embodiments of the methods of the disclosure, the hyperactive Sleeping Beauty (SB100X) transposase enzyme comprises an amino acid sequence at least 75%, 80%, 85%, 90%, 95%, 99% or any percentage in between identical to:

(SEQ ID NO: 14486)

1
MGKSKEISQD LRKKIVIDLHK SGSSLGAISK RLKVPRSSVQ

TIVRKYKHHG TTQPSYRSGR

61
RPVLSPRDER TLVRKVQINP RTTAKDLVKM LEETGTKVSI

STVKRVLYRH NLKGRSARKK

121
PLLQNRHKKP RLRFATAHGD KDRTFWRNVL WSDETKIELF

GHNDHRYVWR KKGEACKPKN

181
TIPTVKHGGG SIMLWGCFAA GGTGAIHKID GIMRKENYVD

ILKQHLKTSV RKLKLGRKWV

241
FQMDNDPKHT SKVVAKWLKD NKVKVLEWPS QSPDLNPIEN

LWAELKKRVR ARRPTNLTQL

301
HQLCQEEWAK IHPTYCGKLV EGYPKRLTQV KQFKGNATKY.

The Helraiser transposon is transposed by the Helitron transposase. Helitron transposases mobilize the Helraiser transposon, an ancient element from the bat genome that was active about 30 to 36 million years ago. An exemplary Helraiser transposon of the disclosure includes Helibat1, which comprises a nucleic acid sequence comprising:

(SEQ ID NO: 17006)

1
TCCTATATAA TAAAAGAGAA ACATGCAAAT TGACCTCCC

TCCGCTACGC TCAAGCCACG

61
CCCACCAGCC AATCAGAAGT GACTATGCAA ATTAACCCAA

CAAAGATGGC AGTTAAATTT

121
GCATACGCAG GTGTCAAGCG CCCCAGGAGG CAACGGCGGC

CGCGGGCTCC CAGGACCTTC

181
GCTGGCCCCG GGAGGCGAGG CCGGCCGCGC CTAGCCACAC

CCGCGGGCTC CCGGGACCTT

241
CGCCAGCAGA GAGCAGAGCG GGAGAGCGGG CGGAGAGCGG

GAGGTTTGGA GGACTTGGCA

301
GAGCAGGAGG CCGCTGGACA TAGAGCAGAG CGAGAGAGAG

GGTGGCTTGG AGGGCGTGGC

361
TCCCTCTGTC ACCCCAGCTT CCTCATCACA GCTGTGGAAA

CTGACAGCAG GGAGGAGGAA

421
GTCCCACCCC CACAGAATCA GCCAGAATCA GCCGTTGGTC

AGACAGCTCT CAGCGGCCTG

481
ACAGCCAGGA CTCTCATTCA CCTGCATCTC AGACCGTGAC

AGTAGAGAGG TGGGACTATG

541
TCTAAAGAAC AACTGTTGAT ACAACGTAGC TCTGCAGCCG

AAAGATGCCG GCGTTATCGA

601
CAGAAAATGT CTGCAGAGCA ACGTGCGTCT GATCTTGAAA

GAAGGCGGCG CCTGCAACAG

661
AATGTATCTG AAGAGCAGCT ACTGGAAAAA CGTCGCTCTG

AAGCCGAAAA ACAGCGGCGT

721
CATCGACAGA AAATGTCTAA AGACCAACGT GCCTTTGAAG

TTGAAAGAAG GCGGTGGCGA

781
CGACAGAATA TGTCTAGAGA ACAGTCATCA ACAAGTACTA

CCAATACCGG TAGGAACTGC

841
CTTCTCAGCA AAAATGGAGT ACATGAGGAT GCAATTCTCG

AACATAGTTG TGGTGGAATG

901
ACTGTTCGAT GTGAATTTTG CCTATCACTA AATTTCTCTG

ATGAAAAACC ATCCGATGGG

961
AAATTTACTC GATGTTGTAG CAAAGGGAAA GTCTGTCCAA

ATGATATACA TTTTCCAGAT

1021
TACCCGGCAT ATTTAAAAAG ATTAATGACA AACGAAGATT

CTGACAGTAA AAATTTCATG

1081
GAAAATATTC GTTCCATAAA TAGTTCTTTT GCTTTTGCTT

CCATGGGTGC AAATATTGCA

1141
TCGCCATCAG GATATGGGCC ATACTGTTTT AGAATACACG

GACAAGTTTA TCACCGTACT

1201
GGAACTTTAC ATCCTTCGGA TGGTGTTTCT CGGAAGTTTG

CTCAACTCTA TATTTTGGAT

1261
ACAGCCGAAG CTACAAGTAA AAGATTAGCA ATGCCAGAAA

ACCAGGGCTG CTCAGAAAGA

1321
CTCATGATCA ACATCAACAA CCTCATGCAT GAAATAAATG

AATTAACAAA ATCGTACAAG

1381
ATGCTACATG AGGTAGALAA GGAAGCCCAA TCTGAAGCAG

CAGCAAAAGG TATTGCTCCC

1441
ACAGAAGTAA CAATGGCGAT TAAATACGAT CGTAACAGTG

ACCCAGGTAG ATATAATTCT

1501
CCCCGTGTAA CCGAGGTTGC TGTCATATTC AGAAACGAAG

ATGGAGAACC TCCTTTTGAA

1561
AGGGACTTGC TCATTCATTG TAAACCAGAT CCCAATAATC

CAAATGCCAC TAAAATGAAA

1621
CAAATCAGTA TCCTGTTTCC TACATTAGAT GCAATGACAT

ATCCTATTCT TTTTCCACAT

1681
GGTGAAAAAG GCTGGGGAAC AGATATTGCA TTAAGACTCA

GAGACAACAG TGTAATCGAC

1741
AATAATACTA GACAALATGT AAGGACACGA GTCACACAAA

TGCAGTATTA TGGATTTCAT

1801
CTCTCTGTGC GGGACACGTT CAATCCTATT TTAAATGCAG

GAAAATTAAC TCAACAGTTT

1861
ATTGTGGATT CATATTCAAA AATGGAGGCC AATCGGATAA

ATTTCATCAA AGCAAACCAA

1921
TCTAAGTTGA GAGTTGAAAA ATATAGTGGT TTGATGGATT

ATCTCAAATC TAGATCTGAA

1981
AATGACAATG TGCCGATTGG TAAAATGATA ATACTTCCAT

CATCTTTTGA GGGTAGTCCC

2041
AGAAATATGC AGCAGCGATA TCAGGATGCT ATGGCAATTG

TAACGAAGTA TGGCAAGCCC

2101
GATTTATTCA TAACCATGAC ATGCAACCCC AAATGGGCAG

ATATTACAAA CAATTTACAA

2161
CGCTGGCAAA AAGTTGAALA CAGACCTGAC TTGGTAGCCA

GAGTTTTTLA TATTAAGCTG

2221
AATGCTCTTT TALATGATAT ATGTAAATTC CATTTATTTG

GCAAAGTAAT AGCTAAAATT

2281
CATGTCATTG AATTTCAGAA ACGCGGACTG CCTCACGCTC

ACATATTATT GATATTAGAT

2341
AGTGAGTCCA AATTACGTTC AGAAGATGAC ATTGACCGTA

TAGTTAAGGC AGAAATTCCA

2401
GATGAAGACC AGTGTCCTCG ACTTTTTCAA ATTGTAAAAT

CAAATATGGT ACATGGACCA

2461
TGTGGAATAC AAAATCCAAA TAGTCCATGT ATGGAAAATG

GAAAATGTTC AAAGGGATAT

2521
CCAAAAGAAT TTCAAAATGC GACCATTGGA AATATTGATG

GATATCCCAA ATACAAACGA

2581
AGATCTGGTA GCACCATGTC TATTGGALAT AAAGTTGTCG

ATAACACTTG GATTGTCCCT

2641
TATAACCCGT ATTTGTGCCT TAAATATAAC TGTCATATAA

ATGTTGAAGT CTGTGCATCA

2701
ATTAAAAGTG TCAAATATTT ATTTAAATAC ATCTATAAAG

GGCACGATTG TGGAAATATT

2761
CAAATTTCTG AAAAAAATAT TATCAATCAT GACGAAGTAC

AGGACTTCAT TGACTCCAGG

2821
TATGTGAGCG CTCCTGAGGC TGTTTGGAGA CTTTTTGCAA

TGCGAATGCA TGACCAATCT

2881
CATGCAATCA CAAGATTAGC TATTCATTTG CCAAATGATC

AGAATTTGTA TTTTCATACC

2941
GATGATTTTG CTGAAGTTTT AGATAGGGCT AAAAGGCATA

ACTCGACTTT GATGGCTTGG

3001
TTCTTATTGA ATAGAGAAGA TTCTGATGCA CGTAATTATT

ATTATTGGGA GATTCCACAG

3061
CATTATGTGT TTAATAATTC TTTGTGGACA AAACGCCGAA

AGGGTGGGAA TAAAGTATTA

3121
GGTAGACTGT TCACTGTGAG CTTTAGAGAA CCAGAACGAT

ATTACCTTAG ACTTTTGCTT

3181
CTGCATGTAA AAGGTGCGAT AAGTTTTGAG GATCTGCGAA

CTGTAGGAGG TGTAACTTAT

3241
GATACATTTC ATGAAGCTGC TAAACACCGA GGATTATTAC

TTGATGACAC TATCTGGAAA

3301
GATACGATTG ACGATGCAAT CATCCTTAAT ATGCCCAAAC

AACTACGGCA ACTTTTTGCA

3361
TATATATGTG TGTTTGGATG TCCTTCTGCT GCAGACAAAT

TATGGGATGA GAATAAATCT

3421
CATTTTATTG AAGATTTCTG TTGGAAATTA CACCGAAGAG

AAGGTGCCTG TGTGAACTGT

3481
GAAATGCATG CCCTTAACGA AATTCAGGAG GTATTCACAT

TGCATGGAAT GAAATGTTCA

3541
CATTTCAAAC TTCCGGACTA TCCTTTATTA ATGAATGCAA

ATACATGTGA TCAATTGTAC

3601
GAGCAACAAC AGGCAGAGGT TTTGATAAAT TCTCTGAATG

ATGAACAGTT GGCAGCCTTT

3661
CAGACTATAA CTTCAGCCAT CGAAGATCAA ACTGTACACC

CCAAATGCTT TTTCTTGGAT

3721
GGTCCAGGTG GTAGTGGAAA AACATATCTG TATAAAGTTT

TAACACATTA TATTAGAGGT

3781
CGTGGTGGTA CTGTTTTACC CACAGCATCT ACAGGAATTG

CTGCAAATTT ACTTCTTGGT

3841
GGAAGAACCT TTCATTCCCA ATATAAATTA CCAATTCCAT

TAAATGAAAC TTCAATTTCT

3901
AGACTCGATA TAAAGAGTGA AGTTGCTAAA ACCATTAAAA

AGGCCCAACT TCTCATTATT

3961
GATGAATGCA CCATGGCATC CAGTCATGCT ATAAACGCCA

TAGATAGATT ACTAAGAGAA

4021
ATTATGAATT TGAATGTTGC ATTTGGTGGG AAAGTTCTCC

TTCTCGGAGG GGATTTTCGA

4081
CAATGTCTCA GTATTGTACC ACATGCTATG CGATCGGCCA

TAGTACAAAC GAGTTTAAAG

4141
TACTGTAATG TTTGGGGATG TTTCAGAAAG TTGTCTCTTA

AAACAAATAT GAGATCAGAG

4201
GATTCTGCTT ATAGTGAATG GTTAGTAAAA CTTGGAGATG

GCAAACTTGA TAGCAGTTTT

4261
CATTTAGGAA TGGATATTAT TGAAATCCCC CATGAAATGA

TTTGTAACGG ATCTATTATT

4321
GAAGCTACCT TTGGAAATAG TATATCTATA GATAATATTA

AAAATATATC TAAACGTGCA

4381
ATTCTTTGTC CAAAAAATCA GCATGTTCAA AAATTAAATG

AAGAAATTTT GGATATACTT

4441
GATGGAGATT TTCACACATA TTTGAGTGAT GATTCCATTG

ATTCAACAGA TGATGCTGAA

4501
AAGGAAAATT TTCCCATCGA ATTTCTTAAT AGTATTACTC

CTTCGGGAAT GGCGTGTCAT

4561
AAATTAAAAT TGAAAGTGGG TGCAATCATC ATGCTATTGA

GAAATCTTLA TAGTAAATGG

4621
GGTCTTTGTA ATGGTACTAG ATTTATTATC AAAAGATTAC

CACCTAACAT TATCGAAGCT

4681
GAAGTATTAA CAGGATCTGC AGAGGGAGAG GTTGTTCTGA

TTCCAAGAAT TGATTTGTCC

4741
CCATCTGACA CTGGCCTCCC ATTTAAATTA ATTCGAAGAC

AGTTTCCCGT GATGCCAGCA

4801
TTTGCGATGA CTATTAATAA ATCACAAGGA CAAACTCTAG

ACAGAGTAGG AATATTCCTA

4861
CCTGAACCCG TTTTCGCACA TGGTCAGTTA TATGTTGCTT

TCTCTCGAGT TCGAAGAGCA

4921
TGTGACGTTA AAGTTAAAGT TGTAAATACT TCATCACAAG

GGAAATTAGT CAAGCACTCT

4981
GAAAGTGTTT TTACTCTTAA TGTGGTATAC AGGGAGATAT

TAGAATAAGT TTAATCACTT

5041
TATCAGTCAT TGTTTGCATC AATGTTGTTT TTATATCATG

TTTTTGTTGT TTTTATATCA

5101
TGTCTTTGTT GTTGTTATAT CATGTTGTTA TTGTTTATTT

ATTAATAAAT TTATGTATTA

5161
TTTTCATATA CATTTTACTC ATTTCCTTTC ATCTCTCACA

CTTCTATTAT AGAGAAAGGG

5221
CAAATAGCAA TATTAAAATA TTTCCTCTAA TTAATTCCCT

TTLAATGTGC ACGAATTTCG

5281
TGCACCGGGC CACTAG.

Unlike other transposases, the Helitron transposase does not contain an RNase-H like catalytic domain, but instead comprises a RepHel motif made up of a replication initiator domain (Rep) and a DNA helicase domain. The Rep domain is a nuclease domain of the HUH superfamily of nucleases.

An exemplary Helitron transposase of the disclosure comprises an amino acid sequence comprising:

(SEQ ID NO: 14501)

1
MSKEQLLIQR SSAAERCRRY RQKMSAEQRA SDLERRRRLQ

QNVSEEQLLE KRRSEAEKQR

61
RHRQKMSKDQ RAFEVERRRW RRQNMSREQS STSTTNTGRN

CLLSKNGVHE DAILEHSCGG

121
MTVRCEFCLS LNFSDEKPSD GKFTRCCSKG KVCPNDIHEP

DYPAYLKRLM TNEDSDSKNF

181
MENIRSINSS FAFASMGANI ASPSGYGPYC FRIHGQVYHR

TGTLHPSDGV SRKFAQIYIL

241
DTAEATSKRL AMPENQGCSE RLMININNLM HEINELTKSY

KMLHEVEKEA QSEAAAKGIA

301
PTEVTMAIKY DRNSDPGRYN SPRVTEVAVI FRNEDGEPPF

ERDLLIHCKP DPNNPNATKM

361
KQISILFPTL DAMTYPILFP HGEKGWGTDI ALRLRDNSVI

DNNTRQNVRT RVTQMQYYGF

421
HLSVRDTFNP ILNAGKLTQQ FIVDSYSKME ANRINFIKAN

QSKLRVEKYS GLMDYLKSRS

481
ENDNVPIGKM IILPSSFEGS PRNMQQRYQD AMAIVTKYGK

PDLFITMTCN PKWADITNNL

541
QRWQKVENRP DLVARVFNIK LNAILNDICK FHLFGKVIAK

IHVIEFQKRG LPHAEILLIL

601
DSESKLRSED DIDRIVKAEI PDEDQCPRLF QIVYSNMVHG

PCGIQNPNSP CMENGKCSKG

661
YPKEFQNATI GNIDGYPKYK RRSGSTMSIG NKVVDNTWIV

PYNPYLCLKY NCHINVEVCA

721
SIKSVKYLFK YIYKGHDCAN IQISEKNIIN HDEVQDFIDS

RYVSAPEAVW RLFAMRMHDQ

781
SHAITRLAIH LPNDQNLYFH TDDFAEVLDR AKRHNSTLMA

WELLNREDSD ARNYYYWEIP

841
QHYVENNSLW TKRRKGGNKV LGRLFTVSFR EPERYYLRLL

LLHVKGAISF EDLRTVGGVT

901
YDTFHEAAKH RGLLLDDTIW KDTIDDAIIL NMPKQLRQLF

AYICVFGCPS AADKLWDENK

961
SHFIEDFCWK LHRREGACVN CEMHALNEIQ EVETLEGMKC

SHFKLPDYPL LMNANTCDQL

1021
YEQQQAEVLI NSINDEQLAA FQTITSAIED QTVHPKCFFL

DGPGGSGKTY LYKVITHYIR

1081
GRGGTVLPTA STGIAANLLL GGRTFHSQYK LPIPLNETSI

SRLDIKSEVA KTIKKAQLLI

1141
IDECTMASSH AINAIDRLLR EIMNLNVAFG GKVILLGGDF

RQCLSIVPHA MRSAIVQTSL

1201
KYCNVWGCFR KLSLKTNMRS EDSAYSEWLV KIGDGKLDSS

FHLGMDIIEI PHEMICNGSI

1261
IEATFGNSIS IDNIKNISKR AILCPKNEHV QKLNEEILDI

LDGDFHTYLS DDSIDSTDDA

1321
EKENFPIEFL NSITPSGMPC HKLKLKVGAI IMILRNLNSK

WGLCNGTRFI IKRIRPNIIE

1381
AEVLTGSAEG EVVLIPPIDL SPSDTGLPFK LIRRQFPVMP

AFAMTINKSQ GQTLDRVGIF

1441
LPEPVFAHGQ LYVAFSRVRR ACDVKVKVVN TSSQGKLVKH

SESVFTLNVV YREILE.

In Helitron transpositions, a hairpin close to the 3′ end of the transposon functions as a terminator. However, this hairpin can be bypassed by the transposase, resulting in the transduction of flanking sequences. In addition, Helraiser transposition generates covalently closed circular intermediates. Furthermore, Helitron transpositions can lack target site duplications. In the Helraiser sequence, the transposase is flanked by left and right terminal sequences termed LTS and RTS. These sequences terminate with a conserved 5′-TC/CTAG-3′ motif. A 19 bp palindromic sequence with the potential to form the hairpin termination structure is located 11 nucleotides upstream of the RTS and consists of the sequence

(SEQ ID NO: 14500)

GTGCACGAATTTCGTGCACCGGCCACTAG.

Tol2 transposons may be isolated or derived from the genome of the medaka fish, and may be similar to transposons of the hAT family. Exemplary Tol2 transposons of the disclosure are encoded by a sequence comprising about 4.7 kilobases and contain a gene encoding the Tol2 transposase, which contains four exons. An exemplary Tol2 transposase of the disclosure comprises an amino acid sequence comprising the following:

(SEQ ID NO: 14502)

1
MEEVCDSSAA ASSTVQNQPQ DQEHPWIPYLR EFFSLSGVNK

DSFKMKCVLC LPLNKEISAF

61
KSSPSNLRKE IERMHPNYLK NYSKLTAQKR KIGTSTHASS

SKQLKVDSVF PVKEVSPVTV

121
NKAILRYIIQ GLHPFSTVDL PSFKELISTL QPGISVITRP

TLRSKIAEAA LIMEQKVTAA

181
MSEVEWIATT TDCWTARRKS FIGVTAHWIN PGSLERHSAA

LACKRLMGSH TFEVLASAMN

241
DIHSEYEIRD KVVCTTTDSG SNFMKAFRVF GVENNDIETE

ARRCESDDTD SEGCGEGSDG

301
VEFQDASRVL DQDDGFEFQL PKHQKCACHL LNLVSSVDAQ

KALSNEHYKK LYRSVFGKCQ

361
ALWNKSSRSA LPAEAVESES RLQLLRPNQT RWNSTFMAVD

RILQICKEAG EGALRNICTS

421
LEVPMFNPAE MLFLTEWANT MRPVAKVLDI LQAETNTQLG

WLLPSVHQLS LKLQRLHHSL

481
RYCDPLVDAI QQGIQTRFKH MFEDPEIIAA AILLPKFRTS

WTNDETIIKR GMDYIRVHLE

541
PLDHKKELAN SSSDDEDFFA SLKPTTHEAS KELDGYLACV

SDTRESLLTF PAICSLSIKT

501
NTPLPASAAC ERLFSTAGLL FSPKPARLDT NNFENQLLLK

LNLREYNFE.

An exemplary Tol2 transposon of the disclosure, including inverted repeats, subterminal sequences and the Tol2 transposase, is encoded by a nucleic acid sequence comprising the following:

(SEQ ID NO: 17007)

1
CAGAGGTGTA AAGTACTTGA GTAATTTTAC TTGATTACTG

TACTTAAGTA TTATTTTTGG

61
GGATTTTTAC TTTACTTGAG TACAATTAAA AATCAATACT

TTTACTTTTA CTTAATTACA

121
TTTTTTTAGA AAAAAAAGTA CTTTTTACTC CTTACAATTT

TATTTACAGT CAAAAAGTAC

181
TTATTTTTTG GAGATCACTT CATTCTATTT TCCCTTGCTA

TTACCAAACC AATTGAATTG

241
CGCTGATGCC CAGTTTAATT TAAATGTTAT TTATTCTGCC

TATGALLATC GTTTTCACAT

301
TATATGAAAT TGGTCAGACA TGTTCATTGG TCCTTTGGAA

GTGACGTCAT GTCACATCTA

361
TTACCACAAT GCACAGCACC TTGACCTGGA AATTAGGGAA

ATTATAACAG TCAATCAGTG

421
GAAGAAAATG GAGGAAGTAT GTGATTCATC AGCAGCTGCG

AGCAGCACAG TCCAAAATCA

481
GCCACAGGAT CAAGAGCACC CGTGGCCGTA TCTTCGCGAA

TTCTTTTCTT TAAGTGGTGT

541
AAATAAAGAT TCATTCAAGA TGAAATGTGT CCTCTGTCTC

CCGCTTAATA AAGAAATATC

601
GGCCTTCAAA AGTTCGCCAT CAAACCTAAG GAAGCATATT

GAGGTAAGTA CATTAAGTAT

661
TTTGTTTTAC TGATAGTTTT TTTTTTTTTT TTTTTTTTTT

TTTTTGGGTG TGCATGTTTT

721
GACGTTGATG GCGCGCCTTT TATATGTGTA GTAGGCCTAT

TTTCACTAAT GCATGCGATT

781
GACAATATAA GGCTCACGTA ATAAAATGCT AAAATGCATT

TGTAATTGGT AACGTTAGGT

841
CCACGGGAAA TTTGGCGCCT ATTGCAGCTT TGAATAATCA

TTATCATTCC GTGCTCTCAT

901
TGTGTTTGAA TTCATGCAAA ACACAAGAAA ACCAAGCGAG

AAATTTTTTT CCAAACATGT

961
TGTATTGTCA AAACGGTAAC ACTTTACAAT GAGGTTGATT

AGTTCATGTA TTAACTAACA

1021
TTAAATAACC ATGAGCAATA CATTTGTTAC TGTATCTGTT

AATGTTTGTT AACGTTAGTT

1081
AATAGAANTA CAGATGTTCA TTGTTTGTTC ATGTTAGTTC

ACAGTGCATT AACTAATGTT

1141
AACAAGATAT AAAGTATTAG TAAATGTTGA AATTAACATG

TATACGTGCA GTTCATTATT

1201
AGTTCATGTT AACTAATGTA GTTAACTAAC GAACCTTATT

GTAAAAGTGT TACCATCAAA

1261
ACTAATGTAA TGAAATCAAT TCACCCTGTC ATGTCAGCCT

TAGAGTCCTG TGTTTTTGTC

1321
AATATAATCA GAAATAAAAT TAATGTTTGA TTGTCACTAA

ATGCTACTGT ATTTCTAAAA

1381
TCAACAAGTA TTTAACATTA TAAAGTGTGC AATTGGCTGC

AAATGTCAGT TTTATTAAAG

1441
GGTTAGTTCA CCCAAAAATG AAAATAATGT CATTAATGAC

TCGCCCTCAT GTCGTTCCAA

1501
GCCCGTAAGA CCTCCGTTCA TCTTCAGAAC ACAGTTTAAG

ATATTTTAGA TTTAGTCCGA

1561
GAGCTTTCTG TGCCTCCATT GAGAATGTAT GTACGGTATA

CTGTCCATGT CCAGAAAGGT

1621
AATAAAAACA TCAAAGTAGT CCATGTGACA TCAGTGGGTT

AGTTAGAATT TTTTGAAGCA

1681
TCGAATACAT TTTGGTCCAA AAATAACAAA ACCTACGACT

TTATTCGGCA TTGTATTCTC

1741
TTCCGGGTCT GTTGTCAATC CGCGTTCACG ACTTCGCAGT

GACGCTACAA TGCTGAATAA

1801
AGTCGTAGGT TTTGTTATTT TTGGACCAAA ATGTATTTTC

GATGCTTCAA ATAATTCTAG

1861
CTAACCCACT GATGTCACAT GGACTACTTT GATGTTTTTA

TTACCTTTCT GGACATGGAC

1921
AGTATACCGT ACATACATTT TCAGTGGAGG GACAGAAAGC

TCTCGGACTA AATCTAAAAT

1981
ATCTTAAACT GTGTTCCGAA GATGAACGGA GGTGTTACGG

GCTTGGAACG ACATGAGGGT

2041
GAGTCATTAA TGACATCTTT TCATTTTTGG GTGAACTAAC

CCTTTAATGC TGTAATCAGA

2101
GAGTGTATGT GTAATTGTTA CATTTATTGC ATACAATATA

AATATTTATT TGTTGTTTTT

2161
ACAGAGAATG CACCCAAATT ACCTCAAAAA CTACTCTAAA

TTGAGAGCAC AGAAGAGAAA

2221
GATCGGGACC TCCACCCATG CTTCCAGCAG TAAGCAACTG

AAAGTTGACT CAGTTTTCCC

2281
AGTCAAAGAT GTGTCTCCAG TCACTGTGAA CAAAGCTATA

TTAAGGTACA TCATTCAAGG

2341
ACTTCATCCT TTCAGCACTG TTGATCTGCC ATCATTTAAA

GAGCTGATTA GTACACTGCA

2401
GCCTGGCATT TCTGTCATTA CAAGGCCTAC TTTACGGTCC

AAGATAGCTG AAGCTGCTCT

2461
GATCATGAAA CAGAAAGTGA CTGCTGCCAT GAGTGAAGTT

GAATGGATTG CAACCACAAC

2521
GGATTGTTGG ACTGCACGTA GAAAGTCATT CATTGGTGTA

ACTGCTCACT GGATCAACCC

2581
TGGAAGTCTT GAAAGACATT CCGCTGCACT TGCCTGCAAA

AGATTAATGG GCTCTCATAC

2641
TTTTGAGGTA CTGGCCAGTG CCATGAATGA TATCCACTCA

GAGTATGAAA TACGTGACAA

2701
GGTTGTTTGC ACAACCACAG ACAGTGGTTC CAACTTTATG

AAGGCTTTCA GAGTTTTTGG

2761
TGTGGAAAAC AATGATATCG AGACTGAGGC AAGAAGGTGT

GAAAGTGATG ACACTGATTC

2821
TGAAGGCTGT GGTGAGGGAA GTGATGGTGT GGAATTCCAA

GATGCCTCAC GAGTCCTGGA

2881
CCAAGACGAT GGCTTCGAAT TCCAGCTACC AAAACATCAA

AAGTGTGCCT GTCACTTACT

2941
TAACCTAGTC TCAAGCGTTG ATGCCCAAAA AGCTCTCTCA

AATGAAGACT ACAAGAAACT

3001
CTACAGATCT GTCTTTGGCA AATGCCAAGC TTTATGGAAT

AAAAGCAGCC GATCGGCTCT

3061
AGCAGCTGAA GCTGTTGAAT CAGAAAGCCG GCTTCAGCTT

TTAAGGCCAA ACCAAACGCG

3121
GTGGAATTCA ACTTTTATGG CTGTTGACAG AATTCTTCAA

ATTTGCAAAG AAGCAGGAGA

3181
AGGCGCAGTT CGGAATATAT GCACCTCTCT TGAGGTTCCA

ATGTAAGTGT TTTTCCCCTC

3241
TATCGATGTA AACAAATGTG GGTTGTTTTT GTTTAATACT

CTTTGATTAT GCTGATTTCT

3301
CCTGTAGGTT TAATCCAGCA GAAATGCTGT TCTTGACAGA

GTGGGCCAAC ACAATGCGTC

3361
CAGTTGCAAA AGTACTCGAC ATCTTGCAAG CGGAAACGAA

TACACAGCTG GGGTGGCTGC

3421
TGCCTAGTGT CCATCAGTTA AGCTTGAAAC TTCAGCGACT

CCACCATTCT CTCAGGTACT

3481
GTGACCCACT TGTGGATGCC CTACAACAAG GAATCCAAAC

ACGATTCAAG CATATGTTTG

3541
AAGATCCTGA GATCATAGCA GCTGCCATCC TTCTCCCTAA

ATTTCGGACC TCTTGGACAA

3601
ATGATGAAAC CATCATAAAA CGAGGTAAAT GAATGCAAGC

AACATACACT TGACGAATTC

3661
TAATCTGGGC AACCTTTGAG CCATACCAAA ATTATTCTTT

TATTTATTTA TTTTTGCACT

3721
TTTTAGGAAT GTTATATCCC ATCTTTGGCT GTGATCTCAA

TATGAATATT GATGTAAAGT

3781
ATTCTTGCAG CAGGTTGTAG TTATCCCTCA GTGTTTCTTG

AAACCAAACT CATATGTATC

3841
ATATGTGGTT TGGAAATGCA GTTAGATTTT ATGCTAAAAT

AAGGGATTTG CATGATTTTA

3901
GATGTAGATG ACTGCACGTA AATGTAGTTA ATGACAAAAT

CCATAALATT TGTTCCCAGT

3961
CAGAAGCCCC TCAACCAAAC TTTTCTTTGT GTCTGCTCAC

TGTGCTTGTA GGCATGGACT

4021
ACATCAGAGT GCATCTGGAG CCTTTGGACC ACAAGAAGGA

ATTGGCCAAC AGTTCATCTG

4081
ATGATGAAGA TTTTTTCGCT TCTTTGAAAC CGACAACACA

TGAAGCCAGC AAAGAGTTGG

4141
ATGGATATCT GGCCTGTGTT TCAGACACCA GGGAGTCTCT

GCTCACGTTT CCTGCTATTT

4201
GCAGCCTCTC TATCAAGACT AATACACCTC TTCCCGCATC

GGCTGCCTGT GAGAGGCTTT

4261
TCAGCACTGC AGGATTGCTT TTCAGCCCCA AAAGAGCTAG

GCTTGACACT AACAATTTTG

4321
AGAATCAGCT TCTACTGAAG TTAAATCTGA GGTTTTACAA

CTTTGAGTAG CGTGTACTGG

4381
CATTAGATTG TCTGTCTTAT AGTTTGATAA TTAAATACAA

ACAGTTCTAA AGCAGGATAA

4441
AACCTTGTAT GCATTTCATT TAATGTTTTT TGAGATTAAA

AGCTTALACA AGAATCTCTA

4501
GTTTTCTTTC TTGCTTTTAC TTTTACTTCC TTAATACTCA

AGTACAATTT TAATGGAGTA

4561
CTTTTTTACT TTTACTCAAG TAAGATTCTA GCCAGATACT

TTTACTTTTA ATTGAGTAAA

4621
ATTTTCCCTA AGTACTTGTA CTTTCACTTG AGTAAAATTT

TTGAGTACTT TTTACACCTC

4681
TG.

Exemplary transposon/transposase systems of the disclosure include, but are not limited to, piggyBac® and piggyBac-like transposons and transposases.

PiggyBac® and piggyBac-like transposases recognizes transposon-specific inverted terminal repeat sequences (ITRs) on the ends of the transposon, and moves the contents between the ITRs into TTAA or TTAT chromosomal sites. The piggyBac or piggyBac-like transposon system has no payload limit for the genes of interest that can be included between the ITRs.

In certain embodiments, and, in particular, those embodiments wherein the transposon is a piggyBac® transposon, the transposase is a piggyBac®, Super piggyBac™ (SPB) transposase. In certain embodiments, and, in particular, those embodiments wherein the transposase is a piggyBac®, Super piggyBac™ (SPB), the sequence encoding the transposase is an mRNA sequence.

In certain embodiments of the methods of the disclosure, the transposase enzyme is a piggyBac® or piggyBac-like transposase enzyme.

In certain embodiments of the methods of the disclosure, the transposase enzyme is a piggyBac® or a piggyBac-like transposase enzyme. The piggyBac® (PB) or piggyBac-like transposase enzyme may comprise or consist of an amino acid sequence at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% or any percentage in between identical to:

(SEQ ID NO: 14487)

1
MGSSLDDEHI LSALLQSDDE LVGEDSDSEI SDHVSEDDVQ

SDTEEAFIDE VHEVQPTSSG

61
SEILDEQNVI EQPGSSLASN RILTLPQPTI RGKNKHCWST

SKSTRRSRVS ALNIVRSQRG

121
PTRMCPNIYD PLLCFKLFFT DEIISEIVKW TNAEISLKRR

ESMTGATFRD TNEDEIYAFF

181
GILVMTAVRK DNHMSTDDLF DRSLSMVYVS VMSRDRFDFL

IRCLRMDDKS IRPTLRENDV

241
FTPVRKIWDL FIHQCIQNYT PGAHLTIDEQ LLGERGRCPF

RMYIPNKPSK YGIKILMMCD

301
SGTKYMINGM PYLGRGTQTN GVPLGEYYVK ELSKPVHGSC

RNITCDNWFT SIPLAKNLLQ

361
EPYKLTIVGT VRSNKREIPE VLKNSRSRPV GTSMFCFDGP

LTLVSYKPKP AKMVYLLSSC

421
DEDASINEST GKPQMVMYYN QTKGGVDTLD QMCSVMTCSR

KTNRWPMALL YGMINIACIN

481
SFIIYSHNVS SKGEKVQSRK KFMPNLYMSL TSSFMRKRLE

APTLKRYLRD NISNILPNEV

541
PGTSDDSTEE PVMKKRTYCT YCPSKIRRKA NASCKKCKKV

ICREHNIDMC QSCF.

In certain embodiments of the methods of the disclosure, the transposase enzyme is a piggyBac® or piggyBac-like transposase enzyme that comprises or consists of an amino acid sequence having an amino acid substitution at one or more of positions 30, 165, 282, or 538 of the sequence:

In certain embodiments, the transposase enzyme is a piggyBac® or piggyBac-like transposase enzyme that comprises or consists of an amino acid sequence having an amino acid substitution at two or more of positions 30, 165, 282, or 538 of the sequence of SEQ ID NO: 14487. In certain embodiments, the transposase enzyme is a piggyBac® or piggyBac-like transposase enzyme that comprises or consists of an amino acid sequence having an amino acid substitution at three or more of positions 30, 165, 282, or 538 of the sequence of SEQ ID NO: 14487. In certain embodiments, the transposase enzyme is a piggyBac® or piggyBac-like transposase enzyme that comprises or consists of an amino acid sequence having an amino acid substitution at each of the following positions 30, 165, 282, and 538 of the sequence of SEQ ID NO: 14487. In certain embodiments, the amino acid substitution at position 30 of the sequence of SEQ ID NO: 14487 is a substitution of a valine (V) for an isoleucine (I). In certain embodiments, the amino acid substitution at position 165 of the sequence of SEQ ID NO: 14487 is a substitution of a serine (S) for a glycine (G). In certain embodiments, the amino acid substitution at position 282 of the sequence of SEQ ID NO: 14487 is a substitution of a valine (V) for a methionine (M). In certain embodiments, the amino acid substitution at position 538 of the sequence of SEQ ID NO: 14487 is a substitution of a lysine (K) for an asparagine (N).

In certain embodiments of the methods of the disclosure, the transposase enzyme is a Super piggyBac™ (SPB) or piggyBac-like transposase enzyme. In certain embodiments, the Super piggyBac™ (SPB) or piggyBac-like transposase enzyme of the disclosure may comprise or consist of the amino acid sequence of the sequence of SEQ ID NO: 14487 wherein the amino acid substitution at position 30 is a substitution of a valine (V) for an isoleucine (I), the amino acid substitution at position 165 is a substitution of a serine (S) for a glycine (G), the amino acid substitution at position 282 is a substitution of a valine (V) for a methionine (M), and the amino acid substitution at position 538 is a substitution of a lysine (K) for an asparagine (N). In certain embodiments, the Super piggyBac™ (SPB) or piggyBac-like transposase enzyme may comprise or consist of an amino acid sequence at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% or any percentage in between identical to:

(SEQ ID NO: 14484)

1
MGSSLDDEHI LSALLQSDDE LVGEDSDSEI SDHVSEDDVQ

SDTEEAFIDE VHEVQPTSSG

61
SEILDEQNVI EQPGSSLASN RILTLPQPTI RGKNKHCWST

SKSTRRSRVS ALNIVRSQRG

121
PTRMCPNIYD PLLCFKLFFT DEIISEIVKW TNAEISLKRR

ESMTGATFRD TNEDEIYAFF

181
GILVMTAVRK DNHMSTDDLF DRSLSMVYVS VMSRDRFDFL

IRCLRMDDKS IRPTLRENDV

241
FTPVRKIWDL FIHQCIQNYT PGAHLTIDEQ LLGERGRCPF

RMYIPNKPSK YGIKILMMCD

301
SGTKYMINGM PYLGRGTQTN GVPLGEYYVK ELSKPVHGSC

RNITCDNWFT SIPLAKNLLQ

361
EPYKLTIVGT VRSNKREIPE VLKNSRSRPV GTSMFCFDGP

LTLVSYKPKP AKMVYLLSSC

421
DEDASINEST GKPQMVMYYN QTKGGVDTLD QMCSVMTCSR

KTNRWPMALL YGMINIACIN

481
SFIIYSHNVS SKGEKVQSRK KFMPNLYMSL TSSFMRKRLE

APTLKRYLRD NISNILPNEV

541
PGTSDDSTEE PVMKKRTYCT YCPSKIRRKA NASCKKCKKV

ICREHNIDMC QSCF.

In certain embodiments of the methods of the disclosure, including those embodiments wherein the transposase comprises the above-described mutations at positions 30, 165, 282 and/or 538, the piggyBac®, Super piggyBac™ or piggyBac-like transposase enzyme may further comprise an amino acid substitution at one or more of positions 3, 46, 82, 103, 119, 125, 177, 180, 185, 187, 200, 207, 209, 226, 235, 240, 241, 243, 258, 296, 298, 311, 315, 319, 327, 328, 340, 421, 436, 456, 470, 486, 503, 552, 570 and 591 of the sequence of SEQ ID NO: 14487 or SEQ ID NO: 14484. In certain embodiments, including those embodiments wherein the transposase comprises the above-described mutations at positions 30, 165, 282 and/or 538, the piggyBac®, Super piggyBac™ or piggyBac-like transposase enzyme may further comprise an amino acid substitution at one or more of positions 46, 119, 125, 177, 180, 185, 187, 200, 207, 209, 226, 235, 240, 241, 243, 296, 298, 311, 315, 319, 327, 328, 340, 421, 436, 456, 470, 485, 503, 552 and 570. In certain embodiments, the amino acid substitution at position 3 of SEQ ID NO: 14487 or SEQ ID NO: 14484 is a substitution of an asparagine (N) for a serine (S). In certain embodiments, the amino acid substitution at position 46 of SEQ ID NO: 14487 or SEQ ID NO: 14484 is a substitution of a serine (S) for an alanine (A). In certain embodiments, the amino acid substitution at position 46 of SEQ ID NO: 14487 or SEQ ID NO: 14484 is a substitution of a threonine (T) for an alanine (A). In certain embodiments, the amino acid substitution at position 82 of SEQ ID NO: 14487 or SEQ ID NO: 14484 is a substitution of a tryptophan (W) for an isoleucine (I). In certain embodiments, the amino acid substitution at position 103 of SEQ ID NO: 14487 or SEQ ID NO: 14484 is a substitution of a proline (P) for a serine (S). In certain embodiments, the amino acid substitution at position 119 of SEQ ID NO: 14487 or SEQ ID NO: 14484 is a substitution of a proline (P) for an arginine (R). In certain embodiments, the amino acid substitution at position 125 of SEQ ID NO: 14487 or SEQ ID NO: 14484 is a substitution of an alanine (A) a cysteine (C). In certain embodiments, the amino acid substitution at position 125 of SEQ ID NO: 14487 or SEQ ID NO: 14484 is a substitution of a leucine (L) for a cysteine (C). In certain embodiments, the amino acid substitution at position 177 of SEQ ID NO: 14487 or SEQ ID NO: 14484 is a substitution of a lysine (K) for a tyrosine (Y). In certain embodiments, the amino acid substitution at position 177 of SEQ ID NO: 14487 or SEQ ID NO: 14484 is a substitution of a histidine (H) for a tyrosine (Y). In certain embodiments, the amino acid substitution at position 180 of SEQ ID NO: 14487 or SEQ ID NO: 14484 is a substitution of a leucine (L) for a phenylalanine (F). In certain embodiments, the amino acid substitution at position 180 of SEQ ID NO: 14487 or SEQ ID NO: 14484 is a substitution of an isoleucine (I) for a phenylalanine (F). In certain embodiments, the amino acid substitution at position 180 of SEQ ID NO: 14487 or SEQ ID NO: 14484 is a substitution of a valine (V) for a phenylalanine (F). In certain embodiments, the amino acid substitution at position 185 of SEQ ID NO: 14487 or SEQ ID NO: 14484 is a substitution of a leucine (L) for a methionine (M). In certain embodiments, the amino acid substitution at position 187 of SEQ ID NO: 14487 or SEQ ID NO: 14484 is a substitution of a glycine (G) for an alanine (A). In certain embodiments, the amino acid substitution at position 200 of SEQ ID NO: 14487 or SEQ ID NO: 14484 is a substitution of a tryptophan (W) for a phenylalanine (F). In certain embodiments, the amino acid substitution at position 207 of SEQ ID NO: 14487 or SEQ ID NO: 14484 is a substitution of a proline (P) for a valine (V). In certain embodiments, the amino acid substitution at position 209 of SEQ ID NO: 14487 or SEQ ID NO: 14484 is a substitution of a phenylalanine (F) for a valine (V). In certain embodiments, the amino acid substitution at position 226 of SEQ ID NO: 14487 or SEQ ID NO: 14484 is a substitution of a phenylalanine (F) for a methionine (M). In certain embodiments, the amino acid substitution at position 235 of SEQ ID NO: 14487 or SEQ ID NO: 14484 is a substitution of an arginine (R) for a leucine (L). In certain embodiments, the amino acid substitution at position 240 of SEQ ID NO: 14487 or SEQ ID NO: 14484 is a substitution of a lysine (K) for a valine (V). In certain embodiments, the amino acid substitution at position 241 of SEQ ID NO: 14487 or SEQ ID NO: 14484 is a substitution of a leucine (L) for a phenylalanine (F). In certain embodiments, the amino acid substitution at position 243 of SEQ ID NO: 14487 or SEQ ID NO: 14484 is a substitution of a lysine (K) for a proline (P). In certain embodiments, the amino acid substitution at position 258 of SEQ ID NO: 14487 or SEQ ID NO: 14484 is a substitution of a serine (S) for an asparagine (N). In certain embodiments, the amino acid substitution at position 296 of SEQ ID NO: 14487 or SEQ ID NO: 14484 is a substitution of a tryptophan (W) for a leucine (L). In certain embodiments, the amino acid substitution at position 296 of SEQ ID NO: 14487 or SEQ ID NO: 14484 is a substitution of a tyrosine (Y) for a leucine (L). In certain embodiments, the amino acid substitution at position 296 of SEQ ID NO: 14487 or SEQ ID NO: 14484 is a substitution of a phenylalanine (F) for a leucine (L). In certain embodiments, the amino acid substitution at position 298 of SEQ ID NO: 14487 or SEQ ID NO: 14484 is a substitution of a leucine (L) for a methionine (M). In certain embodiments, the amino acid substitution at position 298 of SEQ ID NO: 14487 or SEQ ID NO: 14484 is a substitution of an alanine (A) for a methionine (M). In certain embodiments, the amino acid substitution at position 298 of SEQ ID NO: 14487 or SEQ ID NO: 14484 is a substitution of a valine (V) for a methionine (M). In certain embodiments, the amino acid substitution at position 311 of SEQ ID NO: 14487 or SEQ ID NO: 14484 is a substitution of an isoleucine (I) for a proline (P). In certain embodiments, the amino acid substitution at position 311 of SEQ ID NO: 14487 or SEQ ID NO: 14484 is a substitution of a valine for a proline (P). In certain embodiments, the amino acid substitution at position 315 of SEQ ID NO: 14487 or SEQ ID NO: 14484 is a substitution of a lysine (K) for an arginine (R). In certain embodiments, the amino acid substitution at position 319 of SEQ ID NO: 14487 or SEQ ID NO: 14484 is a substitution of a glycine (G) for a threonine (T). In certain embodiments, the amino acid substitution at position 327 of SEQ ID NO: 14487 or SEQ ID NO: 14484 is a substitution of an arginine (R) for a tyrosine (Y). In certain embodiments, the amino acid substitution at position 328 of SEQ ID NO: 14487 or SEQ ID NO: 14484 is a substitution of a valine (V) for a tyrosine (Y). In certain embodiments, the amino acid substitution at position 340 of SEQ ID NO: 14487 or SEQ ID NO: 14484 is a substitution of a glycine (G) for a cysteine (C). In certain embodiments, the amino acid substitution at position 340 of SEQ ID NO: 14487 or SEQ ID NO: 14484 is a substitution of a leucine (L) for a cysteine (C). In certain embodiments, the amino acid substitution at position 421 of SEQ ID NO: 14487 or SEQ ID NO: 14484 is a substitution of a histidine (H) for the aspartic acid (D). In certain embodiments, the amino acid substitution at position 436 of SEQ ID NO: 14487 or SEQ ID NO: 14484 is a substitution of an isoleucine (I) for a valine (V). In certain embodiments, the amino acid substitution at position 456 of SEQ ID NO: 14487 or SEQ ID NO: 14484 is a substitution of a tyrosine (Y) for a methionine (M). In certain embodiments, the amino acid substitution at position 470 of SEQ ID NO: 14487 or SEQ ID NO: 14484 is a substitution of a phenylalanine (F) for a leucine (L). In certain embodiments, the amino acid substitution at position 485 of SEQ ID NO: 14487 or SEQ ID NO: 14484 is a substitution of a lysine (K) for a serine (S). In certain embodiments, the amino acid substitution at position 503 of SEQ ID NO: 14487 or SEQ ID NO: 14484 is a substitution of a leucine (L) for a methionine (M). In certain embodiments, the amino acid substitution at position 503 of SEQ ID NO: 14487 or SEQ ID NO: 14484 is a substitution of an isoleucine (I) for a methionine (M). In certain embodiments, the amino acid substitution at position 552 of SEQ ID NO: 14487 or SEQ ID NO: 14484 is a substitution of a lysine (K) for a valine (V). In certain embodiments, the amino acid substitution at position 570 of SEQ ID NO: 14487 or SEQ ID NO: 14484 is a substitution of a threonine (T) for an alanine (A). In certain embodiments, the amino acid substitution at position 591 of SEQ ID NO: 14487 or SEQ ID NO: 14484 is a substitution of a proline (P) for a glutamine (Q). In certain embodiments, the amino acid substitution at position 591 of SEQ ID NO: 14487 or SEQ ID NO: 14484 is a substitution of an arginine (R) for a glutamine (Q).

In certain embodiments of the methods of the disclosure, including those embodiments wherein the transposase comprises the above-described mutations at positions 30, 165, 282 and/or 538, the piggyBac® or piggyBac-like transposase enzyme or may comprise or the Super piggyBac™ transposase enzyme may further comprise an amino acid substitution at one or more of positions 103, 194, 372, 375, 450, 509 and 570 of the sequence of SEQ ID NO: 14487 or SEQ ID NO: 14484. In certain embodiments of the methods of the disclosure, including those embodiments wherein the transposase comprises the above-described mutations at positions 30, 165, 282 and/or 538, the piggyBac® or piggyBac-like transposase enzyme may comprise or the Super piggyBac™ transposase enzyme may further comprise an amino acid substitution at two, three, four, five, six or more of positions 103, 194, 372, 375, 450, 509 and 570 of the sequence of SEQ ID NO: 14487 or SEQ ID NO: 14484. In certain embodiments, including those embodiments wherein the transposase comprises the above-described mutations at positions 30, 165, 282 and/or 538, the piggyBac® or piggyBac-like transposase enzyme may comprise or the Super piggyBac™ transposase enzyme may further comprise an amino acid substitution at positions 103, 194, 372, 375, 450, 509 and 570 of the sequence of SEQ ID NO: 14487 or SEQ ID NO: 14484. In certain embodiments, the amino acid substitution at position 103 of SEQ ID NO: 14487 or SEQ ID NO: 14484 is a substitution of a proline (P) for a serine (S). In certain embodiments, the amino acid substitution at position 194 of SEQ ID NO: 14487 or SEQ ID NO: 14484 is a substitution of a valine (V) for a methionine (M). In certain embodiments, the amino acid substitution at position 372 of SEQ ID NO: 14487 or SEQ ID NO: 14484 is a substitution of an alanine (A) for an arginine (R). In certain embodiments, the amino acid substitution at position 375 of SEQ ID NO: 14487 or SEQ ID NO: 14484 is a substitution of an alanine (A) for a lysine (K). In certain embodiments, the amino acid substitution at position 450 of SEQ ID NO: 14487 or SEQ ID NO: 14484 is a substitution of an asparagine (N) for an aspartic acid (D). In certain embodiments, the amino acid substitution at position 509 of SEQ ID NO: 14487 or SEQ ID NO: 14484 is a substitution of a glycine (G) for a serine (S). In certain embodiments, the amino acid substitution at position 570 of SEQ ID NO: 14487 or SEQ ID NO: 14484 is a substitution of a serine (S) for an asparagine (N). In certain embodiments, the piggyBac® or piggyBac-like transposase enzyme may comprise a substitution of a valine (V) for a methionine (M) at position 194 of SEQ ID NO: 14487. In certain embodiments, including those embodiments wherein the piggyBac® or piggyBac-like transposase enzyme may comprise a substitution of a valine (V) for a methionine (M) at position 194 of SEQ ID NO: 14487, the piggyBac® or piggyBac-like transposase enzyme may further comprise an amino acid substitution at positions 372, 375 and 450 of the sequence of SEQ ID NO: 14487 or SEQ ID NO: 14484. In certain embodiments, the piggyBac® or piggyBac-like transposase enzyme may comprise a substitution of a valine (V) for a methionine (M) at position 194 of SEQ ID NO: 14487, a substitution of an alanine (A) for an arginine (R) at position 372 of SEQ ID NO: 14487, and a substitution of an alanine (A) for a lysine (K) at position 375 of SEQ ID NO: 14487. In certain embodiments, the piggyBac™ or piggyBac-like transposase enzyme may comprise a substitution of a valine (V) for a methionine (M) at position 194 of SEQ ID NO: 14487, a substitution of an alanine (A) for an arginine (R) at position 372 of SEQ ID NO: 14487, a substitution of an alanine (A) for a lysine (K) at position 375 of SEQ ID NO: 14487 and a substitution of an asparagine (N) for an aspartic acid (D) at position 450 of SEQ ID NO: 14487.

In certain embodiments, the piggyBac® or piggyBac-like transposase enzyme is isolated or derived from an insect. In certain embodiments, the insect is Trichoplusia ni (GenBank Accession No. AAA87375; SEQ ID NO: 16796), Argyrogramma agnata (GenBank Accession No. GU477713; SEQ ID NO: 14534, SEQ ID NO: 16797), Anopheles gambiae (GenBank Accession No. XP_312615 (SEQ ID NO: 16798); GenBank Accession No. XP_320414 (SEQ ID NO: 16799); GenBank Accession No. XP_310729 (SEQ ID NO: 16800)), Aphis gossypii (GenBank Accession No. GU329918; SEQ ID NO: 16801, SEQ ID NO: 16802), Acyrthosiphon pisum (GenBank Accession No. XP_001948139; SEQ ID NO: 16803), Agrotis ipsilon (GenBank Accession No. GU477714; SEQ ID NO: 14537, SEQ ID NO: 16804), Bombyx mori (GenBank Accession No. BAD11135; SEQ ID NO: 14505), Chilo suppressalis (GenBank Accession No. JX294476; SEQ ID NO: 16805, SEQ ID NO: 16806), Drosophila melanogaster (GenBank Accession No. AAL39784; SEQ ID NO: 16807), Helicoverpa armigera (GenBank Accession No. ABS18391; SEQ ID NO: 14525), Heliothis virescens (GenBank Accession No. ABD76335; SEQ ID NO: 16808), Macdunnoughia crassisigna (GenBank Accession No. EU287451; SEQ ID NO: 16809, SEQ ID NO: 16810), Pectinophora gossypiella (GenBank Accession No. GU270322; SEQ ID NO: 14530, SEQ ID NO: 16811), Tribolium castaneum (GenBank Accession No. XP_001814566; SEQ ID NO: 16812), Ctenoplusia agnata (also called Argyrogramma agnata), Messour bouvieri, Megachile rotundata, Bombus impatiens, Manestra brassicae, Mayetiola destructor or Apis mellifera.

In certain embodiments, the piggyBac® or piggyBac-like transposase enzyme is isolated or derived from an insect. In certain embodiments, the insect is Trichoplusia ni (AAA87375).

In certain embodiments, the piggyBac® or piggyBac-like transposase enzyme is isolated or derived from an insect. In certain embodiments, the insect is Bombyx mori (BAD11135).

In certain embodiments, the piggyBac® or piggyBac-like transposase enzyme is isolated or derived from a crustacean. In certain embodiments, the crustacean is Daphnia pulicaria (AAM76342, SEQ ID NO: 16813).

In certain embodiments, the piggyBac® or piggyBac-like transposase enzyme is isolated or derived from a vertebrate. In certain embodiments, the vertebrate is Xenopus tropicalis (GenBank Accession No. BAF82026; SEQ ID NO: 14518), Homo sapiens (GenBank Accession No. NP_689808; SEQ ID NO: 16814), Mus musculus (GenBank Accession No. NP_741958; SEQ ID NO: 16815), Macaca fascicularis (GenBank Accession No. AB179012; SEQ ID NO: 16816, SEQ ID NO: 16817). Rattus norvegicus (GenBank Accession No. XP_220453; SEQ ID NO: 16818) or Myotis lucifugus.

In certain embodiments, the piggyBac® or piggyBac-like transposase enzyme is isolated or derived from a urochordate. In certain embodiments, the urochordate is Ciona intestinalis (GenBank Accession No. XP_002123602; SEQ ID NO: 16819).

In certain embodiments, the piggyBac® or piggyBac-like transposase inserts a transposon at the sequence 5′-TTAT-3′ within a chromosomal site (a TTAT target sequence).

In certain embodiments, the piggyBac® or piggyBac-like transposase inserts a transposon at the sequence 5′-TTAA-3′ within a chromosomal site (a TTAA target sequence).

In certain embodiments, the target sequence of the piggyBac® or piggyBac-like transposon comprises or consists of 5′-CTAA-3′, 5′-TTAG-3′, 5′-ATAA-3′, 5′-TCAA-3′, 5′AGTT-3′. 5′-ATTA-3′, 5′-GTTA-3′, 5′-TTGA-3′. 5′-TTTA-3′, 5′-TTAC-3′, 5′-ACTA-3′, 5′-AGGG-3′, 5′-CTAG-3′, 5′-TGAA-3′, 5′-AGGT-3′, 5′-ATCA-3′, 5′-CTCC-3′, 5′-TAAA-3′, 5′-TCTC-3′, 5′TGAA-3′, 5′-AAAT-3′, 5′-AATC-3′, 5′-ACAA-3′, 5′-ACAT-3, 5′-ACTC-3′, 5′-AGTG-3′, 5′-ATAG-3′, 5′-CAAA-3′, 5′-CACA-3′, 5′-CATA-3′, 5′-CCAG-3′, 5′-CCCA-3′, 5′-CGTA-3-, 5′-GTCC-3′, 5′-TAAG-3′, 5′-TCTA-3′, 5′-TGAG-3′, 5′-TGTT-3′, 5-TTCA-3′5′-TTCT-3′ and 5′-TTTT-3′.

In certain embodiments of the methods of the disclosure, the transposase enzyme is a piggyBac® or piggyBac-like transposase enzyme. In certain embodiments, the piggyBac® or piggyBac-like transposase enzyme is isolated or derived from Bombyx mori. The piggyBac® or piggyBac-like transposase enzyme may comprise or consist of an amino acid sequence at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% or any percentage in between identical to:

(SEQ ID NO: 14504)

1
MDIERQEERI RAMLEEELSD YSDESSSEDE TDHCSEHEVN

YDTEEERIDS VDVPSNSRQE

61
EANAIIANES DSDPDDDLPL SLVRQRASAS RQVSGPFYTS

KDGTKWYKNC QRPNVRLRSE

121
NIVTEQAQVK NIARDASTEY ECWNIFVTSD MLQEILTHTN

SSIRHRQTKT AAENSSAETS

181
FYMQETTLCE LKALIALLYL AGLIKSNRQS LKDLWRTDGT

GVDIFRTTMS LQRFQFLQNN

241
IRFDDKSTRD ERKQTDNMAA FRSIFDQFVQ CCQNAYSPSE

FLTIDEMLLS FRGRCLFRVY

301
IPNKPAKYGI KILALVDAKN FDVVNLEVYA GKQPSGPYAV

SNRPFEVVER LIQPVARSHR

361
NVTFDNWFTG YELMLHLLNE YRLTSVGTVR KNKRQIPESF

IRTDRQPNSS VFGFQKDITL

421
VSYAPKKNKV VVVMSTMHHD NSIDESTGEK QKPEMITFYN

STKAGVDVVD ELSANYNVSR

481
NSKRWPMTLF YGVLNMAAIN ACIIYRPNKN VTIKRTEFIR

SLGLSMIYEH LHSRNKKKNI

541
PTYLRQRIEK QLGEPSPRHV NVPGRYVRCQ DCPYKKDRKT

KHSCNACAKP ICMEHAKFLC

601
ENCAELDSSL.

The piggyBac® (PB) or piggyBac-like transposase enzyme may comprise or consist of an amino acid sequence at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% or any percentage in between identical to:

(SEQ ID NO: 14505)

1
MDIERQEERI RAMLEEELSD YSDESSSEDE TDHCSEHEVN

YDTEEERIDS VDVPSNSRQE

61
EANAIIANES DSDPDDDLPL SLVRQRASAS RQVSGPFYTS

KDGTKWYKNC QRPNVRLRSE

121
NIVTEQAQVK NIARDASTEY ECWNIFVTSD MLQEILTHTN

SSIRHRQTKT AAENSSAETS

181
FYMQETTLCE LKALIALLYL AGLIKSNRQS LKDLWRTDGT

GVDIFRTTMS LQRFQFLQNN

241
IRFDDKSTRD ERKQTDNMAA FRSIFDQFVQ CCQNAYSPSE

FLTIDEMLLS FRGRCLFRVY

301
IPNKPAKYGI KILALVDAKN FYVVNLEVYA GKQPSGPYAV

SNRPFEVVER LIQPVARSHR

361
NVTFDNWFTG YELMLHLLNE YRLTSVGTVR KNKRQIPESF

IRTDRQPNSS VFGFQKDITL

421
VSYAPKKNKV VVVMSTMHHD NSIDESTGEK QKPEMITFYN

STKAGVDVVD ELSANYNVSR

481
NSKRWPMTLF YGVLNMAAIN ACIIYRPNKN VTIKRTEFIR

SLGLSMIYEH LHSRNKKKNI

541
PTYLRQRIEK QLGEPSPRHV NVPGRYVRCQ DCPYKKDRKT

KHSCNACAKP ICMEHAKFLC

601
ENCAELDSSL.

In certain embodiments, the piggyBac® or piggyBac-like transposase is fused to a nuclear localization signal. In certain embodiments, the amino acid sequence of the piggyBac® or piggyBac-like transposase fused to a nuclear localization signal is encoded by a polynucleotide sequence comprising:

(SEQ ID NO: 14629)

1
atggcaccca aaaagaaacg taaagtgatg gacattgaaa

gacaggaaga aagaatcagg

61
gcgatgctcg aagaagaact gagcgactac tccgacgaat

cgtcatcaga ggatgaaacc

121
gaccactgta gcgagcatga ggttaactac gacaccgagg

aggagagaat cgactctgtg

181
gatgtgccct ccaactcacg ccaagaagag gccaatgcaa

ttatcgcaaa cgaatcggac

241
agcgatccag acgatgatct gccactgtcc ctcgtgcgcc

agcgggccag cgcttcgaga

301
caagtgtcag gtccattcta cacttcgaag gacggcacta

agtggtacaa gaattgccag

361
cgacctaacg tcagactccg ctccgagaat atcgtqaccg

aacaggctca ggtcaagaat

421
atcgcccgcg acgcctcgac tgagtacgag tgttggaata

tcttcgtgac ttcggacatg

481
ctgcaagaaa ttctgacgca caccaacagc tcgattaggc

atcgccagac caagactgca

541
gcggagaact catcggccga aacctccttc tatatgcaag

agactactct gtgcgaactg

601
aaggcgctga ttgcactgct gtacttggcc ggcctcatca

aatcaaatag gcagagcctc

661
aaagatctct ggagaacgga tggaactgga gtggatatct

ttcggacgac tatgagcttg

721
cagcggttcc agtttctgca aaacaatatc agattcgacg

acaagtccac ccgggacgaa

781
aggaaacaga ctgacaacat ggctgcgttc cggtcaatat

tcgatcagtt tgtgcagtgc

841
tgccaaaacg cttatagccc atcggaattc ctgaccatcg

acgaaatgct tctctccttc

901
cgggggcgct gcctgttccg agtgtacatc ccgaacaagc

cggctaaata cggaatcaaa

961
atcctggccc tggtggacgc caagaatttc tacgtcgtga

atctcgaagt gtacgcagga

1021
aagcaaccgt cgggaccgta cgctgtttcg aaccgcccgt

ttgaagtcgt cgagcggctt

1081
attcagccgg tggccagatc ccaccgcaat gttaccttcg

acaattggtt caccggctac

1141
gagctgatgc ttcaccttct gaacgagtac cggctcacta

gcgtggggac tgtcaggaag

1201
aacaagcggc agatcccaga atccttcatc cgcaccgacc

gccagcctaa ctcgtccgtg

1261
ttcggatttc aaaaggatat cacgcttgtc tcgtacgccc

ccaagaaaaa caaggtcgtg

1321
gtcgtgatga gcaccatgca tcacgacaac agcatcgacg

agtcaaccgg agaaaagcaa

1381
aagcccgaga tgatcacctt ctacaattca actaaggccg

gcgtcgacgt cgtggatgaa

1441
ctgtgcgcga actataacgt gtcccggaac tctaagcggt

ggcctatgac tctcttctac

1501
ggagtgctga atatggccgc aatcaacgcg tgcatcatct

accgcaccaa caagaacgtg

1561
accatcaagc gcaccgagtt catcagatcg ctgggtttga

gcatgatcta cgagcacctc

1621
cattcacgga acaagaagaa gaatatccct acttacctga

ggcagcgtat cgagaagcag

1681
ttgggagaac caagcccgcg ccacgtgaac gtgccggggc

gctacgtgcg gtgccaagat

1741
tgcccgtaca aaaaggaccg caaaaccaaa agatcgtgta

acgcgtgcgc caaacctatc

1801
tgcatggagc atgccaaatt tctgtgtgaa aattgtgctg

aactcgattc ctccctg.

In certain embodiments, the piggyBac® or piggyBac-like transposase is hyperactive. A hyperactive piggyBac or piggyBac-like transposase is a transposase that is more active than the naturally occurring variant from which it is derived. In certain embodiments, the hyperactive piggyBac or piggyBac-like transposase enzyme is isolated or derived from Bombyx mori. In certain embodiments, the piggyBac® or piggyBac-like transposase is a hyperactive variant of SEQ ID NO: 14505. In certain embodiments, the hyperactive piggyBac or piggyBac-like transposase comprises a sequence that is at least 90% identical to:

(SEQ ID NO: 14576)

1
MDIERQEERI RAMLEEELSD YSDESSSEDE TDHCSEHEVN

YDTEEERIDS VDVPSNSRQE

61
EANAIIANES DSDPDDDLPL SLVRQRASAS RQVSGPFYTS

KDGTKWYKNC QRPNVRLRSE

121
NIVTEQAQVK NIARDASTEY ECWNIFVTSD MLQEILTHTN

SSIRHRQTKT AAENSSAETS

181
FYMQETTLCE LKALIALLYL AGLIKSNRQS LKDLWRTDGT

GVDIFRTTMS LQRFQFLQNN

241
IRFDDKSTRD ERKQTDNMAA FRSIFDQFVQ SCQNAYSPSE

FLTIDEMLLS FRGRCLFRVY

301
IPNKPAKYGI KILALVDAKN FDVVNLEVYA GKQPSGPYAV

SNRPFEVVER LIQPVARSHR

361
NVTFDNWFTG YELMLHLLNE YRLTSVGTVR KNKRQIPESF

IRTDRQPNSS VFGFQKDITL

421
VSYAPKKNKV VVVMSTMHHD NSIDESTGEK QKPEMITFYN

STKAGVDVVD ELSANYNVSR

481
NSKRWPMTLF YGVLNMAAIN ACIIYRPNKN VTIKRTEFIR

SLGLSMIYEH LHSRNKKKNI

541
PTYLRQRIEK QLGEPSPRHV NVPGRYVRCQ DCPYKKDRKT

KHSCNACAKP ICMEHAKFLC

601
ENCAELDSHL.

In certain embodiments, the hyperactive piggyBac or piggyBac-like transposase comprises SEQ ID NO: 14576. In certain embodiments, the hyperactive piggyBac or piggyBac-like transposase comprises a sequence of:

(SEQ ID NO: 14630)

1
MDIERQEERI RAMLEEELSD YSDESSSEDE TDHCSEHEVN

YDTEEERIDS VDVPSNSRQE

61
EANAIIANES DSDPDDDLPL SLVRQRASAS RQVSGPFYTS

KDGTKWYKNC QRPNVRLRSE

121
NIVTEQAQVK NIARDASTEY ECWNIFVTSD MLQEILTHTN

SSIRHRQTKT AAENSSAETS

181
FYMQETTLCE LKALIALLYL AGLIKSNRQS LKDLWRTDGT

GVDIFRTTMS LQRFQFLLNN

241
IRFDDKSTRD ERKQTDNMAA FRSIFDQFVQ SCQNAYSPSE

FLTIDEMLLS FRGRCLFRVY

301
IPNKPAKYGI KILALVDAKN FDVHNLEVYA GKQPSGPYAV

SNRPFEVVER LIQPVARSHR

361
NVTFDNWFTG YEVMLHLLNE YRLTSVGTVR KNKRQIPESF

IRTDRQPNSS VFGFQKDITL

421
VSYAPKKNKV VVVMSTMHHD NSIDESTGEK QKPEMITFYN

STKAGVDVVD ELSANYNVSR

481
NSKRWPMTLF YGVLNMAAIN ACIIYRTNKN VTIKRTEFIR

SLGLSMIYEH LHSRNKKKNI

541
PTYLRQRIEK QLGEPSPRHV NVPGRYVRCQ DCPYKKDRKT

KRSCNACAKP ICMEHAKFLC

601
ENCAHLDS.

In certain embodiments, the hyperactive piggyBac or piggyBac-like transposase comprises a sequence of;

(SEQ ID NO: 14631)

1
MDIERQEERI RAMLEEELSD YSDESSSEDE TDHCSEHEVN

YDTEEERIDS VDVPSNSRQE

61
EANAIIANES DSDPDDDLPL SLVRQRASAS RQVSGPFYTS

KDGTKWYKNC QRPNVRLRSE

121
NIVTEQAQVK NIARDASTEY ECWNIFVTSD MLQEILTHTN

SSIRHRQTKT AAENSSASTS

181
FYMQETTLCE LKALIALLYI AGLIKSNRQS LKDLWRTDGT

GVDIFRTTMS LQRFQFLLNN

241
IRFDDKSTRD ERKQTDNMAA FRSIFDQFVQ SCQNAYSPSE

FLTIDEMLLS FRGRCLFRVY

301
IPNKPAKYGI KILALVDAKN FDVVNLEVYA GKQPSGPYAV

SNRPFEVVER LIQPVARSHR

361
NVTFDNWFTG YELMLHLLNE YRLTSVGTVR KNKRQIPESF

IRTDRQPNSS VFGFQKDITL

421
VSYAPKKNKV VVVMSTMHHD NSIDESTGEK QKPEMITFYN

STKAGVDVVD ELSANYNVSR

481
NSKRWPMTLF YGVLNMAAIN ACIIYRPNKN VTIKRTEFIR

SLGLSMIYEH LHSRNKKKNI

541
PTYLRQRIEK QLGEPSPRHV NVPGRYVRCQ DCPYKKDRKT

KHSCNACAKP ICMEHAKFLC

601
ENCAELDSSL.

In certain embodiments, the hyperactive piggyBac or piggyBac-like transposase comprises a sequence of:

(SEQ ID NO: 14632)

1
MDIERQEERI RAMLEEELSD YSDESSSEDE TDHCSEHEVN

YDTEEERIDS VDVPSNSRQE

61
EANAIIANES DSDPDDDLPL SLVRQRASAS RQVSGPFYTS

KDGTKWYKNC QRPNVRLRSE

121
NIVTEQAQVK NIARDASTEY ECWNIFVTSD MLQEILTHTN

SSIRHRQTKT AAENSSAETS

181
FYMQETTLCE LKALIGLLYI AGLIKSNRQS LKDLWRTDGT

GVDIFRTTMS LQRFQFLLNN

241
IRFDDKSTRD ERKQTDNMAA FRSIFDQFVQ SCQNAYSPSE

FLTIDEMLLS FRGRCLFRVY

301
IPNKPAKYGI KILALVDAKN FYVKNLEVYA GKQPSGPYAV

SNRPFEVVER LIQPVARSHR

361
NVTFDNWFTG YELMLHLLNE YRLTSVGTVR KNKRQIPENF

IRTDRQPNSS VFGFQKDITL

421
VSYAPKKNKV VVVMSTMHHD NSIDESTGEK QKPEMITFYN

STKAGVDVVD ELSANYNVSR

481
NSKRWPMTLF YGVLNMAAIN ACIIYRTNKN VTIKRTEFIR

SLGLSMIYEH LHSRNKKKNI

541
PTYLRQRIEK QLGEPSPRHV NVPGRYVRCQ DCPYKKDRKT

KRSCNACAKP ICMEHAKFLC

601
ENCAELDSSL.

In certain embodiments, the hyperactive piggyBac or piggyBac-like transposase comprises a sequence of:

(SEQ ID NO: 14633)

1
MDIERQEERI RAMLEEELSD YSDESSSEDE TDHCSEHEVN

YDTEEERIDS VDVPSNSRQE

61
EANAIIANES DSDPDDDLPL SLVRQRASAS RQVSGPFYTS

KDGTKWYKNC QRPNVRLRSE

121
NIVTEQAQVK NIARDASTEY ECWNIFVTSD MLQEILTHTN

SSIRHRQTKT AAENSSAETS

181
FYMQETTLCE LKALIGLLYI AGLIKSNRQS LKDLWRTDGT

GVDIFRTTMS LQRFQFLQNN

241
IRFDDKSTRD ERKQTDNMAA FRSIFDQFVQ SCQNAYSPSE

FLTIDEMLLS FRGRCLFRVY

301
IPNKPAKYGI KILALVDAKN FYVKNLEVYA GKQPSGPYAV

SNRPFEVVER LIQPVARSHR

361
NVTFDNWFTG YELMLHLLNE YRLTSVGTVR KNKRQIPESF

IRTDRQPNSS VFGFQKDITL

421
VSYAPKKNKV VVVMSTMHHD NSIDESTGEK QKPEMITFYN

STKAGVDVVD ELCANYNVSR

481
NSKRWPMTLF YGVLNMAAIN ACIIYRTNKN VTIKRTEFIR

SLGLSMIYEH LHSRNKKKNI

541
PTYLRQRIEK QLGEPSPRHV NVPGRYVRCQ DCPYKKDRKT

KRSCNACAKP ICMEHAKFLC

601
ENCAELDSSL.

In certain embodiments, the hyperactive piggyBac or piggyBac-like transposase comprises a sequence of:

(SEQ ID NO: 14634)

1
MDIERQEERI RAMLEEELSD YSDESSSEDE TDHCSEHEVN

YDTEEERIDS VDVPSNSRQE

61
EANAIIANES DSDPDDDLPL SLVRQRASAS RQVSGPFYTS

KDGTKWYKNC QRPNVRLRSE

121
NIVTEQAQVK NIARDASTEY ECWNIFVTSD MLQEILTHTN

SSIRHRQTKT AAENSSAETS

181
FYMQETTLCE LKALIALLYL AGLIKSNRQS LKDLWRTDGT

GVDIFRTTMS LQRFQFLQNN

241
IRFDDKSTRD ERKQTDNMAA FRSIFDQFVQ CCQNAYSPSE

FLTIDEMLLS FRGRCLFRVY

301
IPNKPAKYGI KILALVDAKN DYVVNLEVYA GKQPSGPYAV

SNRPFEVVER LIQPVARSHR

361
NVTFDNWFTG YELMLHLLNE YRLTSVGTVR KNKRQIPESF

IRTDRQPNSS VFGFQKDITL

421
VSYAPKKNKV VVVMSTMHHD NSIDESTGEK QKPEMITFYN

STKAGVDVVD ELSANYNVSR

481
NSKRWPMTLF YGVLNMAAIN ACIIYRTNKN VTIKRTEFIR

SLGLSMIYEH LHSRNKKKNI

541
PTYLRQRIEK QLGEPSPRHV NVKGRYVRCQ DCPYKKDRKT

KRSCNACAKP ICMEHAKFLC

601
ENCAELDSSL.

In certain embodiments, the hyperactive piggyBac or piggyBac-like transposase is more active than the transposase of SEQ ID NO: 14505. In certain embodiments, the hyperactive piggyBac or piggyBac-like transposase is at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% or any percentage in between identical to SEQ ID NO: 14505.

In certain embodiments, the hyperactive piggyBac or piggyBac-like transposase comprises an amino acid substitution at a position selected from 92, 93, 96, 97, 165, 178, 189, 196, 200, 201, 211, 215, 235, 238, 246, 253, 258, 261, 263, 271, 303, 321, 324, 330, 373, 389, 399, 402, 403, 404, 448, 473, 484, 507, 523, 527, 528, 543, 549, 550, 557.601, 605, 607, 609, 610 or a combination thereof (relative to SEQ ID NO: 14505). In certain embodiments, the hyperactive piggyBac or piggyBac-like transposase comprises an amino acid substitution of Q92A, V93L, V93M, P96G, F97H, F97C, H165E, H165W, E178S, E178H, C189P, A196G, L200I, A201Q, L211A, W215Y, G219S, Q235Y, Q235G, Q238L, K246I, K253V, M258V, F261L, S263K, C271S, N303R, F321W, F321D, V324K, V324H, A330V, L373C, L373V, V389L, S399N, R402K, T403L, D404Q, D404S, D404M, N441R, G448W, E449A, V469T, C473Q, R484K T507C, G523A, I527M, Y528K Y543I, E549A, K550M, P557S, E601V, E605H, E605W, D607H, S609H, L610I or any combination thereof. In certain embodiments, the hyperactive piggyBac or piggyBac-like transposase comprises an amino acid substitution of Q92A, V93L, V93M, P96G, F97H, F97C, H165E, H165W, E178S, E178H, C189P, A196G. L200I, A201Q, L211A, W215Y, G219S, Q235Y, Q235G, Q238L, K246I, K253V, M258V, F261L, S263K, C271S, N303R, F321W, F321D, V324K, V324H, A330V, L373C, L373V, V389L, S399N, R402K, T403L, D404Q, D404S, D404M. N441R, G448W, E449A, V469T, C473Q, R484K T507C, G523A, I527M, Y528K Y543I, E549A, K550M, P557S, E601V, E605H, E605W, D607H, S609H and L610I.

In certain embodiments, the hyperactive piggyBac or piggyBac-like transposase comprises one or more substitutions of an amino acid that is not wild type, wherein the one or more substitutions a for wild type amino acid comprises a substitution of E4X, A12X, M13X, L14X, E15X, D20X, E24X, S25X, S26X, S27X, D32X, H33X, E36X, E44X, E45X, E46X, I48X, D49X, R58X, A62X, N63X, A64X, I65X, I66X, N68X, E69X, D71X, S72X, D76X, P79X, R84X, Q85X, A87X, S88X, Q92X, V93X, S94X, G95X, P96X, F97X, Y98X, T99X, I145X, S149X, D150X, L152X, E154X, T157X, N160X, S161X, S162X, H165X, R166X, T168X, K169X, T170X, A171X, E173X, S175X, S176X, E178X, T179X, M183X, Q184X, T186X, T187X, L188X, C189X, L194X, I195X, A196X, L198X, L200X, A201X, L203X, I204X, K205X, A206X, N207X, Q209X, S210X, L21 X, K212X, D213X, L214X, W215X, R216X, T217X, G219X, V222X, D223X, I224X, T227X, M229X, Q235X, L237X, Q238X, N239X, N240X, P302X, N303X, P305X, A306X, K307X, Y308X, I310X, K311X, I312X, L313X, A314X, L315X, V316X, D317X, A318X, K319X, N320X, F321X, Y322X, V323X, V324X, L326X, E327X, V328X, A330X, Q333X, P334X, S335X, G336X, P337X, A339X, V340X, S341X, N342X, R343X, P344X, F345X, E346X, V347X, E349X, I352X, Q353X, V355X, A356X, R357X, N361X, D365X, W367X, T369X, G370X, L373X, M374X, L375X, H376X, N379X, E380X, R382X, V386X, V389X, N392X, R394X, Q395X, S399X, F400X, I401X, R402XT403X, D404X, R405X, Q406X, P407X, N408X, S409X, S410X, V411X, F412X, F414X, Q415X, I418X, T419X, L420X, N428XV432X, M434X, D440X, N441X, S442X, I443X, D444X, E445X, G448X, E449X, Q451X, K452X, M455X, I456X, T457X, F458X, S461X, A464X, V466X, Q468X, V469X, E471X, L472X, C473X, A474X, K483X, W485X, T488X, L489X, Y491X, G492X, V493X, M496X, I499X, C502X, I503X, T507X, K509X, N510X, V511X, T512X, I513X, R515X, E517X, S521X, G523X, L524X, S525X, I527X, Y528X, E529X, H532X, S533X, N535X, K536X, K537X, N539X, I540X, T542X, Y543X, Q546X, E549X, K550X, Q551X, G553X, E554X, P555X, S556X, P557X, R558X, H559X, V560X, N561X, V562X, P563X, G564X, R565X, Y566X, V567X, Q570X, D571X, P573X, Y574X, K576X, K581X, S583X, A586X, A588X, E594X, F598X, L599X, E601X, N602X, C603X, A604X, E605X, L606X, D607X, S608X, S609X or L610X (relative to SEQ ID NO: 14505). A list of hyperactive amino acid substitutions can be found in U.S. Pat. No. 10,041,077, the contents of which are incorporated herein by reference in their entirety.

In certain embodiments, the piggyBac® or piggyBac-like transposase is integration deficient. In certain embodiments, an integration deficient piggyBac or piggyBac-like transposase is a transposase that can excise its corresponding transposon, but that integrates the excised transposon at a lower frequency than a corresponding wild type transposase. In certain embodiments, the piggyBac® or piggyBac-like transposase is an integration deficient variant of SEQ ID NO: 14505.

In certain embodiments, the integration deficient piggyBac or piggyBac-like transposase comprises a sequence of:

(SEQ ID NO: 14606)

1
MDIERQEERI RAMLEEELSD YSDESSSEDE TDHCSEHEVN

YDTEEERIDS VDVPSNSRQE

61
EANAIIANES DSDPDDDLPL SLVRQRASAS RQVSGPFYTS

KDGTKWYKNC QRPNVRLRSE

121
NIVTEQAQVK NIARDASTEY ECWNIFVTSD MLQEILTHTN

SSIRHRQTKT AAENSSAETS

181
FYMQETTLCE LKALIALLYL AGLIKSNRQS LKDLWRTDGT

GVDIFRTTMS LQRFQFLQNN

241
IRFDDKSTRD ERKQTDNMAA FRSIFDQFVQ CCQNAYSPSE

FLTIDEMLLS FRGRCLFRVY

301
IPNKPAKYGI KILALVDAKN FYVVNLEVYA GKQPSGPYAV

SNRPFEVVER LIQPVARSHR

361
NVTFDNWFTG YELMLHLLNE YRLTSVGTVR KNKRQIPESF

IRTDRQPNSS VFGFQKDITL

421
VSYAPKKNKV VVVMSTMHHD NSIDESTGEK QKPEMITFYN

STKAGVDVVD ELCANYNVSR

481
NSKKWPMTLF YGVLNMAAIN ACIIYRTNKN VTIKRTEFIR

SLGLSMMYEH LHSRNKKKNI

541
PTYLRQRIEK QLGEPSPRHV NVPGRYVRCQ DCPYKKDRKT

KHSCNACAKP ICMEHAKFLC

601
ENCAELDSSL.

In certain embodiments, the integration deficient piggyBac or piggyBac-like transposase comprises a sequence of:

(SEQ ID NO: 14607)

1
MDIERQEERI RAMLEEELSD YSDESSSEDE TDHCSEHEVN

YDTEEERIDS VDVPSNSRQE

61
EANAIIANES DSDPDDDLPL SLVRQRASAS RQVSGPFYTS

KDGTKWYKNC QRPNVRLRSE

121
NIVTEQAQVK NIARDASTEY ECWNIFVTSD MLQEILTHTN

SSIRHRQTKT AAENSSAETS

181
FYMQETTLCE LKALIGLLYL AGLIKSNRQS LKDLWRTDGT

GVDIFRTTMS LQRFQFLQNN

241
IRFDDKSTLD ERKQTDNMAA FRSIFDQFVQ SCQNAYSPSE

FLTIDEMLLS FRGRCLFRVY

301
IPNKPAKYGI KILALVDAKN FDVVNLEVYA GKQPSGPYAV

SNRPFEVVER LIQPVARSHR

361
NVTFDNWFTG YELMLHLLNE YRLTSVGTVR KNKRQIPESF

IRTDRQPNSS VFGFQKDITL

421
VSYAPKKNKV VVVMSTMHHD NSIDESTGEK QKPEMITFYN

STKAGVDVVD ELSANYNVSR

481
NSKRWPMTLF YGVLNMAAIN ACIIYRPNKN VTIKRTEFIR

SLGLSMIYEH LHSRNKKKNI

541
PTYLRQRIEK QLGEPSPRHV NVPGRYVRCQ DCPYKKDRKT

KHSCNACAKP ICMEHAKFLC

601
VNCAELDSSL.

In certain embodiments, the piggyBac® or piggyBac-like transposase that is integration deficient comprises a sequence of:

(SEQ ID NO: 14608)

1
MDIERQEERI RAMLEEELSD YSDESSSEDE TDHCSEHEVN

YDTEEERIDS VDVPSNSRQE

61
EANAIIANES DSDPDDDLPL SLVRQRASAS RQVSGPFYTS

KDGTKWYKNC QRPNVRLRSE

121
NIVTEQAQVK NIARDASTEY ECWNIFVTSD MLQEILTHTN

SSIRHRQTKT AAENSSAETS

181
FYMQETTLCE LKALIALLYL AGLIKSNRQS LKDLWRTDGT

GVDIFRTTMS LQRFQFLLNN

241
IRFDDKSTRD ERKQTDNMAA FRSIFDQFVQ CCQNAYSPSE

FLTIDEMLLS FRGRCLFRVY

301
IPNKPAKYGI KILALVDAKN FDVVNLEVYA GKQPSGPYAV

SNRPFEVVER LIQPVARSHR

361
NVTFDNWFTG YECMLHLLNE YRLTSVGTVR KNKRQIPESF

IRTDRQPNSS VFGFQKDITL

421
VSYAPKKNKV VVVMSTMHHD NSIDESTGEK QKPEMITFYN

STKAGVDVVD ELSANYNVSR

481
NSKKWPMTLF YGVLNMAAIN ACIIYRTNKN VTIKRTEFIR

SLGLSMIKEH LHSRNKKKNI

541
PTYLRQRIEK QLGEPSPRHV NVPGRYVRCQ DCPYKKDRKT

KHSCNACAKP ICMEHAKFLC

601
ENCAELDSSL.

In certain embodiments, the integration deficient transposase comprises a sequence that is at least 90% identical to SEQ ID NO: 14608.

In certain embodiments, the piggyBac® or piggyBac-like transposon is isolated or derived from Bombyx mori. In certain embodiments, the piggyBac® or piggyBac-like transposon comprises a sequence of:

(SEQ ID NO: 14506)

1
ttatcccggc gagcatgagg cagggtatct cataccctgg

taaaatttta aagttgtgta

61
ttttataaaa ttttcgtctg acaacactag cgcgctcagt

agctggaggc aggagcgtgc

121
gggaggggat agtggcgtga tcgcagtgtg gcacgggaca

ccggcgagat attcgtgtgc

181
aaacctgttt cgggtatgtt ataccctgcc tcattgttga

cgtatttttt ttatgtaatt

241
tttccgatta ttaatttcaa ctgttttatt ggtattttta

tgttatccat tgttcttttt

301
ttatgattta ctgtatcggt tgtctttcgt tcctttagtt

gagttttttt ttattatttt

361
cagtttttga tcaaa.

In certain embodiments, the piggyBac® or piggyBac-like transposon comprises a sequence of:

(SEQ ID NO: 14507)

1
tcatattttt agtttaaaaa aataattata tgttttataa

tgaaaagaat ctcattatct

61
ttcagtatta ggttgattta tattccaaag aataatattt

ttgttaaatt gttgattttt

121
gtaaacctct aaatgtttgt tgctaaaatt actgtgttta

agaaaaagat taataaataa

181
taataatttc ataattaaaa acttctttca ttgaatgcca

ttaaataaac cattatttta

241
caaaataaga tcaacataat tgagtaaata ataataagaa

caatattata gtacaacaaa

301
atatgggtat gtcataccct gccacattct tgatgtaact

ttttttcacc tcatgctcgc

361
cgggttat.

In certain embodiments, the piggyBac® or piggyBac-like transposon comprises a sequence of:

(SEQ ID NO: 14508)

1
ttatcccggc gagcatgagg cagggtatct cataccctgg

taaaatttta aagttgtgta

61
ttttataaaa ttttcgtctg acaacactag cgcgctcagt

agctggaggc aggagcgtgc

121
gggaggggat agtggcgtga tcgcagtgtg gcacgggaca

ccggcgagat attcgtgtgc

181
aaacctgttt cgggtatgtt ataccctgcc tcat.

In certain embodiments, the piggyBac® (PB) or piggyBac-like transposon comprises a sequence of:

(SEQ ID NO: 14509)

1
taaataataa taatttcata attaaaaact tctttcattg

aatgccatta aataaaccat

61
tattttacaa aataagatca acataattga gtaaataata

ataagaacaa tattatagta

121
caacaaaata tgggtatgtc ataccctgcc acattcttga

tgtaactttt tttcacctca

181
tgctcgccgg gttat.

In certain embodiments, the piggyBac® or piggyBac-like transposon comprises a left sequence corresponding to SEQ ID NO: 14506 and a right sequence corresponding to SEQ ID NO: 14507. In certain embodiments, one piggyBac® or piggyBac-like transposon end is at least 85%, at least 90%, at least 95%, at least 98%, at least 99% identical or any percentage in between identical to SEQ ID NO: 14506 and the other piggyBac® or piggyBac-like transposon end is at least 85%, at least 90%, at least 95%, at least 98%, at least 99% or any percentage in between identical to SEQ ID NO: 14507. In certain embodiments, the piggyBac® or piggyBac-like transposon comprises SEQ ID NO: 14506 and SEQ ID NO: 14507 or SEQ ID NO: 14509. In certain embodiments, the piggyBac® or piggyBac-like transposon comprises SEQ ID NO: 14508 and SEQ ID NO: 14507 or SEQ ID NO: 14509. In certain embodiments, the left and right transposon ends share a 16 bp repeat sequence at their ends of CCCGGCGAGCATGAGG (SEQ ID NO: 14510) immediately adjacent to the 5′-TTAT-3 target insertion site, which is inverted in the orientation in the two ends. In certain embodiments, left transposon end begins with a sequence comprising 5′-TTATCCCGGCGAGCATGAGG-3 (SEQ ID NO: 14511), and the right transposon ends with a sequence comprising the reverse complement of this sequence: 5′-CCTCATGCTCGCCGGGTTAT-3′ (SEQ ID NO: 14512).

In certain embodiments, the piggyBac® or piggyBac-like transposon comprises one end comprising at least 14, 16, 18, 20, 30 or 40 contiguous nucleotides of SEQ ID NO: 14506 or SEQ ID NO: 14508. In certain embodiments, the piggyBac® or piggyBac-like transposon comprises one end comprising at least 14, 16, 18, 20, 30 or 40 contiguous nucleotides of SEQ ID NO: 14507 or SEQ ID NO: 14509. In certain embodiments, the piggyBac® or piggyBac-like transposon comprises one end with at least 90% identity to SEQ ID NO: 14506 or SEQ ID NO: 14508. In certain embodiments, the piggyBac® or piggyBac-like transposon comprises one end with at least 90% identity to SEQ ID NO: 14507 or SEQ ID NO: 14509.

In certain embodiments, the piggyBac® or piggyBac-like transposon comprises a sequence of:

(SEQ ID NO: 14515)

1
ttaacccggc gagcatgagg cagggtatct cataccctgg

taaaatttta aagttgtgta

61
ttttataaaa ttttcgtctg acaacactag cgcgctcagt

agctggaggc aggagcgtgc

121
gggaggggat agtggcgtga tcgcagtgtg gcacgggaca

ccggcgagat attcgtgtgc

181
aaacctgttt cgggtatgtt ataccctgcc tcattgttga

cgtatttttt ttatgtaatt

241
tttccgatta ttaatttcaa ctgttttatt ggtattttta

tgttatccat tgttcttttt

301
ttatgattta ctgtatcggt tgtctttcgt tcctttagtt

gagttttttt ttattatttt

361
cagtttttga tcaaa.

In certain embodiments, the piggyBac® or piggyBac-like transposon comprises a sequence of:

(SEQ ID NO: 14516)

1
tcatattttt agtttaaaaa aataattata tgttttataa

tgaaaagaat ctcattatct

61
ttcagtatta ggttgattta tattccaaag aataatattt

ttgttaaatt gttgattttt

121
gtaaacctct aaatgtttgt tgctaaaatt actgtgttta

agaaaaagat taataaataa

181
taataatttc ataattaaaa acttctttca ttgaatgcca

ttaaataatt cattatttta

241
caaaataaga tcaacataat tgagtaaata ataataagaa

caatattata gtacaacaaa

301
atatgggtat gtcataccct tttttttttt tttttttttt

ttttttcggg tagagggccg

361
aacctcctac gaggtccccg cgcaaaaggg gcgcgcgggg

tatgtgagac tcaacgatct

421
gcatggtgtt gtgagcagac cgcgggccca aggattttag

agcccaccca ctaaacgact

481
cctctgcact cttacacccg acgtccgatc ccctccgagg

tcagaacccg gatgaggtag

541
gggggctacc gcggtcaaca ctacaaccag acggcgcggc

tcaccccaag gacgcccagc

601
cgacggagcc ttcgaggcga atcgaaggct ctgaaacgtc

ggccgtctcg gtacggcagc

661
ccgtcgggcc gcccagacgg tgccgctggt gtcccggaat

accccgctgg accagaacca

721
gcctgccggg tcgggacgcg atacaccgtc gaccggtcgc

tctaatcact ccacggcagc

781
gcgctagagt gctggta.

In certain embodiments, the piggyBac® or piggyBac-like transposon comprises a sequence of CCCGGCGAGCATGAGG (SEQ ID NO: 14510). In certain embodiments, the piggyBac® or piggyBac-like transposon comprises an ITR sequence of SEQ ID NO: 14510. In certain embodiments, the piggyBac® or piggyBac-like transposon comprises a sequence of TTATCCCGGCGAGCATGAGG (SEQ ID NO: 14511). In certain embodiments, the piggyBac® or piggyBac-like transposon comprises at least 16 contiguous nucleotides from SEQ ID NO: 14511. In certain embodiments, the piggyBac® or piggyBac-like transposon comprises a sequence of CCTCATGCTCGCCGGGTTAT (SEQ ID NO: 14512). In certain embodiments, the piggyBac® or piggyBac-like transposon comprises at least 16 contiguous nucleotides from SEQ ID NO: 14512. In certain embodiments, the piggyBac® or piggyBac-like transposon comprises one end comprising at least 16 contiguous nucleotides from SEQ ID NO: 14511 and one end comprising at least 16 contiguous nucleotides from SEQ ID NO: 14512. In certain embodiments, the piggyBac® or piggyBac-like transposon comprises SEQ ID NO: 14511 and SEQ ID NO: 14512. In certain embodiments, the piggyBac® or piggyBac-like transposon comprises a sequence of TTAACCCGGCGAGCATGAGG (SEQ ID NO: 14513). In certain embodiments, the piggyBac® or piggyBac-like transposon comprises a sequence of CCTCATGCTCGCCGGGTTAA (SEQ ID NO: 14514).

In certain embodiments, the piggyBac® or piggyBac-like transposon may have ends comprising SEQ ID NO: 14506 and SEQ ID NO: 14507, or a variant of either or both of these having at least 90% sequence identity to SEQ ID NO: 14506 or SEQ ID NO: 14507, and the piggyBac® or piggyBac-like transposase has the sequence of SEQ ID NO: 14504 or SEQ ID NO: 14505, or a sequence at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% or any percentage in between identity to SEQ ID NO: 14504 or SEQ ID NO: 14505. In certain embodiments, the piggyBac® or piggyBac-like transposon comprises a heterologous polynucleotide inserted between a pair of inverted repeats, where the transposon is capable of transposition by a piggyBac® or piggyBac-like transposase having at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% or any percentage in between identity to SEQ ID NO: 14504 or SEQ ID NO: 14505. In certain embodiments, the transposon comprises two transposon ends, each of which comprises SEQ ID NO: 14510 in inverted orientations in the two transposon ends. In certain embodiments, each inverted terminal repeat (ITR) is at least 90% identical to SEQ ID NO: 14510.

In certain embodiments, the piggyBac® or piggyBac-like transposon is capable of insertion by a piggyBac® or piggyBac-like transposase at the sequence 5′-TTAT-3 within a target nucleic acid. In certain embodiments, one end of the piggyBac® or piggyBac-like transposon comprises at least 16 contiguous nucleotides from SEQ ID NO: 14506 and the other transposon end comprises at least 16 contiguous nucleotides from SEQ ID NO: 14507. In certain embodiments, one end of the piggyBac® or piggyBac-like transposon comprises at least 17, at least 18, at least 19, at least 20, at least 22, at least 25, at least 30 contiguous nucleotides from SEQ ID NO: 14506 and the other transposon end comprises at least 17, at least 18, at least 19, at least 20, at least 22, at least 25, at least 30 contiguous nucleotides from SEQ ID NO: 14507.

In certain embodiments, the piggyBac® or piggyBac-like transposon comprises transposon ends (each end comprising an ITR) corresponding to SEQ ID NO: 14506 and SEQ ID NO: 14507, and has a target sequence corresponding to 5′-TTAT3′. In certain embodiments, the piggyBac® or piggyBac-like transposon also comprises a sequence encoding a transposase (e.g. SEQ ID NO: 14505). In certain embodiments, the piggyBac® or piggyBac-like transposon comprises one transposon end corresponding to SEQ ID NO: 14506 and a second transposon end corresponding to SEQ ID NO: 14516. SEQ ID NO: 14516 is very similar to SEQ ID NO: 14507, but has a large insertion shortly before the ITR. Although the ITR sequences for the two transposon ends are identical (they are both identical to SEQ ID NO: 14510), they have different target sequences: the second transposon has a target sequence corresponding to 5′-TTAA-3′, providing evidence that no change in ITR sequence is necessary to modify the target sequence specificity. The piggyBac® or piggyBac-like transposase (SEQ ID NO: 14504), which is associated with the 5′-TTAA-3′ target site differs from the 5′-TTAT-3′-associated transposase (SEQ ID NO: 14505) by only 4 amino acid changes (D322Y, S473C, A507T, H582R). In certain embodiments, the piggyBac® or piggyBac-like transposase (SEQ ID NO: 14504), which is associated with the 5′-TTAA-3′ target site is less active than the 5′-TTAT-3′-associated piggyBac® or piggyBac-like transposase (SEQ ID NO: 14505) on the transposon with 5′-TTAT-3′ ends. In certain embodiments, piggyBac® or piggyBac-like transposons with 5′-TTAA-3′ target sites can be converted to piggyBac® or piggyBac-like transposases with 5′-TTAT-3 target sites by replacing 5′-TTAA-3′ target sites with 5′-TTAT-3′. Such transposons can be used either with a piggyBac® or piggyBac-like transposase such as SEQ ID NO: 14504 which recognizes the 5′-TTAT-3′ target sequence, or with a variant of a transposase originally associated with the 5′-TTAA-3′ transposon. In certain embodiments, the high similarity between the 5′-TTAA-3′ and 5′-TTAT-3′ piggyBac® or piggyBac-like transposases demonstrates that very few changes to the amino acid sequence of a piggyBac® or piggyBac-like transposase alter target sequence specificity. In certain embodiments, modification of any piggyBac® or piggyBac-like transposon-transposase gene transfer system, in which 5′-TTAA-3′ target sequences are replaced with 5′-TTAT-3′-target sequences, the ITRs remain the same, and the transposase is the original piggyBac® or piggyBac-like transposase or a variant thereof resulting from using a low-level mutagenesis to introduce mutations into the transposase. In certain embodiments, piggyBac® or piggyBac-like transposon transposase transfer systems can be formed by the modification of a 5′-TTAT-3′-active piggyBac® or piggyBac-like transposon-transposase gene transfer systems in which 5′-TTAT-3′ target sequences are replaced with 5′-TTAA-3′-target sequences, the ITRs remain the same, and the piggyBac® or piggyBac-like transposase is the original transposase or a variant thereof.

(SEQ ID NO: 14577)

1
cccggcgagc atgaggcagg gtatctcata ccctggtaaa

attttaaagt tgtgtatttt

61
ataaaatttt cgtctgacaa cactagcgcg ctcagtagct

ggaggcagga gcgtgcggga

121
ggggatagtg gcgtgatcgc agtgtggcac gggacaccgg

cgagatattc gtgtgcaaac

181
ctgtttcggg tatgttatac cctgcctcat tgttgacgta t.

In certain embodiments, the piggyBac® or piggyBac-like transposon comprises a sequence of:

(SEQ ID NO: 14578)

1
tttaagaaaa agattaataa ataataataa tttcataatt

aaaaacttct ttcattgaat

61
gccattaaat aaaccattat tttacaaaat aagatcaaca

taattgagta aataataata

121
agaacaatat tatagtacaa caaaatatgg gtatgtcata

ccctgccaca ttcttgatgt

181
aacttttttt cacctcatgc tcgccggg.

In certain embodiments, the transposon comprises at least 16 contiguous bases from SEQ ID NO: 14577 and at least 16 contiguous bases from SEQ ID NO: 14578, and inverted terminal repeats that are at least 87% identical to CCCGGCGAGCATGAGG (SEQ ID NO: 14510). In certain embodiments, the piggyBac® or piggyBac-like transposon comprises a sequence of:

(SEQ ID NO: 14595)

1
cccggcgagc atgaggcagg gtatctcata ccctggtaaa

attttaaagt tgtgtatttt

61
ataaaatttt cgtctgacaa cactagcgcg ctcagtagct

ggaggcagga gcgtgcggga

121
ggggatagtg gcgtgatcgc agtgtggcac gggacaccgg

cgagatattc gtgtgcaaac

181
ctgtttcggg tatgttatac cctgcctcat tgttgacgta

ttttttttat gtaatttttc

241
cgattattaa tttcaactgt tttattggta tttttatgtt

atccattgtt ctttttttat

301
gatttactgt atcggttgtc tttcgttcct ttagttgagt

ttttttttat tattttcagt

361
ttttgatcaa a.

In certain embodiments, the piggyBac® or piggyBac-like transposon comprises a sequence of:

(SEQ ID NO: 14596)

1
tcatattttt agtttaaaaa aataattata tgttttataa

tgaaaagaat ctcattatct

61
ttcagtatta ggttgattta tattccaaag aataatattt

ttgttaaatt gttgattttt

121
gtaaacctct aaatgtttgt tgctaaaatt actgtgttta

agaaaaagat taataaataa

181
taataatttc ataattaaaa acttctttca ttgaatgcca

ttaaataaac cattatttta

241
caaaataaga tcaacataat tgagtaaata ataataagaa

caatattata gtacaacaaa

301
atatgggtat gtcataccct gccacattct tgatgtaact

ttttttcacc tcatgctcgc

361
cggg.

In certain embodiments, the piggyBac® or piggyBac-like transposon comprises SEQ ID NO: 14595 and SEQ ID NO: 14596, and is transposed by the piggyBac or piggyBac-like transposase of SEQ ID NO: 14505. In certain embodiments, the ITRs of SEQ ID NO: 14595 and SEQ ID: 14596 are not flanked by a 5′-TTAA-3′ sequence. In certain embodiments, the ITRs of SEQ ID NO: 14595 and SEQ ID: 14596 are flanked by a 5′-TTAT-3′ sequence.

In certain embodiments, the piggyBac® or piggyBac-like transposon comprises a sequence of:

(SEQ ID NO: 14597)

1
cccggcgagc atgaggcagg gtatctcata ccctggtaaa

attttaaagt tgtgtatttt

61
ataaaatttt cgtctgacaa cactagcgcg ctcagtagct

ggaggcagga gcgtgcggga

121
ggggatagtg gcgtgatcgc agtgtggcac gggacaccgg

cgagatattc gtgtgcaaac

181
ctgtttcggg tatgttatac cctgcctcat tgttgacgta

ttttttttat gtaatttttc

241
cgattattaa tttcaactgt tttattggta tttttatgtt

atccattgtt ctttttttat

301
g.

In certain embodiments, the piggyBac® or piggyBac-like transposon comprises a sequence of:

(SEQ ID NO: 14598)

1
cagggtatct cataccctgg taaaatttta aagttgtgta

ttttataaaa ttttcgtctg

61
acaacactag cgcgctcagt agctggaggc aggagcgtgc

gggaggggat agtggcgtga

121
tcgcagtgtg gcacgggaca ccggcgagat attcgtgtgc

aaacctgttt cgggtatgtt

181
ataccctgcc tcattgttga cgtatttttt ttatgtattt

tttccgatta ttaatttcaa

241
ctgttttatt ggtattttta tgttatccat tgttcttttt

ttatg.

In certain embodiments, the piggyBac® or piggyBac-like transposon comprises a sequence of:

(SEQ ID NO: 14599)

1
cagggtatct cataccctgg taaaatttta aagttgtgta

ttttataaaa ttttcgtctg

61
acaacactag cgcgctcagt agctggaggc aggagcgtgc

gggaggggat agtggcgtga

121
tcgcagtgtg gcacgggaca ccggcgagat attcgtgtgc

aaacctgttt cgggtatgtt

181
ataccctgcc tcattgttga cgtat.

In certain embodiments, the left end of the piggyBac or piggyBac-like transposon comprises a sequence of SEQ ID NO: 14577, SEQ ID NO: 14595, or SEQ ID NOs: 14597-14599. In certain embodiments, the left end of the piggyBac® or piggyBac-like transposon is preceded by a left target sequence.

In certain embodiments, the piggyBac® or piggyBac-like transposon comprises a sequence of:

(SEQ ID NO: 14600)

1
tcatattttt agtttaaaaa aataattata tgttttataa

tgaaaagaat ctcattatct

61
ttcagtatta ggttgattta tattccaaag aataatattt

ttgttaaatt gttgattttt

121
gtaaacctct aaatgtttgt tgctaaaatt actgtgttta

agaaaaagat taataaataa

181
taataatttc ataattaaaa acttctttca ttgaatgcca

ttaaataaac cattatttta

241
caaaataaga tcaacataat tgagtaaata ataataagaa

caatattata gtacaacaaa

301
atatgggtat gtcataccct gccacattct tgatgtaact

ttttttcacc tcatgctcgc

361
cggg.

In certain embodiments, the piggyBac® or piggyBac-like transposon comprises a sequence of:

(SEQ ID NO: 14601)

1
tttaagaaaa agattaataa ataataataa tttcataatt

aaaaacttct ttcattgaat

61
gccattaaat aaaccattat tttacaaaat aagatcaaca

taattgagta aataataata

121
agaacaatat tatagtacaa caaaatataa gtatgtcata

ccctgccaca ttcttgatgt

181
aacttttttt ca.

In certain embodiments, the piggyBac® or piggyBac-like transposon comprises a sequence of:

(SEQ ID NO: 14602)

1
cccggcgagc atgaggcagg gtatctcata ccctggtaaa

attttaaagt tgtgtatttt

61
ataaaatttt cgtctgacaa cactagcgcg ctcagtagct

ggaggcagga gcgtgcggga

121
ggggatagtg gcgtgatcgc agtgtggcac gggacaccgg

cgagatattc gtgtgcaaac

181
ctgtttcggg tatgttatac cctgcctcat tgttgacgta

tttttttLat gtaatttttc

241
cgattattaa tttcaactgt tttattggta tttttatgtt

atccattgtt ctttttttat

301
gatttactgt atcggttgtc tttcgttcct ttagttgagt

ttttttttat tattttcagt

361
ttttgatcaa a.

In certain embodiments, the right end of the piggyBac® or piggyBac-like transposon comprises a sequence of SEQ ID NO: 14578, SEQ ID NO: 14596, or SEQ ID NOs: 14600-14601. In certain embodiments, the right end of the piggyBac® or piggyBac-like transposon is followed by a right target sequence. In certain embodiments, the transposon is transposed by the transposase of SEQ ID NO: 14505. In certain embodiments, the left and right ends of the piggyBac® or piggyBac-like transposon share a 16 bp repeat sequence of SEQ ID NO: 14510 in inverted orientation and immediately adjacent to the target sequence. In certain embodiments, the left transposon end begins with SEQ ID NO: 14510, and the right transposon end ends with the reverse complement of SEQ ID NO: 14510, 5′-CCTCATGCTCGCCGGG-3′ (SEQ ID NO: 14603). In certain embodiments, the piggyBac® or piggyBac-like transposon comprises an ITR with at least 93%, at least 87%, or at least 81% or any percentage in between identity to SEQ ID NO: 14510 or SEQ ID NO: 14603. In certain embodiments, the piggyBac® or piggyBac-like transposon comprises a target sequence followed by a left transposon end comprising a sequence selected from SEQ ID NOs: 88, 105 or 107 and a right transposon end comprising SEQ ID NO: 14578 or 106 followed by a target sequence, in certain embodiments, the piggyBac® or piggyBac like transposon comprises one end that comprises a sequence that is at least 90%, at least 95% or at least 99% or any percentage in between identical to SEQ ID NO: 14577 and one end that comprises a sequence that is at least 90%, at least 95% or at least 99% or any percentage in between identical to SEQ ID NO: 14578. In certain embodiments, one transposon end comprises at least 14, at least 16, at least 18 or at least 20 contiguous bases from SEQ ID NO: 14577 and one transposon end comprises at least 14, at least 16, at least 18 or at least 20 contiguous bases from SEQ ID NO: 14578.

In certain embodiments, the piggyBac® or piggyBac-like transposon comprises two transposon ends wherein each transposon ends comprises a sequence that is at least 81% identical, at least 87% identical or at least 93% identical or any percentage in between identical to SEQ ID NO: 14510 in inverted orientation in the two transposon ends. One end may further comprise at least 14, at least 16, at least 18 or at least 20 contiguous bases from SEQ ID NO: 14599, and the other end may further comprise at least 14, at least 16, at least 18 or at least 20 contiguous bases from SEQ ID NO: 14601. The piggyBac® or piggyBac-like transposon may be transposed by the transposase of SEQ ID NO: 14505, and the transposase may optionally be fused to a nuclear localization signal.

In certain embodiments, the piggyBac® or piggyBac-like transposon comprises SEQ ID NO: 14595 and SEQ ID NO: 14596 and the piggyBac® or piggyBac-like transposase comprises SEQ ID NO: 14504 or SEQ ID NO: 14505. In certain embodiments, the piggyBac® or piggyBac-like transposon comprises SEQ ID NO: 14597 and SEQ ID NO: 14596 and the piggyBac® or piggyBac-like transposase comprises SEQ ID NO: 14504 or SEQ ID NO: 14505. In certain embodiments, the piggyBac® or piggyBac-like transposon comprises SEQ ID NO: 14595 and SEQ ID NO: 14578 and the piggyBac® or piggyBac-like transposase comprises SEQ ID NO: 14504 or SEQ ID NO: 14505. In certain embodiments, the piggyBac® or piggyBac-like transposon comprises SEQ ID NO: 14602 and SEQ ID NO: 14600 and the piggyBac® or piggyBac-like transposase comprises SEQ ID NO: 14504 or SEQ ID NO: 14505.

In certain embodiments, the piggyBac® or piggyBac-like transposon comprises a left end comprising 1, 2, 3, 4, 5, 6, or 7 sequences selected from ATGAGGCAGGGTAT (SEQ ID NO: 14614), ATACCCTGCCTCAT (SEQ ID NO: 14615), GGCAGGGTAT (SEQ ID NO: 14616), ATACCCTGCC (SEQ ID NO: 14617), TAAAATTTTA (SEQ ID NO: 14618), ATTTTATAAAAT (SEQ ID NO: 14619), TCATACCCTG (SEQ ID NO: 14620) and TAAATAATAATAA (SEQ ID NO: 14621). In certain embodiments, the piggyBac® or piggyBac-like transposon comprises a right end comprising 1, 2 or 3 sequences selected from SEQ ID NO: 14617, SEQ ID NO: 14620 and SEQ ID NO: 14621.

In certain embodiments of the methods of the disclosure, the transposase enzyme is a piggyBac® or piggyBac-like transposase enzyme. In certain embodiments, the piggyBac® or piggyBac-like transposase enzyme is isolated or derived from Xenopus tropicalis. The piggyBac® or piggyBac-like transposase enzyme may comprise or consist of an amino acid sequence at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% or any percentage in between identical to:

(SEQ ID NO: 14517)

1
MAKPEYSAEE AAAHCMASSS EEFSGSDSEY VPPASESDSS

TEESWCSSST VSALEEPMEV

61
DEDVDDLEDQ EAGDRADAAA GGEPAWGPPC NFPPEIPPFT

TVPGVKVDTS NFEPINFFQL

121
FMTEAILQDM VLYTNVYAEQ YLTQNPLPRY ARAHAWHPTD

IAEMKRFVGL TLAMGLIKAN

181
SLESYWDTTT VLSIPVFSAT MSRNRYQLLL RFLHFNNNAT

AVPPDQPGHD RLHKLRPLID

241
SLSERFAAVY TPCQNICIDE SLLLFKGRLQ FRQYIPSKRA

RYGIKFYKLC ESSSGYTSYF

301
LIYEGKDSKL DPPGCPPDLT VSGKINWELI SPLLGQGFHL

YVDNFYSSIP LFTALYCLDT

361
PACGTINRNR KGLPRALLDK KLNRGETYAL RKNELLAIKF

FDKKNVFMLT SIHDESVIRE

421
QRVGRPPKNK PLCSKEYSKY MGGVDRTDQL QHYYNATRKT

RAWYKKVGIY LIQMALRNSY

481
IVYKAAVPGP KLSYYKYQLQ ILPALLFGGV EEQTVPEMPP

SDNVARLIGK HFIDTLPPTP

541
GKQRPOKGCK VCRKRGIRRD TRYYCPKCPR NPGLCFKPCF

EIYHTQLHY.

In some embodiments, the piggyBac® or piggyBac-like transposase is a hyperactive variant of SEQ ID NO: 14517. In certain embodiments, the piggyBac® or piggyBac-like transposase is an integration defective variant of SEQ ID NO: 14517. The piggyBac® or piggyBac-like transposase enzyme may comprise or consist of an amino acid sequence at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% or any percentage in between identical to:

(SEQ ID NO: 14518)

1
MAKRFYSAEE AAAHCMAPSS EEFSGSDSEY VPPASESDSS

TEESWCSSST VSALEEPMEV

61
DEDVDDLEDQ EAGDRADAAA GGEPAWGPPC NFPPEIPPFT

TVPGVKVDTS NFEPINFFQL

121
FMTEAILQDM VLYTNVYAEQ YLTQNPLPRY ARAHAWHPTD

IAEMKRFVGL TLAMGLIKAN

181
SLESYWNTTT VLSIPVFSAT MSRNRYQLLI RELHFNNNAT

AYPPDQPDHD RDHKLPLID

241
SLSERFAAVY TPCQNICIDE SLLLFKGRLR FRQYIPSKRA

RYGIKFYKLC ESSSGYTSYF

301
LIYEGKDSKL DPPGCPPDLT VSGKIVWELI SPLLGQGFHL

YVDNFYSSIP LFTALYCLDT

361
PACGTINRTR KGLPRALLDK KLNRGETYAL RKNELLAIKF

FDKKNVFMLT SIHDESVIRE

421
QRVGRPPKNK PLCSKEYSKY MGGVDRTDQL QHYYNATRKT

SAWYKKVGIY LIQMALRNSY

481
IVYKAAVPGP KLSYYKYQLQ ILPALLFGGV EEQTVPEMLP

SDNVARLIGK HFIDTLPPTP

541
GKQRPQKGCK VCRKRGIRRD TRYYCPKCPR NPGLCFKPCF

EIYHTQLHY.

In certain embodiments, the piggyBac® or piggyBac-like transposase is isolated or derived from Xenopus tropicalis. In certain embodiments, the piggyBac® or piggyBac-like transposase is a hyperactive piggyBac or piggyBac-like transposase. In certain embodiments, the hyperactive piggyBac or piggyBac-like transposase comprises a sequence at least 90% identical to:

(SEQ ID NO: 14572)

1
MAKRFYSAEE AAAHCSASSS EEFSGSDSEY VPPASESDSS

TEESWCSSST VSALEEPMEV

61
DEDVDDLEDQ EAGDRADAAA GGEPAWGPPC NFPPEIPPFT

TVPGVKVDTS NFEPINFFQL

121
FMTEAILOM VLYTNVYAEQ YLTQNPLTRG ARAHAWHPTD

IAEMKRFVGL TLAMGLIKAN

181
SIESYWDTTT VLSIPVFGAT MSRNRYQLLL RFLHFNNNAT

AVPPDQPGHD REEKLRPLID

241
SLSERFANVY TPCQNICIDE SLMLFKGRLQ FRQYIPSKRA

RYGIKFYKLC ESSTGYTSYF

301
LIYEGKDSKL DPPGCPPDLT VSGKIVWELI SPLLGQGFEL

YVDNFYSSIP LFTALYCLNT

361
PACGTINPNR KGLPRALLDK KLNRGETYAL RKNELLAIKF

FDKKNVFMLT SIHDESVIRE

421
QRVGRPPKNK PLCSKEYSKY MGGVDPTDQL QHYYNATRKT

RHWYKKVGIY LIQMALPNSY

481
IVYKAAVPGP KLSYYKYQLQ ILPALLFGGV EEQTVPEMPD

SDNVARLIGK HFIDTLPPTP

541
GKQRPQKGCK VCRKRGIRRD TRYYCPKCPR NPGLCRKPCF

EIYHTQLHY.

In certain embodiments, piggyBac® or piggyBac-like transposase is a hyperactive piggyBac or piggyBac-like transposase. A hyperactive piggyBac or piggyBac-like transposase is a transposase that is more active than the naturally occurring variant from which it is derived. In certain embodiments, a hyperactive piggyBac or piggyBac-like transposase is more active than the transposase of SEQ ID NO: 14517. In certain embodiments, the hyperactive piggyBac or piggyBac-like transposase comprises a sequence of:

(SEQ ID NO: 14572)

1
MAKRFYSAEE AAAHCSASSS EEFSGSDSEY VPPASESDSS

TEESWCSSST VSALEEPMEV

61
DEDVDDLEDQ EAGDRADAAA GGEPAWGPPC NFPPEIPPFT

TVPGVKVDTS NFEPINFFQL

121
FMTEAILQDM VLYTNVYAEQ YLTQPLTRG ARAHAWHPTD

IAEMKRFVGL TLAMGLIKAN

181
SIESYWDTTT VLSIPVFGAT MSRNRYQLLL RFLHFNNNAT

AVPPDQPGHD RLHKLRPLID

241
SLSERFANVY TPCQNICIDE SLMLFKGRLQ FRQYIPSKRA

RYGIKFYKLC ESSTGYTSYF

301
LIYEGKDSKL DPPGCPPDLT VSGKIVWELI SPLLGQGFHL

YVDNFYSSIP LFTALYCLNT

361
PACGTINRNR KGLPRALLDK KLNRGETYAL RKNELLAIKF

FDKKNVFMLT SIHDESVIRE

421
QRVGRPPKNK PLCSKEYSKY MGGVDRTDQL QHYYNATRKT

RHWYKKVGIY LIQMALRNSY

481
IVYKAAVPGP KLSYYKYQLQ ILPALLFGGV EEQTVPEMPD

SDNVARLIGK HFIDTLPPTP

541
GKQRPQKGCK VCRKRGIRRD TRYYCPKCPR NPGLCRKPCF

EIYHTQLHY.

In certain embodiments, the hyperactive piggyBac or piggyBac-like transposase comprises a sequence of.

(SEQ ID NO: 14624)

1
MAKRFYSAEE AAAHCMASSS EEFSGSDSEY VPPASESDSS

TEESWCSSST VSALEEPMEV

61
DEDVDDLEDQ EAGDRADAAA GGEPAWGPPC NFPPEIPPFT

TVPGVKVDTS NFEPINFFQL

121
FMTEAILQDM VLYTNVYAEQ YLTQNPLTRY ARAHAWHPTD

IAEMKRFVGL TLAMGLIKAN

181
SLESYWDTTT VLSIPVFSAT MSRNRYQLLL RFLHFNNNAT

AVPPDQPGHD RLHKLRPLID

241
SLSERFAAVY TPCQNICIDE SLLLFKGRLQ FRQYIPSKRA

RYGIKFYKLC ESSSGYTSYF

301
LIYEGKDSKL DPPGCPPDLT VSGKIVWELI SPLLGQGFHL

YVDNFYSSIP LFTALYCLNT

361
PACGTINRNR KGLPRALLDK KLNRGETYAL RKNELLAIKF

FDKKNVFMLT SIHDESVIRE

421
QRVGRPPKNK PLCSKEYSKY MGGVDRTDQL QHYYNATRKT

RHWYKKVGIY LIQMALRNSY

481
IVYKAAVTGP KLSYYKYQLQ ILPAILFGGV EEQTVTEMPP

SDNVARLIGK HFIDTLPPTP

541
GKQPTQKGCK VCRKRGIRRD TRYYCPKCPR NPGLCRKPCF

EIYHTQLHY.

In certain embodiments, the hyperactive piggyBac or piggyBac-like transposase comprises a sequence of:

(SEQ ID NO: 14625)

1
MAKRFYSAEE AAAHCMASSS EEFSGSDSEY VPPASESDSS

TEESWCSSST VSALEEPMEV

61
DEDVDDDEDQ EAGDRADAAA GGEPAWGPPC NFPPEIPPFT

TVPGVKVDTS NFEPINFFQL

121
FMTEAILQDM VLYTNVYAEQ YLTQNPLPRY ARAHAWHPTD

IAEMKRFVGL TLAMGLIKAN

181
SLESYWDTTT VLSIPVFSAT MSRNRYQLLL KFLHFNNEAT

AVPPDQPGHD RLHKLRPLID

241
SLSERFAAVY TPCQNICIDE SLLLFKGRLQ FRQYIPSKRA

RYGIKFYKLC ESSSGYTSYF

301
LIYEGKDSKL DPPGCPPDLT VSGKIVWELI SPLLGQGFHL

YVDNFYSSIP LFTALYCLNT

361
PACGTINRNR KGLPRALLDK KLNRGETYAL RKNELLAIKF

FDKKNVFMLT SIHDESVIRE

421
QRVGRPPKNK PLCSKEYSKY MGGVDRTDQL QHYYNATRKT

RHWYKKVGIY LIQMALRNSY

481
IVYKAAVPGP KLSYYKYQLQ ILPALLFGGV EEQTVPEMPP

SDNVARLIGK HFIDTLPPTP

541
GKQRPQKGCK VCRKRGIRRD TRYYCPKCPR NPGLCRKPCF

EIYHTQLHY.

In certain embodiments, the hyperactive piggyBac or piggyBac-like transposase comprises a sequence of:

(SEQ ID NO: 14627)

1
MAKRFYSAEE ALAHCMASSS EQTSGSDSEY VPPASESDSS

TEESWCSSST VSALEEPMEV

61
DEDVDDLEDQ EAGDRADAAA GGEPAWGPPC NFPPEIPPFT

TVPGVKVDTS NFEPINFFQL

121
FMTEAILQDM VLYTNVYAEQ YLTQNPLTRY ARAHAWHPTD

IAEMKRFVGL TLAMGLIKAN

181
SIESYWDTTT VLSIPVFGAT MSRNRYQLLL RFLHENNNAT

AYPPDQPGHD RLHKLRPLID

241
SLSERFANVY TPCQNICIDE SLLLFKGRLQ FRQYIPSKRA

RYGIKFYKLC ESSSGYTSYF

301
LIYEGKDSKL DPPGCPPDLT VSGKIVWELI SPLLGQGFHL

YVDNFYSSIP LETALYCLNT

361
PACGTINRNR KGLPRALLDK KLNRGETYAL RKNELLAIKF

FDKKNVFMLT SIHDESVIRE

421
QRVGRKPKNK PLCSKEYSKY MGGVDRTDOL QHYYNATRKT

RHWYKKVGIY LIQMALRNSY

481
IVYKAAVPGP KLSYYKYQLQ ILPALLFGGV EEQTVTEMPP

SDNVARLIGK HFIDTLPPTP

541
GKQRPQKGCK VCRKRGIRRD TRYYCPKCPR NPGLCRKPCF

EIYETQLHY.

In certain embodiments, the hyperactive piggyBac or piggyBac-like transposase comprises a sequence of:

(SEQ ID NO: 14628)

1
MAKREYSAEE AAAECSASSS EEFSGSDSEY VPPASESDSS

TEESWCSSST VSALEEPMEV

61
DEDVDDLEDQ EAGDRADAAA GGEPAWGPPC NFPPEIPPFT

TVPGVKVDTS NEEPINFFQL

121
FMTEAILQM VLYTNVYAEQ YLTQPLTRG ARAHAWHPTD

IAEMKRFVGL TLAMGLIKAN

181
SLESYWDTTT VLSIPVFGAT MSRNRYQLLL RFLHFNNNAT

AVPPDQPGHD RLHKLRPLID

241
SLSERFANVY TPCQNICIDE SLMLFKGRLQ FRQYIPSKRA

RYGIKFYKLC ESSTGYTSYF

301
LIYEGKDSKL DPPGCPPDLT VSGKIVWELI SPLLGOGFHL

YVDNFYSSIP LFTALYCLNT

361
PACGTINPNR KGLPRALLDK KLNRGETYAL RKNELLAIKF

FDKKNVFMLT SIHDESVIRE

421
QRVGRPPKNK PLCSKEYSKY MGGVDRTDQL QHYYNATRKT

RHWYKKVGIY LIQMALRNSY

481
IVYKAAVPGP KLSYYKYQLQ ILPALLFGGV EEQTVPEMPP

SDNVARLIGK HFIDTLPPTP

541
GKQRPQKGCK VCRKRGIRRD TRYYCPKCPR NPGLCRKPCF

EIYHTQLHY.

In certain embodiments, the hyperactive piggyBac or piggyBac-like transposase comprises a sequence of:

(SEQ ID NO: 16820)

1
MAKRFYSAEE AAAHCMASSS EEFSGSDSEY VPPASESDSS

TEESWCSSST VSALEEPMEV

61
DEDVDDDEDQ EAGDRADAAA GGEPAWGPPC NFPPEIPPFT

TVPGVKVDTS NFEPINFFQL

121
FMTEAILQDM VLYTNVYAEQ YLTQNPLTRY ARAHAWHPTD

IAEMKRFVGL TLAMGLIKAN

181
SLESYWDTTT VLSIPVFGAT MSRNRYQLLL RELHFNNNAT

AVPPDQPGHD RLHKLRPLID

241
SLSERFANVY TPCQNICIDE SLLLFKGRLQ FRQYIPSKRA

RYGIKFYKLC ESSSGYTSYF

301
LIYEGKDSKL DPPGCPPDLT VSGKIVWELI SPLLGQGFHL

YVDNFYSSIP LFTALYCLNT

361
PACGTINRNR KGLPRALLDK KLNRGETYAL RKNELLAIKF

FDKKNVFMLT SIHDESVIRE

421
QRVGRPPKNK PLCSKEYSKY MGGVDRTDQL QHYYNATRKT

RHWYKKVGIY LIQMALRNSY

481
IVYKAAVPGP KLSYYKYQLQ ILPALLFGGV EEQTVPEMPP

SDNVARLIGK HFIDTLPPTP

541
GKQRPQKGCK VCRKRGIRRD TRYYCPKCPR NPGLCRKPCF

EIYHTQLHY.

In certain embodiments, the hyperactive piggyBac or piggyBac-like transposase comprises an amino acid substitution at a position selected from amino acid 6, 7, 16, 19, 20, 21, 22, 23, 24, 26, 28, 31, 34, 67, 73, 76, 77, 88, 91, 141, 145, 146, 148, 150, 157, 162, 179, 182, 189, 192, 193, 196, 198, 200, 210, 212, 218, 248, 263, 270, 294, 297, 308, 310, 333, 336, 354.357, 358, 359, 377, 423, 426, 428, 438, 447, 450, 462, 469, 472, 498, 502, 517, 520, 523, 533, 534, 576, 577, 582, 583 or 587 (relative to SEQ ID NO: 14517). In certain embodiments, the hyperactive piggyBac or piggyBac-like transposase comprises an amino acid substitution of Y6C, S7G. M16S, S19G, S20Q, S20G, S20D, E21D, E22Q, F23T, F23P. S24Y, S26V, S28Q, V31K, A34E, L67A, G73H, A76V, D77N, P88A, N91D, Y141Q, Y141A, N145E, N145V, P146T, P146V, P146K, P148T, P148H, Y150G, Y150S, Y150C, H157Y, A162C, A179K, L182I, L182V, T189G, L192H, S193N, S193K, V196I, S198G, T200W, L210H. F212N, N218E, A248N, L263M, Q270L, S294T. T297M, S308R, L310R, L333M, Q336M, A354H, C357V, L358F, D359N, L377I, V 423H, P426K, K428R, S438A. T447G, T447A, L450V, A462H, A462Q, I469V, I472L, Q498M, L502V, E517I, P520D, P520G, N523S, I533E, D534A, F576R, F576E, K577I, I582R, Y583F, L587Y or L587W, or any combination thereof including at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or all of these mutations (relative to SEQ ID NO: 14517).

In certain embodiments, the hyperactive piggyBac or piggyBac-like transposase comprises one or more substitutions of an amino acid that is not wild type, wherein the one or more substitutions a for wild type amino acid comprises a substitution of A2X, K3X, R4X, F5X, Y6X, S7X, A11X, A13X, C15X, M16X, A17X, S18X, S19X, S20X, E21X, E22X, F23X, S24X, G25X, 26X, D27X, S28X, E29X, E42X, E43X, S44X, C46X, S47X, S48X, S49X, T50X, V51X, S52X, A53X, L54X, E55X, E56X, P57X, M58X, E59X, E62X, D63X, V64X, D65X, D66X, L67X, E68X, D69X, Q70X, E71X, A72X, G73X, D74X, R75X, A76X, D77X, A78X, A79X, A80X, G81X, G82X, E83X, P84X, A85X, W86X, G87X, P88X, P89X, C90X, N91X, F92X, P93X, E95X, I96X, P97X, P98X, F99X, T100X, T101X, P103X, G104X, V105X. K106X, V107X, D108X, T109X, N111X, P114X, I115X, N116X. F117X, F118X, Q119X, M122X, T123X, E124X, A125X, I126X, L127X, Q128X, D129X, M130X, L132X, Y133X, V126X, Y127X. A138X, E139X. Q140X, Y141X, L142X. Q144X, N145X, P146X, L147X, P148X, Y150X, A151X, A155X, H157X, P158X, I161X, A162X, V168X, T171X, L172X, A173X, M174X, I177X, A179X, L182X, D187X, T188X, T189X, T190X, L192X, S193X, I194X, P195X, V196X, S198X, A199X, T200X, S202X, L208X, L209X, L210X, R211X, F212X, F215X, N217X, N218X, A219X, T220X, A221X, V222X, P224X, D225X, Q226X, P227X, H229X, R231X, H233X, L235X, P237X, I239X, D240X, L242X, S243X, E244X, R244X, F246X, A247X, A248X, V249X, Y250X, T251X, P252X, C253X, Q254X, I256X, C257X, I258X, D259X, E260X, S261X, L262X, L263X, L264X, F265X, K266X, G267X, R268X, L269X, Q270X, F271X, R272X, Q273X, Y274X, I275X, P276X, S277X, K278X, R279X, A280X, R281X, Y282X, G283X, I284X, K285X, F286X, Y287X, K288X, L289X, C290X, E291X, S292X, S293XS294X, G295X, Y296X, T297X, S298X, Y299X, F300X, E304X, L310X, P313X, G314X, P316X, P317X, D318X, L319X, T320X, V321X. K324X, E328X, I330X, S331X, P332X, L333X, L334X, G335X, Q336X, F338X, L340X, D343X, N344X, F345X, Y346X, S347X, L351X, F352X, A354X, L355X, Y356X, C357X, L358X, D359X, T360X, R422X, Y423X, G424X, P426X, K428X, N429X, K430X, P431X, L432X, S434X, K435X, E436X, S438X, K439X, Y440X, G443X, R446X, T447X, L450X, Q451X. N455X, T460X, R461X, A462X, K465X, V467X, G468X, I469X, Y470X, L471X, I472X, M474X, A475X, L476X, R477X, S479X, Y480X, V482XY483X, K484X, A485X, A486X, V487X, P488X, P490X, K491X, S493X, Y494X, Y495X, K496X, Y497T, Q498X, L499X, Q500X, I501X, L502X, P503X, A504X, L505X, L506X, F507X, G508X, G509X, V510X, E511X, E512X, Q513X, T514X, V515X, E517X, M518X, P519X, P520X, S521X, D522X, N523X, V524X, A525X, L527X, I528X, K530X, H531X, F532X, I533X, D534X, T535X, L536X, T539X, P540X, Q546X, K550X, R553X, K554X, R555X, G556X, I557X, R558X, R559X, D560X, T561X, Y564X, P566X, K567X, P569X, R570X. N571X, L574X, C575X, F576X, K577X, P578X, F580X, E581X, I582X, Y583X, T585X, Q586X, L587X, H588X or Y589X (relative to SEQ ID NO: 14517). A list of hyperactive amino acid substitutions can be found in U.S. Pat. No. 10,041,077, the contents of which are incorporated by reference in their entirety.

In certain embodiments, the piggyBac® or piggyBac-like transposase is integration deficient. In certain embodiments, an integration deficient piggyBac or piggyBac-like transposase is a transposase that can excise its corresponding transposon, but that integrates the excised transposon at a lower frequency than a corresponding naturally occurring transposase. In certain embodiments, the piggyBac® or piggyBac-like transposase is an integration deficient variant of SEQ ID NO: 14517. In certain embodiments, the integration deficient piggyBac or piggyBac-like transposase is deficient relative to SEQ ID NO: 14517.

In certain embodiments, the piggyBac® or piggyBac-like transposase is active for excision but deficient in integration. In certain embodiments, the integration deficient piggyBac or piggyBac-like transposase comprises a sequence that is at least 90% identical to a sequence of:

(SEQ ID NO: 14605)

1
MAKRFYSAEE AAAHCMASSS EEFSGSDSEY VPPASESDSS

TEESWCSSST VSALEEPMEV

61
DEDVDDDEDQ EAGDRADAAA GGEPAWGPPC NFPPEIPPFT

TVPGVKVDTS NFEPINFFQL

121
FMTEAILQDM VLYTNVYAEQ YLTQNPLPRY ARAHAWHPTD

IAEMKRFVGL TLAMGLIKAN

181
SLESYWDTTT VLSIPVFSAT MSRNRYQLLL KFLHFNNEAT

AVPPDQPGHD RLHKLRPLID

241
SLSERFAAVY TPCQNICIDE SLLLFKGRLQ FRQYIPSKRA

RYGIKFYKLC ESSSGYTSYF

301
LIYEGKDSKL DPPGCPPDLT VSGKIVWELI SPLLGQGFHL

YVDNFYSSIP LFTALYCLDT

361
PACGTINRNR KGLPRALLDK KLNRGETYAL RKNELLAIKF

FDKKNVFMLT SIHDESVIRE

421
QRVGRPPKNK PLCSKEYSKY MGGVDRTDQL QHYYNATRKT

RHWYKKVGIY LIQMALRNSY

481
IVYKAAVPGP KLSYYKYQLQ ILPALLFGGV EEQTVPEMPP

SDNVARLIGK HFIDTLPPTP

541
GKQRPQKGCK VCRKRGIRRD TRYYCPKCPR NPGLCRKPCF

EIYHTQLHYG RR.

In certain embodiments, the integration deficient piggyBac or piggyBac-like transposase comprises a sequence that is at least 90% identical to a sequence of:

(SEQ ID NO: 14604)

1
MAKRFYSAEE AAAHCMASSS EEFSGSDSEY VPPASESDSS

TEESWCSSST VSALEEPMEV

61
DEDVDDDEDQ EAGDRADAAA GGEPAWGPPC NFPPEIPPFT

TVPGVKVDTS NFEPINFFQL

121
FMTEAILQDM VLYTNVYAEQ YLTQNPLPRY ARAHAWHPTD

IAEMKRFVGL TLAMGLIKAN

181
SLESYWDTTT VLSIPVFSAT MSRNRYQLLL KFLHFNNEAT

AVPPDQPGHD RLHKLRPLID

241
SLSERFAAVY TPCQNICIDE SLLLFKGRLQ FRQYIPSKRA

RYGIKFYKLC ESSSGYTSYF

301
LIYEGKDSKL DPPGCPPDLT VSGKIVWELI SPLLGQGFHL

YVDNFYSSIP LFTALYCLDT

361
PACGTINRNR KGLPRALLDK KLNRGETYAL RKNELLAIKF

FDKKNVFMLT SIHDESVIRE

421
QRVGRPPKNK PLCSKEYSKY MGGVDRTDQL QHYYNATRKT

RHWYKKVGIY LIQMALRNSY

481
IVYKAAVPGP KLSYYKYQLQ ILPALLFGGV EEQTVPEMPP

SDNVARLIGK HFIDTLPPTP

541
GKQRPQKGCK VCRKRGIRRD TRYYCPKCPR NPGLCRKPCF

EIYHTQLHYG.

In certain embodiments, the integration deficient piggyBac or piggyBac-like transposase comprises a sequence that is at least 90% identical to a sequence of:

(SEQ ID NO: 14611)

1
MAKRFYSAEE AAAHCMASSS EEFSGSDSEY VPPASESDSS

TEESWCSSST VSALEEPMEV

61
DEDVDDDEDQ EAGDRADAAA GGEPAWGPPC NFPPEIPPFT

TVPGVKVDTS NFEPINFFQL

121
FMTEAILQDM VLYTNVYAEQ YLTQNPLPRY ARAHAWHPTD

IAEMKRFVGL TLAMGLIKAN

181
SLESYWDTTT VLSIPVFSAT MSRNRYQLLL KFLHFNNEAT

AVPPDQPGHD RLHKLRPLID

241
SLSERFAAVY TPCQNICIDE SLLLFKGRLQ FRQYIPSKRA

RYGIKFYKLC ESSSGYTSYF

301
LIYEGKDSKL DPPGCPPDLT VSGKIVWELI SPLLGQGFHL

YVDNFYSSIP LFTALYCLDT

361
PACGTINRNR KGLPRALLDK KLNRGETYAL RKNELLAIKF

FDKKNVFMLT SIHDESVIRE

421
QRVGRPPKNK PLCSKEYSKY MGGVDRTDQL QHYYNATRKT

RHWYKKVGIY LIQMALRNSY

481
IVYKAAVPGP KLSYYKYQLQ ILPALLFGGV EEQTVPEMPP

SDNVARLIGK HFIDTLPPTP

541
GKQRPQKGCK VCRKRGIRRD TRYYCPKCPR NPGLCRKPCF

EIYHTQLHYG RR.

In certain embodiments, the integration deficient piggyBac or piggyBac-like transposase comprises SEQ ID NO: 14611. In certain embodiments, the integration deficient piggyBac or piggyBac-like transposase comprises a sequence that is at least 90% identical to a sequence of:

(SEQ ID NO: 14612)

1
MAKRFYSAEE AAAHCMASSS EEFSGSDSEY VPPASESDSS

TEESWCSSST VSALEEPMEV

61
DEDVDDDEDQ EAGDRADAAA GGEPAWGPPC NFPPEIPPFT

TVPGVKVDTS NFEPINFFQL

121
FMTEAILQDM VLYTNVYAEQ YLTQNPLPRY ARAHAWHPTD

IAEMKRFVGL TLAMGLIKAN

181
SLESYWDTTT VLSIPVFSAT MSRNRYQLLL KFLHFNNEAT

AVPPDQPGHD RLHKLRPLID

241
SLSERFAAVY TPCQNICIDE SLLLFKGRLQ FRQYIPSKRA

RYGIKFYKLC ESSSGYTSYF

301
LIYEGKDSKL DPPGCPPDLT VSGKIVWELI SPLLGQGFHL

YVDNFYSSIP LFTALYCLDT

361
PACGTINRNR KGLPRALLDK KLNRGETYAL RKNELLAIKF

FDKKNVFMLT SIHDESVIRE

421
QRVGRPPKNK PLCSKEYSKY MGGVDRTDQL QHYYNATRKT

RHWYKKVGIY LIQMALRNSY

481
IVYKAAVPGP KLSYYKYQLQ ILPALLFGGV EEQTVPEMPP

SDNVARLIGK HFIDTLPPTP

541
GKQRPQKGCK VCRKRGIRRD TRYYCPKCPR NPGLCRKPCF

EIYHTQLHYG RR.

In certain embodiments, the integration deficient piggyBac or piggyBac-like transposase comprises SEQ ID NO: 14612. In certain embodiments, the integration deficient piggyBac or piggyBac-like transposase comprises a sequence that is at least 90% identical to a sequence of;

(SEQ ID NO: 14613)

1
MAKRFYSAEE AAAHCMASSS EEFSGSDSEY VPPASESDSS

TEESWCSSST VSALEEPMEV

61
DEDVDDDEDQ EAGDRADAAA GGEPAWGPPC NFPPEIPPFT

TVPGVKVDTS NFEPINFFQL

121
FMTEAILQDM VLYTNVYAEQ YLTQNPLPRY ARAHAWHPTD

IAEMKRFVGL TLAMGLIKAN

181
SLESYWDTTT VLSIPVFSAT MSRNRYQLLL KFLHFNNEAT

AVPPDQPGHD RLHKLRPLID

241
SLSERFAAVY TPCQNICIDE SLLLFKGRLQ FRQYIPSKRA

RYGIKFYKLC ESSSGYTSYF

301
LIYEGKDSKL DPPGCPPDLT VSGKIVWELI SPLLGQGFHL

YVDNFYSSIP LFTALYCLDT

361
PACGTINRNR KGLPRALLDK KLNRGETYAL RKNELLAIKF

FDKKNVFMLT SIHDESVIRE

421
QRVGRPPKNK PLCSKEYSKY MGGVDRTDQL QHYYNATRKT

RHWYKKVGIY LIQMALRNSY

481
IVYKAAVPGP KLSYYKYQLQ ILPALLFGGV EEQTVPEMPP

SDNVARLIGK HFIDTLPPTP

541
GKQRPQKGCK VCRKRGIRRD TRYYCPKCPR NPGLCRKPCF

EIYHTQLHYG RR.

In certain embodiments, the integration deficient piggyBac or piggyBac-like transposase comprises SEQ ID NO: 14613. In certain embodiments, the integration deficient piggyBac or piggyBac-like transposase comprises an amino acid substitution wherein the Asn at position 218 is replaced by a Glu or an Asp (N218D or N218E) (relative to SEQ ID NO: 14517).

In certain embodiments, the excision competent, integration deficient piggyBac or piggyBac-like transposase comprises one or more substitutions of an amino acid that is not wild type, wherein the one or more substitutions a for wild type amino acid comprises a substitution of A2X, K3X, R4X, F5X, Y6X, S7X, A8X, E9X, E10X, A11X, A12X, A13X, H14X, C15X, M16X, A17X, S18X, S19X, S20X, E2IX, E22X, F23X, S24X, G25X, 26X, D27X, S28X, E29X, V31X, P32X, P33X, A34X, S35X, E36X, S37X, D38X, S39X, S40X, T41X, E42X, E43X, S44X, W45X, C46X, S47X, S48X, S49X, T50X, V51X, S52X, A53X, L54X, E55X, E56X, P57X, M58X, E59X, V60X, M122X, T123X, E124X, A125X, L127X, Q128X, D129X, L132X, Y133X, V126X, Y127X, E139X, Q140X, Y141X, L142X, T143X, Q144X, N145X, P146X, L147X, P148X, R149X, Y150X, A151X, H154X, H157X, P158X, T159X, D160X, I161X, A162X, E163X, M164X, K165X, R166X, F167X, V168X, G169X, L170X, T171X, L172X, A173X, M174X, G175X, L176X, I177X, K178X, A179X, N180X, S181X, L182X, S184X, Y185X, D187X, T188X, T189X, T190X, V191X, L192X, S193X, I194X, P195X, V196X, F197X, S198X, A199X, T200X, M201X, S202X, R203X, N204X, R205X, Y206X, Q207X, L208X, L209X, L210X, R211X, F212X, L213X, H241X, F215X, N216X, N217X, N218X, A219X, T220X, A221X, V222X, P223X, P224X, D225X, Q226X, P227X, G228X, H229X, D230X, R231X, H233X, K234X, L235X, R236X, L238X, I239X, D240X, L242X, S243X, E244X, R244X, F246X, A247X, A248X, V249X, Y250X, T251X, P252X, C253X, Q254X, N255X, I256X, C257X, I258X, D259X, E260X, S261X, L262X, L263X, L264X, F265X, K266X, G267X, R268X, L269X, Q270X, F271X, R272X, Q273X, Y274X, I275X, P276X, S277X, K278X, R279X, A280X, R281X, Y282X, G283X, I284X, K285X, F286X, Y287X, K288X, L289X, C290X, E291X, S292X, S293X, S294X, G295X, Y296X, T297X, S298X, Y299X, F300X, I302X, E304X, G305X, K306X, D307X, S308X, K309X, L310X, D311X, P312X, P313X, G314X, C315X, P316X, P317X, D318X, L319X, T320X, V321X, S322X, G323X, K324X, I325X, V326X, W327X, E328X, L329X, I330X, S331X, P332X, L333X, L334X, G335X, Q336X, F338X, H339X, L340X, V342X, N344X, F345X, Y346X, S347X, S348X, I349X, L351X, T353X, A354X, Y356X, C357X, L358X, D359X, T360X, P361X, A362X, C363X, G364X, I366X, N367X, R368X, D369X, K371X, G372X, L373X, R375X, A376X, L377X, L378X, D379X, K380X, K381X, L382X, N383X, R384XG385X, T387X, Y388X, A389X, L390X, K392X, N393X, E394X, A397X, K399X, F400X, F401X, D402X, N405X, L406X, L409X, R422X, Y423X, G424X, E425X, P426X, K428X, N429X, K430X, P431X, L432X, S434X, K435X, E436X, S438X, K439X, Y440X, G442X, G443X, V444X, R446X, T447X, L450X, Q451X, H452X, N455X, T457X, R458X, T460X, R461X, A462X, Y464X, K465X, V467X, G468X, I469X, L471X, I472X, Q473X, M474X, L476X, R477X, N478X, S479X, Y480X, V482XY483X, K484X, A485X, A486X, V487X, P488X, G489X, P490X, K491X, L492X, S493X, Y494X, Y495X, K496X, Q498X, L499X, Q500X, I501X, L502X, P503X, A504X, L505X, L506X, F507X, G508X, G509X, V510X, E511X, E512X, Q513X, T514X, V515X, E517X, M518X, P519X, P520X, S521X, D522X, N523X, V524X, A525X, L527X, I528X, G529X, K530X, F532X, I533X, D534X, T535X, L536X, P537X, P538X, T539X, P540X, G541X, F542X, Q543X, R544X, P545X, Q546X, K547X, G548X, C549X, K550X, V551X, C552X, R553X, K554X, R555X, G556X, I557X, R558X, R559X, D560X, T561X, R562X, Y563X, Y564X, C565X, P566X, K567X, C568X, P569X, R570X, N571X, P572X, G573X, L574X, C575X, F576X, K577X, P578X, C579X, F580X, E581X, I582X, Y583X, H584X, T585X, Q586X, L587X, H588X or Y589X (relative to SEQ ID NO: 14517). A list of excision competent, integration deficient amino acid substitutions can be found in U.S. Pat. No. 10,041,077, the contents of which are incorporated by reference in their entirety.

In certain embodiments, the piggyBac® or piggyBac-like transposase is fused to a nuclear localization signal. In certain embodiments, SEQ ID NO: 14517 or SEQ ID NO: 14518 is fused to a nuclear localization signal. In certain embodiments, the amino acid sequence of the piggyBac® or piggyBac like transposase fused to a nuclear localization signal is encoded by a polynucleotide sequence comprising:

(SEQ ID NO: 14626)

1
atggcaccca aaaagaaacg taaagtgatg gccaaaagat

tttacagcgc cgaagaagca

61
gcagcacatt gcatggcatc gtcatccgaa gaattctcgg

ggagcgattc cgaatatgtc

121
ccaccqgcct cggaaagcga ttcgagcact gaggagtcgt

ggtgttcctc ctcaactgtc

181
tcggctcttg aggagccgat ggaagtggat gaggatgtgg

acgacttgga ggaccaggaa

241
gccggagaca gggccgacgc tgccgcggga ggggagccgg

cgtggggacc tccatgcaat

301
tttcctcccg aaatcccacc gttcactact gtgccgggag

tgaaggtcga cacgtccaac

361
ttcgaaccga tcaatttctt tcaactcttc atgactgaag

cgatcctgca agatatggtg

421
ctctacacta atgtgtacgc cgagcagtac ctgactcaaa

acccgctgcc tcgctacgcg

481
agagcgcatg cgtggcaccc gaccgatatc gcggagatga

agcggttcgt gggactgacc

541
ctcgcaatgg gcctgatcaa ggccaacagc ctcgagtcat

actgggatac cacgactgtg

601
cttagcattc cggtgttctc cgctaccatg tcccgtaacc

gctaccaact cctgctgcgg

661
ttcctccact tcaacaacaa tgcgaccgct gtgccacctg

accagccagg acacgacaga

721
ctccacaagc tgcggccatt gatcgactcg ctgagcgagc

gattcgccgc ggtgtacacc

781
ccttqccaaa acatttgcat cgacgagtcg cttctgctgt

ttaaaggccg gcttcagttc

841
cgccagtaca tcccatcgaa gcgcgctcgc tatggtatca

aattctacaa actctgcgag

901
tcgtccagcg gctacacgtc atacttcttg atctacgagg

ggaaggactc taagctggac

961
ccaccggggt gtccaccgga tcttactgtc tccggaaaaa

tcgtgtggga actcatctca

1021
cctctcctcg gacaaggctt tcatctctac gtcgacaatt

tctactcatc gatccctctg

1081
ttcaccgccc tctactgcct ggatactcca gcctgtggga

ccattaacag aaaccggaag

1141
ggtctgccga gagcactgct ggataagaag ttgaacaggg

gagagactta cgcgctgaga

1201
aagaacgaac tcctcgccat caaattcttc gacaagaaaa

atgtgtttat gctcacctcc

1261
atccacgacg aatccgtcat ccgggagcag cgcgtgggca

ggccgccgaa aaacaagccg

1321
ctgtgctcta aggaatactc caagtacatg gggggtgtcg

accggaccga tcagctgcag

1381
cattactaca acgccactag aaagacccgg gcctggtaca

agaaagtogg catctacctg

1441
atccaaatgg cactgaggaa ttcgtatatt gtctacaagg

ctgccgttcc gggcccgaaa

1501
ctgtcatact acaagtacca gcttcaaatc ctgccqgcgc

tgctgttcgg tggagtggaa

1561
gaacagactg tgcccgagat gccgccatcc gacaacgtgg

cccggttgat cggaaagcac

1621
ttcattgata ccctgcctcc gacgcctgga aagcagcggc

cacagaaggg atgcaaagtt

1681
tgccgcaagc gcggaatacg gcgcgatacc cgctactatt

gcccgaagtg cccccgcaat

1741
cccggactgt gtttcaagcc ctgttttgaa atctaccaca

cccagttgca ttac.

In certain embodiments, the piggyBac® or piggyBac-like transposon is isolated or derived from Xenopus tropicalis. In certain embodiments, the piggyBa®c or piggyBac-like transposon comprises a sequence of:

(SEQ ID NO: 14519)

1
ttaacctttt tactgccaat gacgcatggg atacgtcgtg

gcagtaaaag ggcttaaatg

61
ccaacgacgc gtcccatacg ttgttggcat tttaagtctt

ctctctgcag cggcagcatg

121
tgccgccgct gcagagagtt tctagcgatg acagcccctc

tgggcaacga gccggggggg

181
ctgtc.

In certain embodiments, the piggyBac® or piggyBac-like transposon comprises a sequence of:

(SEQ ID NO: 14520)

1
tttgcatttt tagacattta gaagcctata tcttgttaca

gaattggaat tacacaaaaa

61
ttctaccata ttttgaaagc ttaggttqtt ctgaaaaaaa

caatatattg ttttcctggg

121
taaactaaaa gtcccctcga ggaaaggccc ctaaagtgaa

acagtgcaaa acgttcaaaa

181
actgtctggc aatacaagtt ccactttgac caaaacggct

ggcagtaaaa gggttaa.

In certain embodiments, the piggyBac® or piggyBac-like transposon comprises SEQ ID NO: 14519 and SEQ ID NO: 14520. In certain embodiments, the piggyBac® or piggyBac-like transposon comprises a sequence of:

(SEQ ID NO: 14521)

1
ttaacccttt gcctgccaat cacgcatggg atacgtcgtg

gcagtaaaag ggcttaaatg

61
ccaacgacgc gtcccatacg ttgttggcat tttaagtctt

ctctctgcag cggcagcatg

121
tgccgccgct gcagagagtt tctagcgatg acagcccctc

tgggcaacga gccggggggg

181
ctgtc.

In certain embodiments, the piggyBac® or piggyBac-like transposon comprises a sequence of;

(SEQ ID NO: 14522)

1
tttgcatttt tagacattta gaagcctata tcttgttaca

gaattggaat tacacaaaaa

61
ttctaccata ttttgaaagc ttaggttgtt ctgaaaaaaa

caatatattg ttttcctggg

121
taaactaaaa gtcccctcga ggaaaggccc ctaaagtgaa

acagtgcaaa acgttcaaaa

181
actgtctggc aatacaagtt ccactttggg acaaatcggc

tggcagtgaa agggttaa.

In certain embodiments, the piggyBac® or piggyBac-like transposon comprises a sequence of;

(SEQ ID NO: 14523)

1
ttaacctttt tactgccaat gacgcatggg atacgtcgtg

gcagtaaaag ggcttaaatg

61
ccaacgacgc gtcccatacg ttgttggcat tttaattctt

ctctctgcag cggcagcatg

121
tgccgccgct gcagagagtt tctagcgatg acagcccctc

tgggcaacga gccggggggg

181
ctgtc.

In certain embodiments, the piggyBac® or piggyBac-like transposon comprises SEQ ID NO: 14520 and SEQ ID NO: 14519, SEQ ID NO: 14521 or SEQ ID NO: 14523. In certain embodiments, the piggyBac® or piggyBac-like transposon comprises SEQ ID NO: 14522 and SEQ ID NO: 14519, SEQ ID NO: 14521 or SEQ ID NO: 14523. In certain embodiments, the piggyBac® or piggyBac-like transposon comprises one end comprising at least 14, 16, 18, 20, 30 or 40 contiguous nucleotides from SEQ ID NO: 14519, SEQ ID NO: 14521 or SEQ ID NO: 14523. In certain embodiments, the piggyBac® or piggyBac-like transposon comprises one end comprising at least 14, 16, 18, 20, 30 or 40 contiguous nucleotides from SEQ ID NO: 14520 or SEQ ID NO: 14522. In certain embodiments, the piggyBac® or piggyBac-like transposon comprises one end with at least 90% identity to SEQ ID NO: 14519, SEQ ID NO: 14521 or SEQ ID NO: 14523. In certain embodiments, the piggyBac® or piggyBac-like transposon comprises one end with at least 90% identity to SEQ ID NO: 14520 or SEQ ID NO: 14522. In one embodiment, one transposon end is at least 90% identical to SEQ ID NO: 14519 and the other transposon end is at least 90% identical to SEQ ID NO: 14520.

In certain embodiments, the piggyBac® or piggyBac-like transposon comprises a sequence of TTAACCTTTACTGCCA (SEQ ID NO: 14524). In certain embodiments, the piggyBac® or piggyBac-like transposon comprises a sequence of TTAACCCTTTGCCTGCCA (SEQ ID NO: 14526). In certain embodiments, the piggyBac® or piggyBac-like transposon comprises a sequence of TTAACCYTTTTACTGCCA (SEQ ID NO: 14527). In certain embodiments, the piggyBac® or piggyBac-like transposon comprises a sequence of TGGCAGTAAAAGGGTTAA (SEQ ID NO: 14529). In certain embodiments, the piggyBac® or piggyBac-like transposon comprises a sequence of TGGCAGTGAAAGGGTTAA (SEQ ID NO: 14531). In certain embodiments, the piggyBac® or piggyBac-like transposon comprises a sequence of TTAACCYTTKMCTGCCA (SEQ ID NO: 14533). In certain embodiments, one end of the piggyBac® or piggyBac-like transposon comprises a sequence selected from SEQ ID NO: 14524, SEQ ID NO: 14526 and SEQ ID NO: 14527. In certain embodiments, one end of the piggyBac® (PB) or piggyBac-like transposon comprises a sequence selected from SEQ ID NO: 14529 and SEQ ID NO: 14531. In certain embodiments, each inverted terminal repeat of the piggyBac® or piggyBac-like transposon comprises a sequence of ITR sequence of CCYTTTKMCTGCCA (SEQ ID NO: 14563). In certain embodiments, each end of the piggyBac® (PB) or piggyBac-like transposon comprises SEQ ID NO: 14563 in inverted orientations. In certain embodiments, one ITR of the piggyBac® or piggyBac-like transposon comprises a sequence selected from SEQ ID NO: 14524, SEQ ID NO: 14526 and SEQ ID NO: 14527. In certain embodiments, one ITR of the piggyBac® or piggyBac-like transposon comprises a sequence selected from SEQ ID NO: 14529 and SEQ ID NO: 14531. In certain embodiments, the piggyBac® or piggyBac-like transposon comprises SEQ ID NO: 14533 in inverted orientation in the two transposon ends.

In certain embodiments, The piggyBac® or piggyBac-like transposon may have ends comprising SEQ ID NO: 14519 and SEQ ID NO: 14520 or a variant of either or both of these having at least 90% sequence identity to SEQ ID NO: 14519 or SEQ ID NO: 14520, and the piggyBac® or piggyBac-like transposase has the sequence of SEQ ID NO: 14517 or a variant showing at least %, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% or any percentage in between sequence identity to SEQ ID NO: 14517 or SEQ ID NO: 14518. In certain embodiments, one piggyBac® or piggyBac-like transposon end comprises at least 14 contiguous nucleotides from SEQ ID NO: 14519, SEQ ID NO: 14521 or SEQ ID NO: 14523, and the other transposon end comprises at least 14 contiguous nucleotides from SEQ ID NO: 14520 or SEQ ID NO: 14522. In certain embodiments, one transposon end comprises at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 22, at least 25, at least 30 contiguous nucleotides from SEQ ID NO: 14519, SEQ ID NO: 14521 or SEQ ID NO: 14523, and the other transposon end comprises at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 22, at least 25 or at least 30 contiguous nucleotides from SEQ ID NO: 14520 or SEQ ID NO: 14522.

In certain embodiments, the piggyBac® or piggyBac-like transposase recognizes a transposon end with a left sequence corresponding to SEQ ID NO: 14519, and a right sequence corresponding to SEQ ID NO: 14520. It will excise the transposon from one DNA molecule by cutting the DNA at the 5′-TTAA-3′ sequence at the left end of one transposon end to the 5′-TTAA-3′ at the right end of the second transposon end, including any heterologous DNA that is placed between them, and insert the excised sequence into a second DNA molecule. In certain embodiments, truncated and modified versions of the left and right transposon ends will also function as part of a transposon that can be transposed by the piggyBac® or piggyBac-like transposase. For example, the left transposon end can be replaced by a sequence corresponding to SEQ ID NO: 14521 or SEQ ID NO: 14523, the right transposon end can be replaced by a shorter sequence corresponding to SEQ ID NO: 14522. In certain embodiments, the left and right transposon ends share an 18 bp almost perfectly repeated sequence at their ends (5′-TTAACCYTTTKMCTGCCA: SEQ ID NO: 14533) that includes the 5′-TTAA-3′ insertion site, which sequence is inverted in the orientation in the two ends. That is in (SEQ ID NO: 14519) and SEQ ID NO: 14523 the left transposon end begins with the sequence 5′-TTAACCTTTTTACTGCCA-3′ (SEQ ID NO: 14524), or in (SEQ ID NO: 14521) the left transposon end begins with the sequence 5′-TTAACCCTTTGCCTGCCA-3′ (SEQ ID NO: 14526); the right transposon ends with approximately the reverse complement of this sequence: in SEQ ID NO: 14520 it ends 5′ TGGCAGTAAAAGGGTTAA-3′ (SEQ ID NO: 14529), in (SEQ ID NO: 14522) it ends 5′-TGGCAGTGAAAGGGTTAA-3′ (SEQ ID NO: 14531.) One embodiment of the disclosure is a transposon that comprises a heterologous polynucleotide inserted between two transposon ends each comprising SEQ ID NO: 14533 in inverted orientations in the two transposon ends. In certain embodiments, one transposon end comprises a sequence selected from SEQ ID NOS: 14524, SEQ ID NO: 14526 and SEQ ID NO: 14527. In some embodiments, one transposon end comprises a sequence selected from SEQ ID NO: 14529 and SEQ ID NO: 14531.

In certain embodiments, the piggyBac® (PB) or piggyBac-like transposon is isolated or derived from Xenopus tropicalis. In certain embodiments, the piggyBac or piggyBac-like transposon comprises at a sequence of:

(SEQ ID NO: 14573)

1
ccctttgcct gccaatcacg catgggatac gtcgtggcag

taaaagggct taaatgccaa

61
cgacgcgtcc catacgtt.

In certain embodiments, the piggyBac® or piggyBac-like transposon comprises at a sequence of:

(SEQ ID NO: 14574)

1
cctgggtaaa ctaaaagtcc cctcgaggaa aggcccctaa

agtgaaacag tgcaaaacgt

61
tcaaaaactg tctggcaata caagttccac tttgggacaa

atcggctggc agtgaaaggg.

In certain embodiments, the piggyBac® or piggyBac-like transposon comprises at least 16 contiguous bases from SEQ ID NO: 14573 or SEQ ID NO: 14574, and inverted terminal repeat of CCYTTTBMCTGCCA (SEQ ID NO: 14575).

In certain embodiments, the piggyBac® or piggyBac-like transposon comprises at a sequence of:

(SEQ ID NO: 14579)

1
ccctttgcct gccaatcacg catgggatac gtcgtggcag

taaaagggct taaatgccaa

61
cgacgcgtcc catacgttgt tggcatttta agtcttctct

ctgcagcggc agcatgtgcc

121
gccgctgcag agagtttcta gcgatgacag cccctctggg

caacgagccg ggggggctgt

181
c.

In certain embodiments, the piggyBac® or piggyBac-like transposon comprises at a sequence of:

(SEQ ID NO: 14580)

1
cctttttact gccaatgacg catgggatac gtcgtggcag

taaagggct taaatgccaa

61
cgacgcgtcc catacgttgt tggcatttta attcttctct

ctgcagcggc agcatgtgcc

121
gccgctgcag agagtttcta gcgatgacag cccctctggg

caacgagccg ggggggctgt

181
c.

In certain embodiments, the piggyBac® or piggyBac-like transposon comprises at a sequence of:

(SEQ ID NO: 14581)

1
cctttttact gccaatgacg catgggatac gtcgtggcag

taaaagggct taaatgccaa

61
cgacgcgtcc catacgttgt tggcatttta agtcttctct

ctgcagcggc agcatgtgcc

121
gccgctgcag agagtttcta gcgatgacag cccctctggg

caacgagccg ggggggctgt

131
c.

In certain embodiments, the piggyBac® or piggyBac-like transposon comprises at a sequence of:

(SEQ ID NO: 14582)

1
cctttttact gccaatgacg catgggatac gtcgtggcag

taaaagggct taaatgccaa

61
cgacgcgtcc catacgttgt tggcatttta agtcttctct

ctgcagcggc agcatgtgcc

121
gccgctgcag agag.

In certain embodiments, the piggyBac® or piggyBac-like transposon comprises at a sequence of:

(SEQ ID NO: 14583)

1
cctttttact gccaatgacg catgggatac gtcgtggcag

taaaagggct taaatgccaa

61
cgacgcgtcc catacgttgt tggcatttta agtctt.

In certain embodiments, the piggyBac® or piggyBac-like transposon comprises at a sequence of:

(SEQ ID NO: 14584)

1
ccctttgcct gccaatcacg catgggatac gtcgtggcag

taaaagggct taaatgccaa

61
cgacgcgtcc catacgttgt tggcatttta agtctt.

In certain embodiments, the piggyBac® or piggyBac-like transposon comprises at a sequence of:

(SEQ ID NO: 14585)

1
ttatcctttt tactgccaat gacgcatggg atacgtcgtg

gcagtaaaag ggcttaaatg

61
ccaacgacgc gtcccatacg ttgttggcat tttaagtctt

ctctctgcag cggcagcatg

121
tgccgccgct gcagagagtt tctagcgatg acagcccctc

tgggcaacga gccggggggg

131
ctgtc.

In certain embodiments, the piggyBac® or piggyBac-like transposon comprises at a sequence of:

(SEQ ID NO: 14586)

1
tttgcatttt tagacattta gaaacctata tcttgttaca

gaattggaat tacacaaaaa

61
ttctaccata ttttaaaagc ttaggttgtt ctgaaaaaaa

caatatattg ttttcctggg

121
taaactaaaa atcccctcga ggaaaagccc ctaaagtgaa

acagtgcaaa acgttcaaaa

181
actgtctggc aatacaagtt ccactttggg acaaatcggc

tggcagtaaa aggg.

In certain embodiments, the piggyBac® or piggyBac-like transposon comprises a left transposon end sequence selected from SEQ ID NO: 14573 and SEQ ID NOs: 14579-14585. In certain embodiments, the left transposon end sequence is preceded by a left target sequence. In certain embodiments, the piggyBac® or piggyBac-like transposon comprises at a sequence of:

(SEQ ID NO: 14587)

1
tttgcatttt tagacattta gaagcctata tcttgttaca

gaattggaat tacacaaaaa

61
ttctaccata ttttgaaagc ttaggttgtt ctgaaaaaaa

caatatattg ttttcctggg

121
taaactaaaa gtcccctcga ggaaaggccc ctaaagtgaa

acagtgcaaa acgttcaaaa

181
actgtctggc aatacaagtt ccactttgac caaaacggct

ggcagtaaaa ggg.

In certain embodiments, the piggyBac® or piggyBac-like transposon comprises at a sequence of:

(SEQ ID NO: 14588)

1
ttgttctgaa aaaaacaata tattgttttc ctgggtaaac

taaaagtccc ctcgaggaaa

61
ggcccctaaa gtgaaacagt gcaaaacgtt caaaaactgt

ctggcaatac aagttccact

121
ttgaccaaaa cggctggcag taaaaggg.

In certain embodiments, the piggyBac® or piggyBac-like transposon comprises at a sequence of:

(SEQ ID NO: 14589)

1
tttgcatttt tagacattta gaagcctata tcttgttaca

gaattggat tacacaaaaa

61
ttctaccata ttttgaaagc ttaggttgtt ctgaaaaaaa

caatatattg ttttcctggg

121
taaactaaaa gtcccctcga ggaaaggccc ctaaagtgaa

acagtgcaaa acgttcaaaa

181
actgtctggc aatacaagtt ccactttgac caaaacggct

ggcagtaaaa gggttat.

In certain embodiments, the piggyBac® or piggyBac-like transposon comprises at a sequence of:

(SEQ ID NO: 14590)

1
ttgttctgaa aaaaacaata tattgttttc ctgggtaaac

taaaagtccc ctcgaggaaa

61
ggcccctaaa gtgaaacagt gcaaaacgtt caaaaactgt

ctggcaatac aagttccact

121
ttgggacaaa tcggctgga gtgaaaggg.

In certain embodiments, the piggyBac® or piggyBac-like transposon comprises a right transposon end sequence selected from SEQ ID NO: 14574 and SEQ ID NOs: 14587-14590. In certain embodiments, the right transposon end sequence is followed by a right target sequence. In certain embodiments, the left and right transposon ends share a 14 repeated sequence inverted in orientation in the two ends (SEQ ID NO: 14575) adjacent to the target sequence. In certain embodiments, the piggyBac® or piggyBac-like transposon comprises a left transposon end comprising a target sequence and a sequence that is selected from SEQ ID NOs: 14582-14584 and 14573, and a right transposon end comprising a sequence selected from SEQ ID NOs: 14588-14590 and 14574 followed by a right target sequence.

In certain embodiments, the left transposon end of the piggyBac® or piggyBac-like transposon comprises

(SEQ ID NO: 14591)

1
atcacgcatg ggatacgtcg tggcagtaaa agggcttaaa

tgccaacgac gcgtcccata

61
cgtt,

and an ITR. In certain embodiments, the left transposon end comprises

(SEQ ID NO: 14592)

1
atgacgcatg ggatacgtcg tggcagtaaa agggcttaaa

tgccaacgac gcgtcccata

61
cgttgttggc attttaagtc tt

and an ITR. In certain embodiments, the right transposon end of the piggyBac® or piggyBac-like transposon comprises

(SEQ ID NO: 14593)

1
cctgggtaaa ctaaaagtcc cctcgaggaa aggcccctaa

agtgaaacag tgcaaaacgt

61
tcaaaaactg tctggcaata caagttccac tttgggacaa

atcggc

and an ITR. In certain embodiments, the right transposon end comprises

(SEQ ID NO: 14594)

1
ttgttctgaa aaaaacaata tattgttttc ctgggtaaac

taaaagtccc ctcgaggaaa

61
ggcccctaaa gtgaaacagt gcaaaacgtt caaaaactgt

ctggcaatac aagttccact

121
ttgaccaaaa cggc

and an ITR.

In certain embodiments, one transposon end comprises a sequence that is at least 90%, at least 95%, at least 99% or any percentage in between identical to SEQ ID NO: 14573 and the other transposon end comprises a sequence that is at least 90%, at least 95%, at least 99% or any percentage in between identical to SEQ ID NO: 14574. In certain embodiments, one transposon end comprises at least 14, at least 16, at least 18, at least 20 or at least 25 contiguous nucleotides from SEQ ID NO: 14573 and one transposon end comprises at least 14, at least 16, at least 18, at least 20 or at least 25 contiguous nucleotides from SEQ ID NO: 14574. In certain embodiments, one transposon end comprises at least 14, at least 16, at least 18, at least 20 from SEQ ID NO: 14591, and the other end comprises at least 14, at least 16, at least 18, at least 20 from SEQ ID NO: 14593. In certain embodiments, each transposon end comprises SEQ ID NO: 14575 in inverted orientations.

In certain embodiments, the piggyBac® or piggyBac-like transposon comprises a sequence selected from of SEQ ID NO: 14573, SEQ ID NO: 14579, SEQ ID NO: 14581, SEQ ID NO: 14582, SEQ ID NO: 14583, and SEQ ID NO: 14588, and a sequence selected from SEQ ID NO: 14587, SEQ ID NO: 14588, SEQ ID NO: 14589 and SEQ ID NO: 14586 and the piggyBac® or piggyBac-like transposase comprises SEQ ID NO: 14517 or SEQ ID NO: 14518.

In certain embodiments, the piggyBac® or piggyBac-like transposon comprises ITRs of CCCTTTGCCTGCCA (SEQ ID NO: 14622) (left ITR) and TGGCAGTGAAAGGG (SEQ ID NO: 14623) (right ITR) adjacent to the target sequences.

In certain embodiments of the methods of the disclosure, the transposase enzyme is a piggyBac® or piggyBac-like transposase enzyme. In certain embodiments, the piggyBac® or piggyBac-like transposase enzyme is isolated or derived from Helicoverpa armigera. The piggyBac® or piggyBac-like transposase enzyme may comprise or consist of an amino acid sequence at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% or any percentage in between identical to:

(SEQ ID NO: 14525)

1
MASRQRLNHD EIATILENDD DYSPLDSESE KEDCVVEDDV

WSDNEDAIVD FVEDTSAQED

61
PDNNIASRES PNLEVTSLTS HRIITLPQRS IRGKNNHVWS

TTKGRTTGRT SAINIIRTNR

121
GPTPMCRNIV DPLLCFQLFI TDEIIHEIVK WTNVEIIVKR

QNLKDISASY RDTNTMEIWA

181
LVGILTLTAV MKDNHLSTDE LFDATFSGTR YVSVMSRERF

EFLIRCIRMD DKTLRPTLRS

241
DDAFLPVRKI WEIFINQCRQ NHVPGSNLTV DEQLLGFRGR

CPFRMYIPNK PDKYGIKFPM

301
MCAAATKYMI DAIPYLGKST KTNGLPLGEF YVKDLTKTVH

GTNRNITCDN WFTSIPLAKN

361
MLQAPYNLTI VGTIRSNKRE MPEEIKNSRS RPVGSSMFCF

DGPLTLVSYK PKPSKMVFLL

421
SSCDENAVIN ESNGKPDMIL FYNQTKGGVD SFDQMCKSMS

ANRKTNRWPM AVFYGMLNMA

481
FVNSYIIYCH NKINKQEKPI SRKEFMKKLS IQLTTPWMQE

RLQAPTLKRT LRDNITNVLK

541
NVVPASSENI SNEPEPKKRR YCGVCSYKKR RMTKAQCCKC

KKAICGEHNI DVCQDCI.

In certain embodiments, the piggyBac® or piggyBac-like transposon is isolated or derived from Helicoverpa armigera. In certain embodiments, the piggyBac® or piggyBac-like transposon comprises a sequence of:

(SEQ ID NO: 14570)

1
ttaaccctag aagcccaatc tacgtaaatt tgacgtatac

cgcggcgaaa tatctatgtc

61
tctttcatgt ttaccgtcgg atcgccgcta acttctgaac

caactcagta gccattggga

121
cctcgcagga cacagttgcg tcatctcggt aagtgccgcc

attttgttgt actctctatt

181
acaacacacg tcacgtcacg tcgttgcacg tcattttgac

gtataattgg gctttgtgta

241
acttttgaat ttgtttcaaa ttttttatgt ttgtgattta

tttgagttaa tcgtattgtt

301
tcgttacatt tttcatataa taataatatt ttcaggttga

gtacaaa.

In certain embodiments, the piggyBac® or piggyBac-like transposon comprises a sequence of:

(SEQ ID NO: 11528)

1
agactgtttt tttctaagag acttctaaaa tattattacg

agttgattta attttatgaa

61
aacatttaaa actagttgat tttttttata attacataat

tttaagaaaa agtgttagag

121
gcttgatttt tttgttgatt ttttctaaga tttgattaaa

gtgccataat agtattaata

181
aagagtattt tttaacttaa aatgtatttt atttattaat

taaaacttca attatgataa

241
ctcatgcaaa aatatagttc attaacagaa aaaaatagga

aaactttgaa gttttgtttt

301
tacacgtcat ttttacgtat gattgggctt tatagctagt

taaatatgat tgggcttcta

361
gggttaa.

in certain embodiments of the methods of the disclosure, the transposase enzyme is a piggyBac® or piggyBac-like transposase enzyme. In certain embodiments, the piggyBac® or piggyBac-like transposase enzyme is isolated or derived from Pectinophora gossypiella. The piggyBac® or piggyBac-like transposase enzyme may comprise or consist of an amino acid sequence at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% or any percentage in between identical to:

(SEQ ID NO: 14530)

1
MDLRKQDEKI RQWLEQDIEE DSKGESDNSS SETEDIVEME

VHKNTSSESE VSSESDYEPV

51
CPSKRQRTQI IESEESDNSE SIRPSRPQTS RVIDSDETDE

DVWSSTPQNI PRNPNVIQPS

121
SRFLYGKNKH KNSSAAKPSS VRTSRRNIIH FIPGRKERAR

EVSEPIDIFS LFISEDMLQQ

181
VVTFPNAEML IRKNKYKTET FTVSPTNLEE IRALLGLLFN

AAAMKSNHLP TRMLFNTHRS

241
GTIFKACMSA ERLNFLIKCL RFDDKLTRNV RQRDDRFAPI

RDLWQALISN FQKWYTPGSY

301
ITVDEQLVGF RGRCSFRMYI PNKPNKYGIK LVMAADVNSK

YIVNAIPYLG KGTDPQNQPL

361
ATFFIKEITS TLHGTNRNIT MDNWFTSVPL ANELLMAPYN

LTLVGTLRSN KREIPEKLKN

421
SKSRAIGTSM FCYDGDKTLV SYKAKSNKVV FILSTIHDQP

DINQETGKPE MIHFYNSTKG

481
AVDTVDQMCS SISTNRKTQR WPLCVFYNML NLSIINAYVV

YVYNNVRNNK KPMSRRDFVI

541
KLGDQLMEPW LRQRLQTVTL RRDIKVMIQD ILGESSDLEA

PVPSVSNVRK IYYLCPSKAR

601
RMTKHRCIKC KQAICGPHNI DICSRCIE.

In certain embodiments, the piggyBac® or piggyBac-like transposon is isolated or derived from Pectinophora gossypiella. In certain embodiments, the piggyBac® or piggyBac-like transposon comprises a sequence of:

(SEQ ID NO: 14532)

1
ttaaccctag ataactaaac attogtccgc tcgacgacgc

gctatgccgc gaaattgaag

61
tttacctatt attccgcgtc ccccgccccc gccgcttttt

ctagcttcct gatttgcaaa

121
atagtgcatc gcgtgacacg ctcgaggtca cacgacaatt

aggtcgaaag ttacaggaat

181
ttcgtogtcc gctcgacgaa agtttagtaa ttacgtaagt

ttggcaaagg taagtgaatg

241
aagtattttt ttataattat tttttaattc tttatagtga

taacgtaagg tttatttaaa

301
tttattactt ttatagttat ttagccaatt gttataaatt

ccttgttatt gctgaaaaat

361
ttgcctgttt tagtcaaaat ttattaactt ttcgatcgtt

ttttag.

In certain embodiments, the piggyBac® or piggyBac-like transposon comprises a sequence of:

(SEQ ID NO: 14571)

1
tttcactaag taattttgtt cctatttagt agataagtaa

cacataatta ttgtgatatt

61
caaaacttaa gaggtttaat aaataataat aaaaaaaaaa

tggtttttat ttcgtagtct

121
gctcgacgaa tgtttagtta ttacgtaacc gtgaatatag

tttagtagtc tagggttaa.

In certain embodiments of the methods of the disclosure, the transposase enzyme is a piggyBac® or piggyBac-like transposase enzyme. In certain embodiments, the piggyBac® or piggyBac-like transposase enzyme is isolated or derived from Ctenoplusia agnata. The piggyBac® or piggyBac-like transposase enzyme may comprise or consist of an amino acid sequence at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% or any percentage in between identical to:

(SEQ ID NO: 14534)

1
MASRQHLYQD EIANILENED DYSPHDTDSE MEDCVTQDDV

RSDVEDEMVD NIGNGTSPAS

61
RHEDPETPDP SSEASNLEVT LSSHRIIILP QRSIREKNNH

IWSTTKGQSS GRTAAINIVR

121
TNRGPTRMCR NIVDPEECFQ LFIKEEIVEE IVKWTNVEMV

QKRVNEKDIS ASYRDTNEME

181
IWAIISMLTL SAVMKDNHLS TDELFNVSYG TRYVSVMSRE

RFEFLLRLLR MGDKLLRPNL

241
RQEDAFTPVR KIWEIFINQC RLNYVPGTNL TVDEQLLGFR

GRCPFRMYIP NKPDKYGIKF

301
PMVCDAATKY MVDAIPYLGK STKTQGLPLG EFYVKELTQT

VHGTNRNVTC DNWFTSVPLA

361
KSLLNSPYNL TLVGTIRSNK REIPEEVKNS RSRQVGSSMF

CFDGPLTLVS YKPKPSKMVF

421
LLSSCNEDAV VNQSNGKPDM ILFYNQTKGG VDSFDQMCSS

MSTNRKTNRW PMAVFYGMLN

481
MAFVNSYIIY CHNMLAKKEK PLSRKDFMKK LSTDLTTPSM

QKRLEAPTLK RSLRDNITNV

541
LKIVPQAAID TSFDEPEPKK RRYCGFCSYK KKRMTKTQCF

KCKKPVCGEH NIDVCQDCI.

In certain embodiments, the piggyBac® or piggyBac-like transposon is isolated or derived from Ctenoplusia agnata. In certain embodiments, the piggyBac® or piggyBac-like transposon comprises a sequence of:

(SEQ ID NO: 14535)

1
ttaaccctag aagcccaatc tacgtcattc tgacqtqtat

gtcgccgaaa atactctgtc

61
tctttctcct gcacgatcgg attgccgcga acgctcgatt

caacccagtt ggcgccgaga

121
tctattggag gactgcggcg ttgattcggt aagtcccgcc

attttgtcat agtaacagta

181
ttgcacgtca gcttgacqta tatttgggct ttgtgttatt

tttgtaaatt ttcaacgtta

241
gtttattatt gcatcttttt gttacattac tggtttattt

gcatgtatta ctcaaatatt

301
atttttattt tagcgtagaa aataca.

In certain embodiments, the piggyBac® or piggyBac-like transposon comprises a sequence of:

(SEQ ID NO: 14536)

1
agactgtttt ttttgtattt gcattatata ttatattcta

aagttgattt aattctaaga

61
aaaacattaa aataagtttc tttttgtaaa atttaattaa

ttataagaaa aagtttaagt

121
tgatctcatt ttttataaaa atttgcaatg tttccaaagt

tattattgta aaagaataaa

181
taaaagtaaa ctgagtttta attgatgttt tattatatca

ttatactata tattacttaa

241
ataaaacaat aactgaatgt atttctaaaa ggaatcacta

gaaaatatag tgatcaaaaa

301
tttacacgtc atttttgcgt atgattgggc tttataggtt

ctaaaaatat gattgggcct

361
ctagggttaa.

In certain embodiments, the piggyBac or piggyBac-like transposon comprises an ITR sequence of CCCTAGAAGCCCAATC (SEQ ID NO: 14564).

In certain embodiments of the methods of the disclosure, the transposase enzyme is a piggyBac® or piggyBac-like transposase enzyme. In certain embodiments, the piggyBac® or piggyBac-like transposase enzyme is isolated or derived from Agrotis ipsilon. The piggyBac® (PB) or piggyBac-like transposase enzyme may comprise or consist of an amino acid sequence at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% or any percentage in between identical to:

(SEQ ID NO: 14537)

1
MESRQPINQD EIATILENDD DYSPLDSDSE AEDRVVEDDV

WSDNEDAMID YVEDTSRQED

61
PDNNIASQES ANTEVTSLTS HRIISLPQRS ICGKNNHVWS

TTKGRTTGRT SAINIIRTNR

121
GPTRMCRNIV DPLLCFQLFI TDEIIHEIVK WTNVEMIVKR

QNLIDISASY RDTNTMEMWA

181
LVGILTLTAV MKDNHLSTDE LFDATFSGTR YVSVMSPERF

EFLIRCMRMD DKTLRPTLRS

241
DDAFIPVRKL WEIFINQCRL NYVPGGNLTV DEQLLGFRGR

CPFRMYIPNK PDKYGIRFPM

301
MCDAATKYMI DAIPYLGKST KTNGLPLGEF YVKELTKTVH

GTNRNVTCDN WFTSIPLAKN

361
MLQAPYNLTI VGTIRSNKRE IPEEIKNSRS RPVGSSMECF

DGPLTLVSYK PKPSRMVFLL

421
SSCDENAVIN ESNGKPDMIL FYNQTKGGVD SFDQMCKSMS

ANRKTNRWPM AVFYGMLNMA

481
FVNSYIIYCH NKINKQKKPI NRKEFMKNLS TDLTTPWMQE

RLKAPTLKRT LRDNITNVLK

541
NVVPPSPANN SEEPGPKKRS YCGFCSYKKR RMTKTQFYKC

KKAICGEHNI DVCQDCV.

In certain embodiments, the piggyBac® or piggyBac-like transposon is isolated or derived from Agrotis ipsilon. In certain embodiments, the piggyBac® or piggyBac-like transposon comprises a sequence of:

(SEQ ID NO: 14538)

1
ttaaccctag aagcccaatc tacgtaaatt tgacgtatac

cgcggcgaaa tatatctgtc

61
tctttcacgt ttaccgtcgg attcccgcta acttcggaac

caactcagta gccattgaga

121
actcccagga cacagttgcg tcatctcggt aagtgccgcc

attttgttgt aatagacagg

181
ttgcacgtca ttttgacgta taattgggct ttgtgtaact

tttqaaatta tttataattt

241
ttattgatgt gatttatttg agttaatcgt attgtttcgt

tacatttttc atatgatatt

301
aatattttca gattgaatat aaa.

In certain embodiments, the piggyBac® or piggyBac-like transposon comprises a sequence of:

(SEQ ID NO: 14539)

1
agactgtttt ttttaaaagg cttataaagt attactattg

cgtgatttaa ttttataaaa

61
atatttaaaa ccagttgatt tttttaataa ttacctaatt

ttaagaaaaa atgttagaag

121
cttgatattt ttgttgattt ttttctaaga tttgattaaa

aggccataat tgtattaata

181
aagagtattt ttaacttcaa atttatttta tttattaatt

aaaacttcaa ttatgataat

241
acatgcaaaa atatagttca tcaacagaaa aatataggaa

aactctaata gttttatttt

301
tacacgtcat ttttacgtat gattgggctt tataqctagt

caaatatgat tgggcttcta

361
gggttaa.

In certain embodiments of the methods of the disclosure, the transposase enzyme is a piggyBac® or piggyBac-like transposase enzyme. In certain embodiments, the piggyBac or piggyBac-like transposase enzyme is isolated or derived from Megachile rotundata. The piggyBac® (PB) or piggyBac-like transposase enzyme may comprise or consist of an amino acid sequence at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% or any percentage in between identical to:

(SEQ ID NO: 14540)

1
MNGKDSLGEF YLDDLSDCLD CRSASSTDDE SDSSNIAIRK

RCPIPLIYSD SEDEDMNNNV

61
EDNNHFVKES NRYHYQIVEK YKITSKTKKW KDVTVTEMKK

FLGLIILMGQ VKKDVLYDYW

121
STDPSIETPF FSKVMSRNRF LQIMQSWHFY NNNDISPNSH

RIVKIQPVID YFKEKFNNVY

181
KSDQQLSLDE CLIPWRGRLS IKTYNPAKIT KYGILVRVLS

EARTGYVSNF CVYAADGKKI

241
EETVLSVIGP YKNMWHHVYQ DNYYNSVNIA KIFLKNKLRV

CGTIRKNRSL PQILQTVKLS

301
RGQHQFLRNG HTLLEVWNNG KRNVNMISTI HSAQMAESRN

RSPTSDCPIQ KPISIIDYNK

361
YMKGVDRADQ YISYYSIFRK TKKWTKRVVM FFINCALFNS

FKVITTLNGQ KITYKNFLHK

421
AALSLIEDCG TEEQGTDLPN SEPTTTRTTS RVDHPGRLEN

FGKHKLVNIV TSGQCKKPLR

481
QCRVCASKKK LSRTGFACKY CNVPLHKGDC FERYHSLKKY.

In certain embodiments, the piggyBac® or piggyBac-like transposon is isolated or derived from Megachile rotundata. In certain embodiments, the piggyBac® or piggyBac-like transposon comprises a sequence of:

(SEQ ID NO: 14541)

1
ttaaataatg cccactctag atgaacttaa cactttaccg

accggccgtc gattattcga

61
cgtttgctcc ccagcgctta ccgaccggcc atcgattatt

cgacgtttgc ttcccagcgc

121
ttaccgaccg gtcatcgact tttgatcttt ccgttagatt

tggttaggtc agattgacaa

181
gtagcaagca tttcgcattc tttattcaaa taatcggtgc

ttttttctaa gctttagccc

241
ttagaa.

In certain embodiments, the piggyBac® or piggyBac-like transposon comprises a sequence of:

(SEQ ID NO: 14542)

1
acaacttctt ttttcaacaa atattgttat atggattatt

tatttattta tttatttatg

61
gtatatttta tgtttattta tttatggtta ttatggtata

ttttatgtaa ataataaact

121
gaaaacgatt gtaatagatg aaataaatat tgttttaaca

ctaatataat taaagtaaaa

181
gattttaata aatttcgtta ccctacaata acacgaagcg

tacaatttta ccagagttta

241
ttaa.

In certain embodiments of the methods of the disclosure, the transposase enzyme is a piggyBac® or piggyBac-like transposase enzyme. In certain embodiments, the piggyBac® or piggyBac-like transposase enzyme is isolated or derived from Bombus impatiens. The piggyBac (PB) or piggyBac-like transposase enzyme may comprise or consist of an amino acid sequence at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% or any percentage in between identical to:

(SEQ ID NO: 14543)

1
MNEKNGIGEF YLDDLSDCPD SYSRSNSGDE SDGSDTIIRK

RGSVIPPRYS DSEDDEINNV

61
EDNANNVENN DDIWSTNDEA IILEPFEGSP GLKIMPSSAE

SVTDNVNLFF GDDFFEHLVR

121
ESNRYHYQVM EKYKIPSKAK KWTDITVPEM KKFLGLIVLM

GQIKKDVLYD YWSTDPSIET

181
PFFSQVMSRN RFVQIMQSWH FCNNDNIPHD SHRLAKIQPV

IDYFRRKFND VYKPCQQLSL

241
DESIIPWRGR LSIKTYNPAK ITKYGILVRV LSEAVTGYVC

NFDVYAADGK KLEDTAVIEP

301
YKNIWHQIYQ DNYYNSVKMA RILLKNKVRV CGTIRKNRGL

PRSLKTIQLS RGQYEFRRNH

361
QILLEVWNNG RPNVNMISTI HSAQLMESRS KSKRSDVPIQ

KPNSIIDYNK YMKGVDRADQ

421
YLAYYSIFRK TKKWTKRTVM FFINCALENS FRVYTILNGK

NITYKNFLHK VAVSWIEDGE

481
TNCTEQDDNL PNSEPTRRAP RLDHPGPLSN YGKHKLINIV

TSGRSLKPQR QCRVCAVQKK

541
RSRTCFVCKF CNVPLHKGDC FERYHTLKKY.

In certain embodiments, the piggyBac® or piggyBac-like transposon is isolated or derived from Bombus impatiens. In certain embodiments, the piggyBac® or piggyBac-like transposon comprises a sequence of:

(SEQ ID NO: 14544)

1
ttaatttttt aacattttac cgaccgatag ccgattaatc

gggtttttgc cgctgacgct

61
taccgaccga taacctatta atcggctttt tgtcgtcgaa

gcttaccaac ctatagccta

121
cctataqtta atcggttgcc atggcgataa acaatctttc

tcattatatg agcagtaatt

181
tgttatttag tactaaggta ccttgctcag ttgcgtcagt

tgcgttgctt tgtaagctcc

241
cacagtttta taccaattcg aaaaacttac cgttcgcg.

In certain embodiments, the piggyBac® or piggyBac-like transposon comprises a sequence of:

(SEQ ID NO: 14545)

1
actatttcac atttgaacta aaaaccgttg taatagataa

aataaatata atttagtatt

61
aatattatgg aaacaaaaga ttttattcaa tttaattatc

ctatagtaac aaaaagcggc

121
caattttatc tgagcatacg aaaagcacag atactcccgc

ccgacagtct aaaccgaaac

181
agagccggcg ccagggagaa tctgcgcctg agcagccggt

cggacgtgcg tttgctgttg

241
aaccgctagt ggtcagtaaa ccagaaccag tcagtaagcc

agtaactgat cagttaacta

301
gattgtatag ttcaaattga acttaatcta gtttttaagc

gtttgaatgt tgtctaactt

361
cgttatatat tatattcttt ttaa.

In certain embodiments of the methods of the disclosure, the transposase enzyme is a piggyBac® or piggyBac-like transposase enzyme. In certain embodiments, the piggyBac® or piggyBac-like transposase enzyme is isolated or derived from Mamestra brassicae. The piggyBac® (PB) or piggyBac-like transposase enzyme may comprise or consist of an amino acid sequence at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% or any percentage in between identical to:

(SEQ ID NO: 14546)

1
MFSFVPNKEQ TPTVLIFCFH LKTTAAESHR PLVEAFGEQV

PTVKTCERWF QRFKSGDFDV

61
DDKEEGKPPK RYEDAELQAL LDEDDAQTQK QLAEQLEVSQ

QAVSNRLREG GKIQKVGRWV

121
PHELNERQRE RRKNTCEILL SRYKRKSFLH RIVTGEEKWI

FFVNPKRKKS YVDPGQPATS

181
TARPNRFGKK TRLCVWWDQS GVIYYELLKP GETVNTARYQ

QQLINLNRAL QRKRPEYQKR

241
QHRVIFLHDN APSHTARAVR DTLETLNWEV LPHAAYSPDL

APSDYHLFAS MGHALAEQRF

301
DSYESVEEWL DEWFAAKDDE FYWRGIHKLP ERWDNCVASD

GKYFE.

In certain embodiments, the piggyBac® or piggyBac-like transposon is isolated or derived from Mamestra brassicae. In certain embodiments, the piggyBac® or piggyBac-like transposon comprises a sequence of:

(SEQ ID NO: 14547)

1
ttattgggtt gcccaaaaag taattgcgga tttttcatat

acctgtcttt taaacgtaca

61
tagggatcga actcagtaaa actttgacct tgtgaaataa

caaacttgac tgtccaacca

121
ccatagtttg gcgcgaattg agcgtcataa ttgttttgac

tttttgcagt caac.

In certain embodiments, the piggyBac® or piggyBac-like transposon comprises a sequence of:

(SEQ ID NO: 14548)

1
atgatttttt ctttttaaac caattttaat tagttaattg

atataaaaat ccgcaattac

61
tttttgggca acccaataa.

In certain embodiments of the methods of the disclosure, the transposase enzyme is a piggyBac® or piggyBac-like transposase enzyme. In certain embodiments, the piggyBac® or piggyBac-like transposase enzyme is isolated or derived from Mayetiola destructor. The piggyBac® (PB) or piggyBac-like transposase enzyme may comprise or consist of an amino acid sequence at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% or any percentage in between identical to:

(SEQ ID NO: 14549)

1
MENFENWRKR RHLREVLLGH FEAKKMAES HRLLVEVYGE

HALAKTQCFE WFQRFKSGDF

61
DTEDKERPGQ PKKEEDEELE ALLDEDCCQT QEELAKSLGV

TQQAISKRLK AAGYIQKQGN

121
WVPHELKPRD VERRFCMSEM LLQRHKKKSF LSRIITGDEK

WIHYDNSKRK KSYVKRGGRA

181
KSTPKSNLHG AKVMLCIWWD QRGVLYYELL EPGQTITGDL

YRTQLIRLKQ ALAEKRPEYA

241
KRHGAVIFHH DNARPHVALP VKNYLENSGW EVLPHPPYSP

DLAPSDYHLF RSMQNDLAGK

301
RFTSEQGIRK WLDSFLAAKP AKFFEKGIHE LSERWEKVIA

SDGQYFE.

In certain embodiments, the piggyBac® or piggyBac-like transposon is isolated or derived from Mayetiola destructor. In certain embodiments, the piggyBac® or piggyBac-like transposon comprises a sequence of:

(SEQ ID NO: 14550)

1
taagacttcc aaaatttcca cccgaacttt accttccccg

cgcattatgt ctctcttttc

61
accctctgat ccctggtatt gttgtcgagc acgatttata

ttgggtgtac aacttaaaaa

121
ccggaattgg acgctagatg tccacactaa cgaatagtgt

aaaagcacaa atttcatata

181
tacgtcattt tgaaggtaca tttgacagct atcaaaatca

gtcaataaaa ctattctatc

241
tgtgtgcatc atattttttt attaact.

In certain embodiments, the piggyBac® or piggyBac-like transposon comprises a sequence of:

(SEQ ID NO: 14551)

1
tgcattcatt cattttgtta tcgaaataaa gcattaattt

tcactaaaaa attccggttt

61
ttaagttgta cacccaatat catccttagt gacaattttc

aaatggcttt cccattgagc

121
tgaaaccgtg gctctagtaa gaaaaacgcc caacccgtca

tcatatgcct tttttttctc

181
aacatccg.

In certain embodiments of the methods of the disclosure, the transposase enzyme is a piggyBac® or piggyBac-like transposase enzyme. In certain embodiments, the piggyBac® or piggyBac-like transposase enzyme is isolated or derived from Apis mellifera. The piggyBac® (PB) or piggyBac-like transposase enzyme may comprise or consist of an amino acid sequence at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% or any percentage in between identical to:

(SEQ ID NO: 14552)

1
MENQKEHYRH ILLFYFRKGE NASQAHKKLC AVYGDEAIKE

RQCQNWFDKF PSGDFSLKDE

61
KRSGRPVEVD DDLIKAIIDS DRHSTTREIA EKLEIVSHTCI

ENHLKQLGYV QKLDTWVPHE

121
LKEKHLTQRI NSCDLLKKRN ENDPFLKRLI TGDEKWVVYN

NIKRKRSWSR PREPAQTTSK

181
AGIHRKKVLL SVWWDYKGIV YFELLPPNRT INSVVYIEQL

TKLNNAVEEK RPELTNRKGV

241
VFHHDNARPH TSLVTRQKLL ELGWDVLPHP PYSPDLAPSD

YFLFRSLQNS LNGKNFNNDD

301
DIKSYLIQFF ANKNQKFYER GIMMLPERWQ KVIDQNGQHI TE.

In certain embodiments, the piggyBac® or piggyBac-like transposon is isolated or derived from Apis mellifera. In certain embodiments, the piggyBac® or piggyBac-like transposon comprises a sequence of:

(SEQ ID NO: 14553)

1
ttgggttggc aactaagtaa ttgcggattt cactcataga

tggcttcagt tgaattttta

61
ggtttgctgg cgtagtccaa atgtaaaaca cattttgtta

tttgatagtt ggcaattcag

121
ctgtcaatca gtaaaaaaag ttttttgatc ggttgcgtag

ttttcgtttg gcgttcgttg

181
aaaa.

In certain embodiments, the piggyBac® or piggyBac-like transposon comprises a sequence of:

(SEQ ID NO: 14554)

1
agttatttag ttccatgaaa aaattgtctt tgattttcta

aaaaaaatcc gcaattactt

61
agttgccaat ccaa.

In certain embodiments of the methods of the disclosure, the transposase enzyme is a piggyBac® or piggyBac-like transposase enzyme. In certain embodiments, the piggyBac® or piggyBac-like transposase enzyme is isolated or derived from Messor bouvieri. The piggyBac® (PB) or piggyBac-like transposase enzyme may comprise or consist of an amino acid sequence at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% or any percentage in between identical to:

(SEQ ID NO: 14555)

1
MSSEVPENVH LRHALLFLFH QKKRAAESHR LLVETYGEHA

PTIRTCETWF RQFKCGDENV

61
QDKERPGRPK TFEDAELQEL LDEDSTQTQK QLAEKLNVSR

VAICERLQAM GKIQKMGRWV

121
PHELNDRQME NRKIVSEMLL QRYERKSFLH RIVTGDEKWI

YFENPKRKKS WLSPGEAGPS

181
TARPNRFGRK TMLCVWWDQI GVVYYELLKP GETVNTDRYR

QQMINLNCAL IEKRPQYAQR

241
HDKVILQHDN APSHTAKPVK EMLKSLGWEV LSHPPYSPDL

APSDYHLFAS MGHALAEQHF

301
ADFEEVKKWL DEWFSSKEKL FFWNGIHKLS ERWTKCIESN

GQYFE.

In certain embodiments, the piggyBac® or piggyBac-like transposon is isolated or derived from Messor bouvieri. In certain embodiments, the piggyBac® or piggyBac-like transposon comprises a sequence of:

(SEQ ID NO: 14556)

1
agtcagaaat gacacctcga tcgacgacta atcgacgtct

aatcgacgtc gattttatgt

61
caacatgtta ccaggtgtgt cggtaattcc tttccggttt

ttccggcaga tgtcactagc

121
cataagtatg aaatgttatg atttgataca tatgtcattt

tattctactg acattaacct

181
taaaactaca caagttacgt tccgccaaaa taacagcgtt

atagatttat aattttttga

241
aa.

In certain embodiments, the piggyBac® or piggyBac-like transposon comprises a sequence of:

(SEQ ID NO: 14557)

1
ataaatttga actatccatt ctaagtaacg tgttttcttt

aacgaaaaaa ccggaaaaga

61
attaccgaca ctcctggtat gtcaacatgt tattttcgac

attgaatcgc gtcgattcga

121
agtcgatcga ggtgtcattt ctgact.

In certain embodiments of the methods of the disclosure, the transposase enzyme is a piggyBac or piggyBac-like transposase enzyme. In certain embodiments, the piggyBac® or piggyBac-like transposase enzyme is isolated or derived from Trichoplusia ni. The piggyBac® (PB) or piggyBac-like transposase enzyme may comprise or consist of an amino acid sequence at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% or any percentage in between identical to:

(SEQ ID NO: 14558)

1
MGSSLDDEHI LSALLQDDE LVGEDSDSEV SDHVSEDDVQ

SDTEEAFIDE VHEVQPTSSG

61
SEILDEQNVI EQPGSSLASN RILTLPQRTI RGKNKHCWST

SKSTRRSRVS ALNIVRSQRG

121
PTRMCRNIYD PLLCFKLFFT DEIISEIVKW TNAEISLKRR

ESMTSATFRD TNEDEIYAFF

181
GILVMTAVRK DNHMSTDDLF DPSLSMVYVS VMSRDRFDFL

IRCIRMDDKS IRPTLRENDV

241
FTPVRKIWDL FIHQCIQNYT PGAHLTIDEQ LLGFRGRCPF

RVYIPNKPSK YGIKILMMCD

301
SGTKYMINGM PYLGRGTQTN GVPIGEYYVK ELSKPVHGSC

RNITCDNWFT SIPLAKNLLQ

361
EPYKLTIVGT VRSNKREIPE VLKNSRSRPV GTSMFCFDGP

LTLVSYKPKP AKMVYLLSSC

421
DEDASINEST GKPQMVMYYN QTKGGVDTLD QMCSVMTCSR

KTNRWPMALL YGMINIACIN

481
SFIIYSHNVS SKGEKVQSRK KFMRNLYMSL TSSFMRKRLE

APTLKRYLRD NISNILPKEV

541
PGTSDDSTEE PVMKKRTYCT YCPSKIRRKA NASCKKCKKV

ICREHNIDMC QSCF.

In certain embodiments, the piggyBac® or piggyBac-like transposon is isolated or derived from Trichoplusia ni. In certain embodiments, the piggyBac or piggyBac-like transposon comprises a sequence of:

(SEQ ID NO: 14559)

1
ttaaccctag aaagatagtc tgcgtaaaat tgacgcatgc

attcttgaaa tattgctctc

61
tctttctaaa tagcgcgaat ccgtcgctgt gcatttagga

catctcagtc gccgcttgga

121
gctcccgtga ggcgtgcttg tcaatgcggt aagtgtcact

gattttgaac tataacgacc

181
gcgtgagtca aaatgacgca tgattatctt ttacgtgact

ttaagattt aactcatacg

241
ataattatat tgttatttca tgttctactt acgtgataac

ttattata tatattttct

301
tgttatagat atc.

In certain embodiments, the piggyBac® or piggyBac-like transposon comprises a sequence of:

(SEQ ID NO: 14560)

1
tttgttactt tatagaagaa attttgagtt tttgtttttt

tttaataaat aaataaacat

61
aaataaattg tttgttgaat ttattattag tatgtaagtg

taaatataat aaaacttaat

121
atctattcaa attaataaat aaacctcgat atacagaccg

ataaaacaca tgcgtcaatt

181
ttacgcatga ttatctttaa cgtacgtcac aatatgatta

tctttctagg gttaa.

In certain embodiments, the piggyBac® or piggyBac-like transposon comprises a sequence of:

(SEQ ID NO: 14561)

1
ccctagaaag atagtctgcg taaaattgac gcatgcattc

ttgaaatatt gctctctctt

61
tctaaatagc gcgaatccgt cgctgtgcat ttaggacatc

tcagtcgccg cttggagctc

121
ccgtgaggcg tgcttgtcaa tgcggtaagt gtcactgatt

ttgaactata acgaccgcgt

181
gagtcaaaat gacgcatgat tatcttttac gtgactttta

agatttaact catacgataa

241
ttatattgtt atttcatgtt ctacttacgt gataacttat

tatatatata ttttcttgtt

301
atagatatc.

In certain embodiments, the piggyBac® or piggyBac-like transposon comprises a sequence of:

(SEQ ID NO: 14562)

1
tttgttactt tatagaagaa attttgagtt tttgtttttt

tttaataaat aaataaacat

61
aaataaattg tttgttgaat ttattattag tatgtaagtg

taaatataat aaaacttaat

121
atctattcaa attaataaat aaacctcgat atacagaccg

ataaaacaca tgcgtcaatt

181
ttacgcatga ttatctttaa cgtacgtcac aatatgatta

tctttctagg g.

In certain embodiments, the piggyBac® or piggyBac-like transposon comprises a sequence of:

(SEQ ID NO: 14609)

1
tctaaatagc gcgaatccgt cgctgtgcat ttaggacatc

tcagtcgccg cttggagctc

61
ccgtgaggcg tgcttgtcaa tgcggtaagt gtcactgatt

ttgaactata acgaccgcgt

121
gagtcaaaat gacgcatgat tatcttttac gtgactttta

agatttaact catacgataa

181
ttatattgtt atttcatgtt ctacttacgt gataacttat

tatatatata ttttcttgtt

241
atagatatc.

In certain embodiments, the piggyBac® or piggyBac-like transposon comprises a sequence of;

(SEQ ID NO: 14610)

1
tttgttactt tatagaagaa attttgagtt tttgtttttt

tttaataaat aaataaacat

61
aaataaattg tttgttgaat ttattattag tatgtaagtg

taaatataat aaaacttaat

121
atctattcaa attaataaat aaacctcgat atacagaccg

ataaaacaca tgcgtcaatt

181
ttacgcatga ttatctttaa cgtacgtcac aatatgatta

tctttctagg g.

In certain embodiments, the piggyBac® or piggyBac-like transposon comprises SEQ ID NO: 14561 and SEQ ID NO: 14562, and the piggyBac® or piggyBac-like transposase comprises SEQ ID NO: 14558. In certain embodiments, the piggyBac® or piggyBac-like transposon comprises SEQ ID NO: 14609 and SEQ ID NO: 14610, and the piggyBac® or piggyBac-like transposase comprises SEQ ID NO: 14558.

In certain embodiments, the piggyBac® or piggyBac-like transposon is isolated or derived from Aphis gossypii. In certain embodiments, the piggyBac® or piggyBac-like transposon comprises an ITR sequence of CCTTCCAGCGGGCGCGC (SEQ ID NO: 14565).

In certain embodiments, the piggyBac® or piggyBac-like transposon is isolated or derived from Chilo suppressalis. In certain embodiments, the piggyBac or piggyBac-like transposon comprises an ITR sequence of CCCAGATTAGCCT (SEQ ID NO: 14566).

In certain embodiments, the piggyBac® or piggyBac-like transposon is isolated or derived from Heliothis virescens. In certain embodiments, the piggyBac® or piggyBac-like transposon comprises an ITR sequence of CCCTTAATTACTCGCG (SEQ ID NO: 14567).

In certain embodiments, the piggyBac® or piggyBac-like transposon is isolated or derived from Anopheles stephensi. In certain embodiments, the piggyBac® or piggyBac-like transposon comprises an ITR sequence of CCCTAGAAAGATA (SEQ ID NO: 14569).

DNA transposons in the hAT family are widespread in plants and animals. A number of active hAT transposon systems have been identified and found to be functional, including but not limited to, the Hermes transposon, Ac transposon, hobo transposon, and the Tol2 transposon. The hAT family is composed of two families that have been classified as the AC subfamily and the Buster subfamily, based on the primary sequence of their transposases. Members of the hAT family belong to Class II transposable elements. Class II mobile elements use a cut and paste mechanism of transposition. hAT elements share similar transposases, short terminal inverted repeats, and an eight base-pairs duplication of genomic target.

Compositions and methods of the disclosure may comprise a TcBuster transposon and/or a TcBuster transposase.

Compositions and methods of the disclosure may comprise a TcBuster transposon and/or a hyperactive TcBuster transposase. A hyperactive TcBuster transposase demonstrates an increased excision and/or increased insertion frequency when compared to an excision and/or insertion frequency of a wild type TcBuster transposase. A hyperactive TcBuster transposase demonstrates an increased transposition frequency when compared to a transposition frequency of a wild type TcBuster transposase.

In some embodiments of the compositions and methods of the disclosure, a wild type TcBuster transposase comprises or consists of the amino acid sequence of:

(GenBank Accession No. ABF20545

and SEQ ID NO: 17090)

1
MMLNWLKSGK LESQSQEQSS CYLENSNCLP PTLDSTDIIG

EENKAGTTSR KKRKYDEDYL

61
NFGFTWTGDK DEPNGLCVIC EQVVNNSSLN PAKLKRHLDT

KHPTLKGKSE YFKRKCNELN

121
QKKETFERYV RDDNKNLLKA SYLVSLRIAK QGEAYTIAEK

LIKPCTKDLT TCVFGEKFAS

181
KVDLVPLSDT TISPRIEDMS YFCEAVLVNR LENAKCGFTL

QMDESTDVAG LAILLVFVRY

241
IHESSFEEDM LFCKALPTQT TGEEIFNLLN AYFEKHSIPW

NLCYHICTDG AKAMVGVIKG

301
VIARIKKLVP DIKASHCCLH RHALAVKRIP NALHEVLNDA

VKMINFIKSR PLNARVFALL

361
CDDLGSLHKN LLLHTEVRWL SRGKVLTRFW ELRDEIRIFF

NEREFAGKLN DTSWLQNLAY

421
IADIFSYLNE VNLSLQGPNS TIFKVNSRIN SIKSKLKLWE

ECITKNNTEC FANLNDFLET

481
SNTALDPNLK SNIKEHLNGL KNTFLEYFPP TCNNISWVEN

PFNECGNVDT LPIKEREQLI

541
DIRTDTTLKS SFVPDGIGPF WIKLMDEFPE ISKRAVKELM

PFVTTYLCEK SFSVYVATKT

601
KYRNRLDAED DMRLQLTTIH PDIDNLCNNK QAQKSH.

In some embodiments of the compositions and methods of the disclosure, a TcBuster Transposase comprises or consists of a sequence having at least 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 99% or any percentage identity in between to a wild type TcBuster transposase comprising or consisting of the amino acid sequence of:

In some embodiments of the compositions and methods of the disclosure, a wild type TcBuster transposase is encoded by a nucleic acid sequence comprising or consisting of:

(GenBank Accession No. DQ481197

and SEQ ID NO: 17091)

1
atgatgttga attggctgaa aagtggaaag cttgaaagtc

aatcacagga acagagttcc

61
tgctaccttg agaactctaa ctgcctgcca ccaacgctcg

attctacaga tattatcggt

121
gaagagaaca aagctggtac cacctctcgc aagaagcgga

aatatgacga ggactatctg

181
aacttcggtt ttacatggac tggcgacaag gatgagccca

acggactttg tgtgatttgc

241
gagcaggtag tcaacaattc ctcacttaac ccggccaaac

tgaaacgcca tttggacaca

301
aagcatccga cgcttaaagg caagagcgaa tacttcaaaa

gaaaatgtaa cgagctcaat

361
caaaagaagc atacttttga gcgatacgta agggacgata

acaagaacct cctgaaagct

421
tcttatctcg tcagtttgag aatagctaaa cagggcgagg

catataccat agcggaqaag

481
ttgatcaagc cttgcaccaa ggatctgaca acttgcgtat

ttggagaaaa attcgcgagc

541
aaagttgatc tcgtccccct gtccgacacg actatttcaa

ggcgaatcga agacatgagt

601
tacttctgtg aagccgtgct ggtgaacagg ttgaaaaatg

ctaaatgtgg gtttacgctg

661
cagatggacg agtcaacaga tgttgccggt cttgcaatcc

tgcttgtgtt tgttaggtac

721
atacatgaaa gctcttttga ggaggatatg ttgttctgca

aagcacttcc cactcagacg

781
acaggggagg agattttcaa tcttctcaat gcctatttcg

aaaagcactc catcccatgg

841
aatctgtgtt accacatttg cacagacggt gccaaggcaa

tggtaggagt tattaaagga

901
gtcatagcga gaataaaaaa actcgtccct gatataaaag

ctagccactg ttgcctgcat

961
cgccacgctt tggctgtaaa gcgaataccg aatgcattgc

acgaggtgct caatgacgct

1021
gttaaaatga tcaacttcat caagtctcgg ccgttgaatg

cgcgcgtctt cgctttgctg

1081
tgtgacgatt tggggagcct gcataaaaat cttcttcttc

ataccgaagt gaggtggctg

1141
tctagaggaa aggtgctgac ccgattttgg gaactgagag

atgaaattag aattttcttc

1201
aacgaaaggg aatttgccgg gaaattgaac gacaccagtt

ggttgcaaaa tttggcatat

1261
atagctgaca tattcagtta tctgaatgaa gttaatcttt

ccctgcaagg gccgaatagc

1321
acaatcttca aggtaaatag ccgcattaac agtattaaat

caaagttgaa gttgtgggaa

1381
gagtgtataa cgaaaaataa cactgagtgt tttgcgaacc

tcaacgattt tttggaaact

1441
tcaaacactg cgttggatcc aaacctgaag tctaatattt

tggaacatct caacggtctt

1501
aagaacacct ttctggagta ttttccacct acgtgtaata

atatctcctg ggtggagaat

1561
cctttcaatg aatgcggtaa cgtcgataca ctcccaataa

aagagaggga acaattgatt

1621
gacatacgga ctgatacgac attgaaatct tcattcgtgc

ctgatggtat aggaccattc

1681
tggatcaaac tgatggacga atttccagaa attagcaaac

gagctgtcaa agagctcatg

1741
ccatttgtaa ccacttacct ctgtgagaaa tcattttccg

tctatgtagc cacaaaaaca

1801
aaatatcgaa atagacttga tgctgaagac gatatgcgac

tccaacttac tactatccat

1861
ccagacattg acaacctttg taacaacaag caggctcaga

aatcccactg a.

In some embodiments of the compositions and methods of the disclosure, a TcBuster Transposase comprises or consists of a sequence having at least 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 99% or any percentage identity in between to a wild type TcBuster transposase encoded by a nucleic acid sequence comprising or consisting of.

In some embodiments of the compositions and methods of the disclosure, a TcBuster Transposase comprises or consists of a naturally occurring amino acid sequence.

In some embodiments of the compositions and methods of the disclosure, a TcBuster Transposase comprises or consists of a non-naturally occurring amino acid sequence.

In some embodiments of the compositions and methods of the disclosure, a TcBuster Transposase is encoded by a sequence comprising or consisting of a naturally occurring nucleic acid sequence.

In some embodiments of the compositions and methods of the disclosure, a TcBuster Transposase is encoded by a sequence comprising or consisting of a non-naturally occurring nucleic acid sequence.

In some embodiments of the compositions and methods of the disclosure, a mutant TcBuster Transposase comprises one or more sequence variations when compared to a wild type TcBuster Transposase. In some embodiments, the wild type TcBuster Transposase comprises or consists of the amino acid sequence of SEQ ID NO: 17090. In some embodiments, the wild type TcBuster Transposase is encoded by a sequence comprising or consisting of the nucleic acid sequence of SEQ ID NO: 17091. In some embodiments, the one or more sequence variations comprises one or more of a substitution, inversion, insertion, deletion, transposition, and frameshift. In some embodiments, the one or more sequence variations comprises a modified, synthetic, artificial or non-naturally occurring amino acid. In some embodiments, the one or more sequence variations comprises a modified, synthetic, artificial or non-naturally occurring nucleic acid.

In some embodiments of the compositions and methods of the disclosure, a mutant TcBuster Transposase comprises one or more sequence variations when compared to a wild type TcBuster Transposase. In some embodiments, the one or more sequence variations comprises an amino acid substitution that increases a net charge a neutral pH when compared to a wild type TcBuster Transposase. In some embodiments, the wild type TcBuster Transposase comprises or consists of the amino acid sequence of SEQ ID NO: 17090. In some embodiments, the wild type TcBuster Transposase is encoded by a sequence comprising or consisting of the nucleic acid sequence of SEQ ID NO: 17091. In some embodiments, the one or more sequence variations comprises an amino acid substitution of the aspartic acid (D) at position 223 (D223), the aspartic acid (D) at position 289 (D289) and the aspartic acid (E) at position 589 (E289) of SEQ ID NO: 17090. In some embodiments, the one or more sequence variations comprises an amino acid substitution within 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100 or any number of amino acids in between of position 223, 289 and/or 289 of SEQ ID NO: 17090. In some embodiments, the one or more sequence variations comprises an amino acid substitution within 70 amino acids of position 223, 289 and/or 289 of SEQ ID NO: 17090. In some embodiments, the one or more sequence variations comprises an amino acid substitution within 80 amino acids of position 223, 289 and/or 289 of SEQ ID NO: 17090. In some embodiments, the one or more sequence variations comprises an amino acid substitution of an aspartic acid (D) or a aspartic acid (E) to a neutral amino acid, a lysine (L) or an arginine (R) (e.g. D223L, D223R, D289L, D289R, E289L, E289R of SEQ ID NO: 17090).

In some embodiments of the compositions and methods of the disclosure, a mutant TcBuster Transposase comprises one or more sequence variations when compared to a wild type TcBuster Transposase. In some embodiments, the one or more sequence variations comprises one or more of Q82E, N85S, D99A, D132A, Q151S, Q151A, E153K, E153R, A154P, Y155H, E159A, T171K, T171R, K177E, D183K, D183R, D189A, T191E, S193K, S193R, Y201A, F202D, F202K, C203I, C203V, Q221T, M222L, I223Q, E224G, S225W, D227A, R239H, E243A, E247K, P257K, P257R, Q258T, E263A, E263K, E263R, E274K, E274R, S278K, N281E, L282K, L282R, K292P, V297K, K299S, A303T, H322E, A332S, A358E, A358K, A358S, D376A, V377T, L380N, I398D, I398S, I398K, F400L, V431L, S447E, N450K, N450R, I452F, E469K, K469K, P510D, P510N, E517R, R536S, V553S, P554T, P559D, P559S, P559K, K573E, E578L, K590T, Y595L, V596A, T598I, K599A, Q615A, T618K, T618K, T618R, D622K and D622R of SEQ ID NO: 17090. In some embodiments, the one or more sequence variations comprises an amino acid substitution within 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100 or any number of amino acids in between of position 154, 155, 159, 171, 177, 183, 189, 191, 193, 201, 202, 203, 221, 223, 224, 225, 227, 239, 243, 247, 257, 258, 263, 274, 278, 281, 282, 292, 297, 299, 303, 322, 332, 358, 376, 377, 380, 398, 400, 431, 447, 450, 452, 469, 510, 517, 536, 553, 554, 559, 573, 578, 590, 595, 596, 598, 599, 615, 618, and 622 of SEQ ID NO: 17090.

In some embodiments of the compositions and methods of the disclosure, a mutant TcBuster Transposase comprises one or more sequence variations when compared to a wild type TcBuster Transposase. In some embodiments, the one or more sequence variations comprises one or more of E247K, V297K, A358K, S278K, E247R, E274R, V297R, A358R, S278R, T171R, D183R, S193R, P257K, E263R, L282K, T618K, D622R, E153K, N450K, T171K, D183K, S193K, P257R, E263K, L282R, T618R, D622K, E153R and N450R of SEQ ID NO: 17090. In some embodiments, the one or more sequence variations comprises an amino acid substitution within 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100 or any number of amino acids in between of position 153, 171, 183, 193, 247, 257, 263, 274, 278, 282, 297, 358, 450, 618, 622 of SEQ ID NO: 17090.

In some embodiments of the compositions and methods of the disclosure, a mutant TcBuster Transposase comprises one or more sequence variations when compared to a wild type TcBuster Transposase. In some embodiments, the one or more sequence variations comprises one or more of V377T/E469K, V377T/E469K/R536S, A332S, V553S/P554T, E517R, K299S, Q615A/T618K, S278K, A303T, P510D, P510N, N281S, N281E, K590T, Q258T, E247K, S447E, N85S, V297K, A358K, I452F, V377T/E469K/D189A, K573E/E578L, I452FN377T/E469K/D189A, A358K/V377T/E469K/D189A, K573E/E578L/V377T/E469K-D189A, T171R, D183R, S193R, P257K, E263R, L282K, T618K, D622R, E153K, N450K, T171K, D183K, S193K, P257R, E263K, L282R, T618R, D622K, E153R, N450R, E247K/E274K/V297K/A358K of SEQ ID NO: 17090. In some embodiments, the one or more sequence variations comprises an amino acid substitution within 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100 or any number of amino acids in between of position 85, 153, 171, 189, 193, 247, 257, 258, 263, 274, 278, 281, 282, 297, 299, 303, 332, 358, 377, 450, 469, 447, 452, 469, 510, 517, 536, 553, 554, 573, 578, 590, 615, 618, 622 of SEQ ID NO: 17090.

In some embodiments of the compositions and methods of the disclosure, a mutant TcBuster Transposase comprises one or more sequence variations when compared to a wild type TcBuster Transposase. In some embodiments, the one or more sequence variations comprises one or more of V377T/E469K, V377T/E469K-R536S, V553S/P554T, Q615A/T618K, S278K, A303T, P510D, P510N, N281 S, N281E, K590T, Q258T, E247K, S447E, N85S, V297K, A358K, I452F, V377T/E469K/D189A and K573E/E578L. In some embodiments, the one or more sequence variations comprises an amino acid substitution within 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100 or any number of amino acids in between of position 85, 189, 247, 258, 278, 281, 297, 303, 358, 377, 447, 452, 469, 510, 536, 553, 554, 573, 578, 590, 615, 618 of SEQ ID NO: 17090.

In some embodiments of the compositions and methods of the disclosure, a mutant TcBuster Transposase comprises one or more sequence variations when compared to a wild type TcBuster Transposase. In some embodiments, the one or more sequence variations comprises one or more of Q151S, Q11A, A154P, Q615A, V553S, Y155H, Y201A. F202D, F202K, C203I, C203V, F400L, I398D, I398S, I398K, V431L, P559D, P559S, P559K, M222L of SEQ ID NO: 17090. In some embodiments, the one or more sequence variations comprises an amino acid substitution within 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100 or any number of amino acids in between of position 151, 154, 615, 553, 155, 201, 202, 203, 400, 398, 431, 559, 222 of SEQ ID NO: 17090.

In some embodiments, the mutant TcBuster transposase comprises amino acid substitution 1452K, when numbered in accordance with SEQ ID NO: 17090.

In some embodiments of the compositions and methods of the disclosure, a TcBuster Transposase recognizes a 5′ inverted repeat comprising or consisting of the sequence of:

(SEQ ID NO: 17092)

1
Cagtgttctt caacctttgc catccggcgg aaccctttgt

cgagatattt ttttttatgg

61
aacccttcat ttagtaatac acccagatga gattttaggg

acagctgcgt tgacttgtta

121
cgaacaaggt gagcccgtgc tttggtctag ccaagggcat

ggtaaagact atattcgcgg

181
cgttgtgaca atttaccgaa caactccgcg gccgggaagc

cgatctcggc ttgaacgaat

241
tgttaggtgg cggtacttgg gtcgatatca aagtgcatca

cttcttcccg tatgcccaac

301
tttgtataga gagccactgc gggatcgtca ccgtaatctg

cttgcacgta gatcacataa

361
gcaccaagcg cgttggcctc atgottgagg agattgatga

gcgcggtggc aatgccctgc

421
ctccggtgct cgccggagac tgcgagatca tagatata.

In some embodiments of the compositions and methods of the disclosure, a TcBuster Transposase recognizes a 3′ inverted repeat comprising or consisting of the sequence of:

(SEQ ID NO: 17094)

1
gatatcaagc ttatcqatac cgtcgacctc gagatttctg

aacgattcta ggttaggatc

61
aaacaaaata caatttattt taaaactgta agttaactta

cctttgcttg tctaaaccaa

121
aaacaacaac aaaactacga ccacaagtac agttacatat

ttttgaaaat taaggttaag

181
tgcagtgtaa gtcaactatg cgaatggata acatgtttca

acatgaaact ccgattgacg

241
catgtgcatt ctgaagagcg gcgcggccga cgtctctcga

attgaagcaa tgactcgcgg

301
aaccccgaaa gcctttgggt ggaaccctag ggttccgcgg

aacacaggtt gaagaacact

361
g

In some embodiments of the compositions and methods of the disclosure, a TcBuster Transposase recognizes a 5′ inverted repeat comprising or consisting of the sequence of SEQ ID NO: 17092 and a 3′ inverted repeat comprising or consisting of the sequence of SEQ ID NO: 17093.

In some embodiments of the compositions and methods of the disclosure, a TcBuster Transposase recognizes a 5′ inverted repeat comprising or consisting of the sequence of:

(SEQ ID NO: 17094)

1
Cctgcaggag tgttcttcaa cctttgccat ccggcggaac

cctttgtcga gatatttttt

61
tttatggaac ccttcattta gtaatacacc cagatgagat

tttagggaca gctgcgttga

121
cttqttacga acaaggtgag cccgtgcttt ggtaataaaa

actctaaata agatttaaat

181
ttgcatttat ttaaacaaac tttaaacaaa aagataaata

ttccaaataa aataatatat

241
aaaataaaaa ataaaaatta atgacttttt tgcgcttgct

tattattgca caaattatca

301
atatcgggat ggatcgttgt ttttt.

In some embodiments of the compositions and methods of the disclosure, a TcBuster Transposase recognizes a 3′ inverted repeat comprising or consisting of the sequence of:

(SEQ ID NO: 17095)

1
Gagccaattc agcatcatat ttctgaacga ttctaggtta

ggatcaaaca aaatacaatt

61
tattttaaaa ctgtaagtta acttaccttt gcttgtctaa

acctaaaaca acaacaaaac

121
tacgaccaca aqtacagtta catatttttg aaaattaagg

ttaagtgcag tgtaaqtcaa

181
ctatgcgaat ggataacatg tttcaacatg aaactccgat

tgacgcatgt gcattctgaa

241
gagcggcgcg gccgacgtct ctcgaattga agcaatgact

cgcggaaccc cgaaagcctt

301
tgggtggaac cctagggttc cgcggaacac aggttgaaga

acactg.

In some embodiments of the compositions and methods of the disclosure, a TcBuster Transposase recognizes a 5′ inverted repeat comprising or consisting of the sequence of SEQ ID NO: 17094 and a 3′ inverted repeat comprising or consisting of the sequence of SEQ ID NO: 17095.

In some embodiments of the compositions and methods of the disclosure, a TcBuster Transposase recognizes an inverted repeat comprising or consisting of a sequence having at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95,% 97%, 99% or any percentage identify in between to one or more of SEQ ID NO: 17092, 17093, 17094 or 17095.

In some embodiments of the compositions and methods of the disclosure, a TcBuster Transposase recognizes an inverted repeat comprising or consisting of a sequence having at least In some embodiments of the compositions and methods of the disclosure, a TcBuster Transposase recognizes an inverted repeat comprising or consisting of a sequence having at least 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 97, 99 or any number of contiguous nucleotides in between having between 90 and 100% identity to SEQ ID NO: 17092, 17093, 17094 or 17095 or any portion thereof.

In some embodiments of the compositions and methods of the disclosure, a TcBuster Transposase recognizes an inverted repeat comprising or consisting of a sequence having at least 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 97, 99 or any number of discontinuous nucleotides in between having between 90 and 100% identity to SEQ ID NO: 17092, 17093, 17094 or 17095 or any portion thereof.

In some embodiments of the compositions and methods of the disclosure, a TcBuster transposon comprises a 3′ inverted repeat and a 5′ inverted repeat. In some embodiments of the compositions and methods of the disclosure, a TcBuster Transposase recognizes a TcBuster transposon comprising a 3′ inverted repeat and a 5′ inverted repeat comprising or consisting of a sequence having at least 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80 85, 90, 95, 97, 99 or any number of discontinuous nucleotides in between having between 90 and 100% identity to SEQ ID NO: 17092, 17093, 17094 or 17095 or any portion thereof.

“Modulation” or “regulation” of gene expression refers to a change in the activity of a gene. Modulation of expression can include, but is not limited to, gene activation and gene repression.

The terms “specifically bind” and “specific binding” as used herein refer to the ability of an antibody, an antibody fragment or a nanobody to preferentially bind to a particular antigen that is present in a homogeneous mixture of different antigens. In certain embodiments, a specific binding interaction will discriminate between desirable and undesirable antigens in a sample. In certain embodiments more than about ten- to 100-fold or more (e.g., more than about 1000- or 10,000-fold). “Specificity” refers to the ability of an immunoglobulin or an immunoglobulin fragment, such as a nanobody, to bind preferentially to one antigenic target versus a different antigenic target and does not necessarily imply high affinity.

A “target site” or “target sequence” is a nucleic acid sequence that defines a portion of a nucleic acid to which a binding molecule will bind, provided sufficient conditions for binding exist.

The terms “nucleic acid” or “oligonucleotide” or “polynucleotide” refer to at least two nucleotides covalently linked together. The depiction of a single strand also defines the sequence of the complementary strand. Thus, a nucleic acid may also encompass the complementary strand of a depicted single strand. A nucleic acid of the disclosure also encompasses substantially identical nucleic acids and complements thereof that retain the same structure or encode for the same protein.

Probes of the disclosure may comprise a single stranded nucleic acid that can hybridize to a target sequence under stringent hybridization conditions. Thus, nucleic acids of the disclosure may refer to a probe that hybridizes under stringent hybridization conditions.

Nucleic acids of the disclosure may be single- or double-stranded. Nucleic acids of the disclosure may contain double-stranded sequences even when the majority of the molecule is single-stranded. Nucleic acids of the disclosure may contain single-stranded sequences even when the majority of the molecule is double-stranded. Nucleic acids of the disclosure may include genomic DNA, cDNA, RNA, or a hybrid thereof. Nucleic acids of the disclosure may contain combinations of deoxyribo- and ribo-nucleotides. Nucleic acids of the disclosure may contain combinations of bases including uracil, adenine, thymine, cytosine, guanine, inosine, xanthine hypoxanthine, isocytosine and isoguanine. Nucleic acids of the disclosure may be synthesized to comprise non-natural amino acid modifications. Nucleic acids of the disclosure may be obtained by chemical synthesis methods or by recombinant methods.

Nucleic acids of the disclosure, either their entire sequence, or any portion thereof, may be non-naturally occurring. Nucleic acids of the disclosure may contain one or more mutations, substitutions, deletions, or insertions that do not naturally-occur, rendering the entire nucleic acid sequence non-naturally occurring. Nucleic acids of the disclosure may contain one or more duplicated, inverted or repeated sequences, the resultant sequence of which does not naturally-occur, rendering the entire nucleic acid sequence non-naturally occurring. Nucleic acids of the disclosure may contain modified, artificial, or synthetic nucleotides that do not naturally-occur, rendering the entire nucleic acid sequence non-naturally occurring.

Given the redundancy in the genetic code, a plurality of nucleotide sequences may encode any particular protein. All such nucleotides sequences are contemplated herein.

As used throughout the disclosure, the term “operably linked” refers to the expression of a gene that is under the control of a promoter with which it is spatially connected. A promoter can be positioned 5′ (upstream) or 3′ (downstream) of a gene under its control. The distance between a promoter and a gene can be approximately the same as the distance between that promoter and the gene it controls in the gene from which the promoter is derived. Variation in the distance between a promoter and a gene can be accommodated without loss of promoter function.

As used throughout the disclosure, the term “promoter” refers to a synthetic or naturally-derived molecule which is capable of conferring, activating or enhancing expression of a nucleic acid in a cell. A promoter can comprise one or more specific transcriptional regulatory sequences to further enhance expression and/or to alter the spatial expression and/or temporal expression of same. A promoter can also comprise distal enhancer or repressor elements, which can be located as much as several thousand base pairs from the start site of transcription. A promoter can be derived from sources including viral, bacterial, fungal, plants, insects, and animals. A promoter can regulate the expression of a gene component constitutively or differentially with respect to cell, the tissue or organ in which expression occurs or, with respect to the developmental stage at which expression occurs, or in response to external stimuli such as physiological stresses, pathogens, metal ions, or inducing agents. Representative examples of promoters include the bacteriophage T7 promoter, bacteriophage T3 promoter, SP6 promoter, lac operator-promoter, tac promoter, SV40 late promoter, SV40 early promoter, RSV-LTR promoter, CMV IE promoter, EF-1 Alpha promoter, CAG promoter, SV40 early promoter or SV40 late promoter and the CMV IE promoter.

As used throughout the disclosure, the term “substantially complementary” refers to a first sequence that is at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98% or 99% identical to the complement of a second sequence over a region of 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 180, 270, 360, 450, 540, or more nucleotides or amino acids, or that the two sequences hybridize under stringent hybridization conditions.

As used throughout the disclosure, the term “substantially identical” refers to a first and second sequence are at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98% or 99% identical over a region of 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 180, 270, 360, 450, 540 or more nucleotides or amino acids, or with respect to nucleic acids, if the first sequence is substantially complementary to the complement of the second sequence.

As used throughout the disclosure, the term “variant” when used to describe a nucleic acid, refers to (i) a portion or fragment of a referenced nucleotide sequence: (ii) the complement of a referenced nucleotide sequence or portion thereof; (iii) a nucleic acid that is substantially identical to a referenced nucleic acid or the complement thereof; or (iv) a nucleic acid that hybridizes under stringent conditions to the referenced nucleic acid, complement thereof, or a sequences substantially identical thereto.

As used throughout the disclosure, the term “vector” refers to a nucleic acid sequence containing an origin of replication. A vector can be a viral vector, bacteriophage, bacterial artificial chromosome or yeast artificial chromosome. A vector can be a DNA or RNA vector. A vector can be a self-replicating extrachromosomal vector, and preferably, is a DNA plasmid. A vector may comprise a combination of an amino acid with a DNA sequence, an RNA sequence, or both a DNA and an RNA sequence.

As used throughout the disclosure, the term “variant” when used to describe a peptide or polypeptide, refers to a peptide or polypeptide that differs in amino acid sequence by the insertion, deletion, or conservative substitution of amino acids, but retain at least one biological activity. Variant can also mean a protein with an amino acid sequence that is substantially identical to a referenced protein with an amino acid sequence that retains at least one biological activity.

A conservative substitution of an amino acid, i.e., replacing an amino acid with a different amino acid of similar properties (e.g., hydrophilicity, degree and distribution of charged regions) is recognized in the art as typically involving a minor change. These minor changes can be identified, in part, by considering the hydropathic index of amino acids, as understood in the art. Kyte et al., J. Mol. Biol. 157: 105-132 (1982). The hydropathic index of an amino acid is based on a consideration of its hydrophobicity and charge. Amino acids of similar hydropathic indexes can be substituted and still retain protein function. In one aspect, amino acids having hydropathic indexes of +2 are substituted. The hydrophilicity of amino acids can also be used to reveal substitutions that would result in proteins retaining biological function. A consideration of the hydrophilicity of amino acids in the context of a peptide permits calculation of the greatest local average hydrophilicity of that peptide, a useful measure that has been reported to correlate well with antigenicity and immunogenicity. U.S. Pat. No. 4,554,101, incorporated fully herein by reference.

Substitution of amino acids having similar hydrophilicity values can result in peptides retaining biological activity, for example immunogenicity. Substitutions can be performed with amino acids having hydrophilicity values within +2 of each other. Both the hyrophobicity index and the hydrophilicity value of amino acids are influenced by the particular side chain of that amino acid. Consistent with that observation, amino acid substitutions that are compatible with biological function are understood to depend on the relative similarity of the amino acids, and particularly the side chains of those amino acids, as revealed by the hydrophobicity, hydrophilicity, charge, size, and other properties.

As used herein, “conservative” amino acid substitutions may be defined as set out in Tables A, B, or C below. In some embodiments, fusion polypeptides and/or nucleic acids encoding such fusion polypeptides include conservative substitutions have been introduced by modification of polynucleotides encoding polypeptides of the disclosure. Amino acids can be classified according to physical properties and contribution to secondary and tertiary protein structure. A conservative substitution is a substitution of one amino acid for another amino acid that has similar properties. Exemplary conservative substitutions are set out in Table A.

TABLE A

Conservative Substitutions I

Side chain characteristics

Amino Acid

Aliphatic
Non-polar
G A P I L V F

Polar - uncharged
C S T M N Q

Polar - charged
D E K R

Aromatic
H F W Y

Other
N Q D E

Alternately, conservative amino acids can be grouped as described in Lehninger. (Biochemistry, Second Edition; Worth Publishers, Inc. NY, N.Y. (1975), pp. 71-77) as set forth in Table B.

TABLE B

Conservative Substitutions II

Side Chain Characteristic

Amino Acid

Non-polar (hydrophobic)
Aliphatic:
A L I V P

Aromatic:
F W Y

Sulfur-containing:
M

Borderline:
G Y

Uncharged-polar
Hydroxyl:
S T Y

Amides:
N Q

Sulfhydryl:
C

Borderline:
G Y

Positively Charged (Basic):
K R H

Negatively Charged (Acidic):
D E

Alternately, exemplary conservative substitutions are set out in Table C.

TABLE C

Conservative Substitutions III

Original Residue
Exemplary Substitution

Ala (A)
Val Leu Ile Met

Arg (R)
Lys His

Asn (N)
Gln

Asp (D)
Glu

Cys (C)
Ser Thr

Gln (Q)
Asn

Glu (E)
Asp

Gly (G)
Ala Val Leu Pro

His (H)
Lys Arg

Ile (I)
Leu Val Met Ala Phe

Leu (L)
Ile Val Met Ala Phe

Lys (K)
Arg His

Met (M)
Leu Ile Val Ala

Phe (F)
Trp Tyr Ile

Pro (P)
Gly Ala Val Leu Ile

Ser (S)
Thr

Thr (T)
Ser

Trp (W)
Tyr The Ile

Tyr (Y)
Trp Phe Thr Ser

Val (V)
Ile Leu Met Ala

It should be understood that the polypeptides of the disclosure are intended to include polypeptides bearing one or more insertions, deletions, or substitutions, or any combination thereof, of amino acid residues as well as modifications other than insertions, deletions, or substitutions of amino acid residues. Polypeptides or nucleic acids of the disclosure may contain one or more conservative substitution.

As used throughout the disclosure, the term “more than one” of the aforementioned amino acid substitutions refers to 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 or more of the recited amino acid substitutions. The term “more than one” may refer to 2, 3, 4, or 5 of the recited amino acid substitutions.

Polypeptides and proteins of the disclosure, either their entire sequence, or any portion thereof, may be non-naturally occurring. Polypeptides and proteins of the disclosure may contain one or more mutations, substitutions, deletions, or insertions that do not naturally-occur, rendering the entire amino acid sequence non-naturally occurring. Polypeptides and proteins of the disclosure may contain one or more duplicated, inverted or repeated sequences, the resultant sequence of which does not naturally-occur, rendering the entire amino acid sequence non-naturally occurring. Polypeptides and proteins of the disclosure may contain modified, artificial, or synthetic amino acids that do not naturally-occur, rendering the entire amino acid sequence non-naturally occurring.

As used throughout the disclosure, “sequence identity” may be determined by using the stand-alone executable BLAST engine program for blasting two sequences (bl2seq), which can be retrieved from the National Center for Biotechnology Information (NCBI) ftp site, using the default parameters (Tatusova and Madden, FEMS Microbiol Lett., 1999, 174, 247-250; which is incorporated herein by reference in its entirety). The terms “identical” or “identity” when used in the context of two or more nucleic acids or polypeptide sequences, refer to a specified percentage of residues that are the same over a specified region of each of the sequences. The percentage can be calculated by optimally aligning the two sequences, comparing the two sequences over the specified region, determining the number of positions at which the identical residue occurs in both sequences to yield the number of matched positions, dividing the number of matched positions by the total number of positions in the specified region, and multiplying the result by 100 to yield the percentage of sequence identity. In cases where the two sequences are of different lengths or the alignment produces one or more staggered ends and the specified region of comparison includes only a single sequence, the residues of single sequence are included in the denominator but not the numerator of the calculation. When comparing DNA and RNA, thymine (T) and uracil (U) can be considered equivalent. Identity can be performed manually or by using a computer sequence algorithm such as BLAST or BLAST 2.0.

As used throughout the disclosure, the term “endogenous” refers to nucleic acid or protein sequence naturally associated with a target gene or a host cell into which it is introduced.

As used throughout the disclosure, the term “exogenous” refers to nucleic acid or protein sequence not naturally associated with a target gene or a host cell into which it is introduced, including non-naturally occurring multiple copies of a naturally occurring nucleic acid, e.g., DNA sequence, or naturally occurring nucleic acid sequence located in a non-naturally occurring genome location.

The disclosure provides methods of introducing a polynucleotide construct comprising a DNA sequence into a host cell. By “introducing” is intended presenting to the plant the polynucleotide construct in such a manner that the construct gains access to the interior of the host cell. The methods of the disclosure do not depend on a particular method for introducing a polynucleotide construct into a host cell, only that the polynucleotide construct gains access to the interior of one cell of the host. Methods for introducing polynucleotide constructs into bacteria, plants, fungi and animals are known in the art including, but not limited to, stable transformation methods, transient transformation methods, and virus-mediated methods.

Homologous Recombination

In certain embodiments of the methods of the disclosure, a modified CAR-T_SCMor CAR-T_CMof the disclosure is produced by introducing an antigen receptor into a primary human T cell of the disclosure by homologous recombination. In certain embodiments of the disclosure, the homologous recombination is induced by a single or double strand break induced by a genomic editing composition or construct of the disclosure. Homologous recombination methods of the disclosure comprise contacting a genomic editing composition or construct of the disclosure to a genomic sequence to induce at least one break in the sequence and to provide an entry point in the genomic sequence for an exogenous donor sequence composition. Donor sequence compositions of the disclosure are integrated into the genomic sequence at the induced entry point by the cell's native DNA repair machinery.

In certain embodiments of the methods of the disclosure, homologous recombination introduces a sequence encoding an antigen receptor and/or a donor sequence composition of the disclosure into a “genomic safe harbor” site. In certain embodiments, a mammalian genomic sequence comprises the genomic safe harbor site. In certain embodiments, a primate genomic sequence comprises the genomic safe harbor site. In certain embodiments, a human genomic sequence comprises the genomic safe harbor site.

Genomic safe harbor sites are able to accommodate the integration of new genetic material in a manner that ensures that the newly inserted genetic elements function reliably (for example, are expressed at a therapeutically effective level of expression) and do not cause deleterious alterations to the host genome that cause a risk to the host organism. Potential genomic safe harbors include, but are not limited to, intronic sequences of the human albumin gene, the adeno-associated virus site 1 (AAVS1), a naturally occurring site of integration of AAV virus on chromosome 19, the site of the chemokine (C-C motif) receptor 5 (CCR5) gene and the site of the human ortholog of the mouse Rosa26 locus.

In certain embodiments of the methods of the disclosure, homologous recombination introduces a sequence encoding an antigen receptor and/or a donor sequence composition of the disclosure into a sequence encoding one or more components of an endogenous T-cell receptor or a major histocompatibility complex (MHC). In certain embodiments, inducing homologous recombination within a genomic sequence encoding the endogenous T-cell receptor or the MHC disrupts the endogenous gene, and optionally, replaces part of the coding sequence of the endogenous gene with a donor sequence composition of the disclosure. In certain embodiments, inducing homologous recombination within a genomic sequence encoding the endogenous T-cell receptor or the MHC disrupts the endogenous gene, and optionally, replaces the entire coding sequence of the endogenous gene with a donor sequence composition of the disclosure. In certain embodiments of the methods of the disclosure, introduction of a sequence encoding an antigen receptor or a donor sequence composition of the disclosure by homologous recombination operably links the antigen receptor to an endogenous T cell promoter. In certain embodiments of the methods of the disclosure, introduction of a sequence encoding an antigen receptor or a donor sequence composition of the disclosure by homologous recombination operably links the antigen receptor or the therapeutic protein to a transcriptional or translational regulatory element. In certain embodiments of the methods of the disclosure, introduction of a sequence encoding an antigen receptor or a donor sequence composition of the disclosure by homologous recombination operably links the antigen receptor or the therapeutic protein to a transcriptional regulatory element. In certain embodiments, the transcriptional regulatory element comprises an endogenous T cell 5′ UTR.

In certain embodiments of the introduction step comprising a homologous recombination, a genomic editing composition contacts a genomic sequence of at least one primary T cell of the plurality of T cells. In certain embodiments of the introduction step comprising a homologous recombination, a genomic editing composition contacts a genomic sequence of a portion of primary T cells of the plurality of T cells. In certain embodiments, the portion of primary T cells is at least 1%, 2%, 5%, 7%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 99% or any percentage in between of the total number of primary T cells in the plurality of T cells. In certain embodiments of the introduction step comprising a homologous recombination, a genomic editing composition contacts a genomic sequence of each primary T cell of the plurality of T cells. In certain embodiments of the introduction step comprising a homologous recombination, a genomic editing composition induces a single strand break. In certain embodiments of the introduction step comprising a homologous recombination, a genomic editing composition induces a double strand break. In certain embodiments of the introduction step comprising a homologous recombination, the introduction step further comprises a donor sequence composition. In certain embodiments, the donor sequence composition comprises a sequence encoding the antigen receptor. In certain embodiments, the donor sequence composition comprises a sequence encoding the antigen receptor, a 5′ genomic sequence and a 3′ genomic sequence, wherein the 5′ genomic sequence is homologous or identical to a genomic sequence of the primary T cell that is 5′ to the break point induced by the genomic editing composition and the 3′ genomic sequence is homologous or identical to a genomic sequence of the primary T cell that is 3′ to the break point induced by the genomic editing composition. In certain embodiments, the 5′ genomic sequence and/or the 3′ genomic sequence comprises at least 50 bp, 100 bp, at least 200 bp, at least 300 bp, at least 400 bp, at least 500 bp, at least 600 bp, at least 700 bp, at least 800 bp, at least 900 bp, at least 1000 bp, at least 1100 bp, at least 1200 bp, at least 1300 bp, at least 1400, or at least 1500 bp, at least 1600 bp, at least 1700 bp, at least 1800 bp, at least 1900 bp, at least 2000 bp in length or any length of base pairs (bp) in between, inclusive of the end points. In certain embodiments of the introduction step comprising a homologous recombination, the genomic editing composition and donor sequence composition are contacted with the genomic sequence simultaneously or sequentially. In certain embodiments of the introduction step comprising a homologous recombination, the genomic editing composition and donor sequence composition are contacted with the genomic sequence sequentially, and the genomic editing composition is provided first. In certain embodiments of the introduction step comprising a homologous recombination, the genomic editing composition comprises a sequence encoding a DNA binding domain and a sequence encoding a nuclease domain. In certain embodiments of the introduction step comprising a homologous recombination, the genomic editing composition comprises a DNA binding domain and a nuclease domain. In certain embodiments of the genomic editing composition, the DNA binding domain comprises a guide RNA (gRNA). In certain embodiments of the genomic editing composition, the DNA binding domain comprises a DNA-binding domain of a TALEN. In certain embodiments of the genomic editing composition, the DNA binding domain comprises a DNA-binding domain of a ZFN. In certain embodiments of the genomic editing composition, the nuclease domain comprises a Cas9 nuclease or a sequence thereof. In certain embodiments of the genomic editing composition, the nuclease domain comprises an inactive Cas9 (SEQ ID NO: 17009, comprising a substitution of a Alanine (A) for Aspartic Acid (D) at position 10 (D10A) and a substitution of Alanine (A) for Histidine (H) at position 840 (H840A)). In certain embodiments of the genomic editing composition, the nuclease domain comprises a short and inactive Cas9 (SEQ ID NO: 17008, comprising a substitution of an Alanine (A) for an Aspartic Acid (D) at position 10 (D10A) and a substitution of an Alanine (A) for an Asparagine (N) at position 540 (N540A)). In certain embodiments of the genomic editing composition, the nuclease domain comprises or further comprises a type I1S endonuclease. In certain embodiments of the genomic editing composition, the type IIS endonuclease comprises AciI, MnlI, AlwI, BbvI, BccI, BceAI, BsmAI, BsmFI, BspCNI, BsrI, BtsCI, HgaI, HphI, HpyAV, MbolI, MylI, PleI, SfaNI, AcuI, BciVI, BfuAI, BmgBI, BmrI, BpmI, BpuEI, BsaI, BseRI, BsgI, BsmI, BspMI, BsrBI, BsrBI, BsrDI, BtgZI, BtsI, EarI, EciI, MmeI, NmeAIII, BbvCI, Bpu10I, BspQI, SapI, BaeI, BsaXI, CspCI, BfiI, MboII, Acc361, FokI or Clo051. In certain embodiments, the type IIS endonuclease comprises Clo051. In certain embodiments of the genomic editing composition, the nuclease domain comprises or further comprises a TALEN or a nuclease domain thereof. In certain embodiments of the genomic editing composition, the nuclease domain comprises or further comprises a ZFN or a nuclease domain thereof. In certain embodiments of the introduction step comprising a homologous recombination, the genomic editing composition induces a break in a genomic sequence and the donor sequence composition is inserted using the endogenous DNA repair mechanisms of the primary T cell. In certain embodiments of the introduction step comprising a homologous recombination, the insertion of the donor sequence composition eliminates a DNA binding site of the genomic editing composition, thereby preventing further activity of the genomic editing composition.

In certain embodiments of the methods of homologous recombination of the disclosure, the nuclease domain of a genomic editing composition or construct is capable of introducing a break at a defined location in a genomic sequence of the primary human T cell, and, furthermore, may comprise, consist essentially of or consist of, a homodimer or a heterodimer. In certain embodiments, the nuclease is an endonuclease. Effector molecules, including those effector molecules comprising a homodimer or a heterodimer, may comprise, consist essentially of or consist of, a Cas9, a Cas9 nuclease domain or a fragment thereof. In certain embodiments, the Cas9 is a catalytically inactive or “inactivated” Cas9 (dCas9). In certain embodiments, the Cas9 is a catalytically inactive or “inactivated” nuclease domain of Cas9. In certain embodiments, the dCas9 is encoded by a shorter sequence that is derived from a full length, catalytically inactivated, Cas9, referred to herein as a “small” dCas9 or dSaCas9.

In certain embodiments, the inactivated, small, Cas9 (dSaCas9) operatively-linked to an active nuclease. In certain embodiments, the disclosure provides a fusion protein comprising, consisting essentially of or consisting of a DNA binding domain and molecule nuclease, wherein the nuclease comprises a small, inactivated Cas9 (dSaCas9). In certain embodiments, the dSaCas9 of the disclosure comprises the mutations D10A and N580A (underlined and bolded) which inactivate the catalytic site. In certain embodiments, the dSaCas9 of the disclosure comprises the amino acid sequence of:

(SEQ ID NO: 17008)

1
MRPNYILGLA IGITSVGYGI IDYETRDVID AGVRLFKEAN

VENNEGRRSK RGARRLKRRR

61
RHRIQRVKKL LFDYNLLTDH SELSGINPYE ARVKGLSQKL

SEEEFSAALL HLAKRRGVHN

121
VNEVEEDTGN ELSTKEQISR NSKALEEKYV AELQLERLKK

DGEVRGSINR FKTSDYVKEA

181
KQLLKVQKAY HQLDQSFIDT YIDIIETRRT YYEGPGEGSP

FGWKDIKEWY EMLNIGHCTYF

241
PEELRSVKYA YNADLYNALN DLNNLVITRD ENEKLEYYEK

FQIIENVFKQ KKKPTLKQIA

301
KEILVNEEDI KGYRVTSTGK PEFTNLKVYE DIKDITARKE

IIENAELLDQ IAKILTIYQS

361
SEDIQEELTN LNSELTQEEI EQISNLKGYT GTHNLSLKAI

NLILDELWHT NDNQIAIFNR

421
LKLVPKKVDL SQQKEIPTTL VDDFILSPVV KRSFIQSIKV

INAIIKKYGL PNDIIIELAR

481
EKNSKDAQKM INEMQKRNRQ TNERIEEIIR TTGKENAKYL

IEKIKLHDMQ EGKCLYSLEA

541
IPLEDLLNNP ENYEVDHIIP RSVSEDNSEN NKVLVKQEEA

SKKGNRTPFQ YLSSSDSKIS

601
YETFKKHILN LAKGKGRISK TKKEYLLEER DINRFSVQKD

FINRHLVDTR YATRGLMNLL

661
RSYFRVNNLD VKVKSINGGF TSFLRRKWKF KKERNKGYKE

HAEDALIIAN ADFIFKEWKK

721
LDKAKKVMEN QMFEEKQAES MPEIETEQEY KEIFITPHQI

KHIKDFKDYK YSHRVDKKPN

781
RELINDTLYS TRKDDKGNTL IVNNLNGLYD KDNDKLKKLI

NKSPEKLLMY HHDPQTYQKL

841
KLIMEQYGDE KNPLYKYYEE TGNYLTKYSK KDNGPVIKKI

KYYGNKLNAH LDITDDYPNS

901
RNKVVKLSLK PYRFDVYLDN GVYKFVTVKN LDVIKKENYY

EVNSKCYEEA KKLKKISKA

961
EFIASFYNND LIKINGELYR VIGVNNULLN RIEVNMIDIT

YREYIENMND KRPPRIIKTI

1021
ASKTQSIKKY STDILGNLYE VKSKKHPQII KKG.

(SEQ ID NO: 17009)

1
XDKKYSIGLA IGTNSVGWAV ITDEYKVPSK KFKVLGNTDR

HSIKKNLIGA LLFDSGETAE

61
ATRLKRTARR RYTRRKNRIC YLQEIFSNEM AKVDDSFFER

LEESFLVEED KKHERHPIFG

121
NIVDEVAYHE KYPTIYHLRK KLVDSTDKAD LRLIYLALAH

MIKFRGHFLI EGDLNPDNSD

181
VDKLFIQLVQ TYNQLFEENP INASGVDAKA ILSARLSKSR

RLENLIAQLP GEKKNGLFGN

241
LIALSLGLTP NFKSNFDLAE DAKLQLSKDT YDDDLDNLLA

QIGDQYADLF LAAKNLSDAI

301
LLSDILRVNT EITKAPLSAS MIKRYDEHHQ DLTLLKALVR

QQPLEKYKEI FFDQSKNGYA

361
GYIDGGASQE EFYKFIKPIL EKMDGTEELL VKLNREDLLR

KQRTFDNGSI PHQIHLGELH

421
AILRRQEDFY PFLKDNREKI EKILTFRIPY YVGPLARGNS

RFAWMTRKSE ETITPWNFEE

481
VVDKGASAQS FIERMTNFDK NLPNEKVLPK HSLLYEYFTV

YNELTKVKYV TEGMRKPAFL

541
SGEQKKAIVD LLFKTNRKVT VKQLKEDYFK KIECFDSVEI

SGVEDRFNAS LGTYHDLLKI

601
IKDKDFLDNE ENEDILEDIV LTLTLFEDRE MIEERLKTYA

HLFDDKVMKQ LKRRRYTGWG

661
RLSRKLINGI RDKQSGKTIL DFLKSDGEAN RNEMQLIHDD

SLTFKEDIQK AQVSGOGDSL

721
HEHIANLAGS PAIKKGILQT VKVVDELVKV MGRHKPENIV

IEMARENQTT QKGQKNSRER

781
MKRIEEGIKE LGSQILKEHP VENTQLQNEK LYLYYLONGR

DMYVDQELDI NRLSDYDVDA

841
IVPQSFLKDD SIDNKVLTRS DKNRGKSDNV PSEEVVKKMK

NYWRQLLNAK LITQRKFDNL

901
TKAERGGLSE LDKAGFIKRQ LVETRQITKH VAQILDSRMN

TKYDENDKLI REVKVITLKS

961
KLVSDFRKDF QFYKVREINN YHHAHDAYLN AVVGTALIKK

YPKLESEFVY GDYKVYDVRK

1021
MIAKSEQEIG KATAKYFFYS NIMNFFKTEI TLANGEIRKR

PLIETNGETG EIVWDKGRDF

1081
ATVRKVLSMP QVNIVKKTEV QTGGFSKESI LPKPNSDKLI

ARKKDWDPKK YGGFDSPTVA

1141
YSVLVVAKVE KGKSKKLKSV KELLGITIME RSSFEKNPID

FLEAKGYKEV KKDLIIKLPK

1201
YSLFELENGR KRMLASAGEL QKGNELALPS KYVNFLYLAS

HYEKLKGSPE DNEQKQLFVE

1261
OHKHIIDEII EOISEFSKRV ILADANLDKV LSAYNKERDK

PIREQAENII HLFTLTNLGA

1321
PAAFKYFDTT IDRKRYTSTK EVLDATLIHQ SITGLYETRI

DLSQLGGD.

In certain embodiments of the disclosure, the nuclease domain may comprise, consist essentially of or consist of a dCas9 or a dSaCas9 and a type IIS endonuclease. In certain embodiments of the disclosure, the nuclease domain may comprise, consist essentially of or consist of a dSaCas9 and a type 11S endonuclease, including, but not limited to, AciI, Mn1I, AlwI, BbvI, BccI, BceAI, BsmAI, BsmFI, BspCNI, BsrI, BtsCI, HgaI, HphI, HpyAV, Mbo1I, My1I, PleI, SfaNI, AcuI, BciVI, BMuAI, BmgBI, BmrI, BpmI, BpuEI, BsaI, BseRI, BsgI, BsmI, BspMI, BsrBI, BsrBI, BsrDI, BtgZI, BtsI, EarI, EciI, MmeI, NmeAIII, BbvCI, Bpu10I, BspQI, SapI, BaeI, BsaXI, CspCI, BfiI, MboII, Acc36I, FokI or Clo051. In certain embodiments of the disclosure, the nuclease domain may comprise, consist essentially of or consist of a dSaCas9 and Clo051. An exemplary Clo51 nuclease domain may comprise, consist essentially of or consist of, the amino acid sequence of:

(SEQ ID NO: 17010)

EGIKSNISLLKDELRGQISHISHEYLSLIDLAFDSKQNRLFEMKVLELLVN

EYGFKGRHLGGSRKPDGIVYSTTLEDNFGIIVDTKAYSEGYSLPISQADEM

ERYVRENSNRDEEVNPNKWWENFSEEVKKYYFVFISGSFKGKFEEQLRRLS

MTTGVNGSAVNVVNLLLGAEKIRSGEMTIEELERAMFNNSEFILKY.

An exemplary dCas9-Clo051 nuclease domain may comprise, consist essentially of or consist of, the amino acid sequence of (Clo051 sequence underlined, linker bold italics, dCas9 sequence in italics):

(SEQ ID NO: 17011)

MAPKKKRKVEGISKSNISLLKDELRGQISHISHEYLSLIDLAFDSKQNRLF

EMKVLELLVNEYGFKGRHLGGSRKPDGIVYSTTLEDNFGIIVDTKAYSEGY

SLPISQADEMERYVRENSNRDEEVNPNKWWENFSEEVKKYYFVFISGSFKG

KFEEQLRRLSMTTGVNGSAVNVVNLLLGAEKIRSGEMTIEELERAMFNNSE

FILKY
custom-character

DKKYSIGLAIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHS

IKKNLIGALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKV

DDSFFHRLEESFLVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVD

STDKADLRLIYLALAHMIKFRGHFLIEGDLNPDNSDVDFLFIQLVQTYNQL

FEENPINASGVDAKAILSARLSKSRRLENLIAQLPGEKKNGLFGNLIALSL

GLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQIGDQYADLFLAAKNLS

DAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVRQQLPEKYK

EIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLL

RKQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPY

YVGPLARGNSRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKN

LPNEKVLPKHSLLYEYFTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLL

FKTNRKVTVKQLKEDYFKKIECFDSVEISGVEDRFNASLGTYHDLLKIIKD

KDFLDNEENEDILEDIVLTLTLFEDREMIEERLKTYAHLFDDKVMKQLKRR

RYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFMQLIHDDSLTFK

EDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGRHKP

ENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQ

NEKLYLYYLQNGRDMYVDQELDINRLSDYDVDAIVPQSFLKDDSIDNKVLT

RSDKNRGKSDNVFSEEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLS

ELDKAGFIKRQLVETRQITKHVAQILDSRMNTKYDENDKLIREVKVITLKS

KLVSDFRKDFQFYKVREINNYHHAHDAYLNAVVGTALIKKYPKLESEFVYG

DYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKTEITLANGEIRKRPL

IETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSKESILPK

RNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELL

GITIMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLA

SAGELQKGNELALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYL

DEIIEQISEFSKRVILADANLDKVLSAYNKHRDKPIREQAENIIHLFTLTN

LGAPAAFKYFDTTIDRKRYTSTKEVLDATLIHQSITGLYETRIDLSQLGGD

GSPKKKRKVSS.

In certain embodiments, the nuclease capable of introducing a break at a defined location in the genomic DNA of the primary human T cell may comprise, consist essentially of or consist of, a homodimer or a heterodimer. Nuclease domains of the genomic editing compositions or constructs of the disclosure may comprise, consist essentially of or consist of a nuclease domain isolated, derived or recombined from a transcription-activator-like effector nuclease (TALEN). TALENs are transcription factors with programmable DNA binding domains that provide a means to create designer proteins that bind to pre-determined DNA sequences or individual nucleic acids. Modular DNA binding domains have been identified in transcriptional activator-like (TAL) proteins, or, more specifically, transcriptional activator-like effector nucleases (TALENs), thereby allowing for the de novo creation of synthetic transcription factors that bind to DNA sequences of interest and, if desirable, also allowing a second domain present on the protein or polypeptide to perform an activity related to DNA. TAL proteins have been derived from the organisms Xanthomonas and Ralstonia.

In certain embodiments of the disclosure, the nuclease domain of the genomic editing composition or construct may comprise, consist essentially of or consist of a nuclease domain isolated, derived or recombined from a TALEN and a type IIS endonuclease. In certain embodiments of the disclosure, the type IIS endonuclease may comprise, consist essentially of or consist of AciI, Mn1I, AlwI, BbvI, BccI, BceAI, BsmAI, BsmFI, BspCNI, BsrI, BtsCI, HgaI, HphI, HpyAV, Mbo1I, My1I, PleI, SfaNI, AcuI, BciVI, BfuAI, BmgBI, BmrI, BpmI, BpuEI, BsaI, BseRI, BsgI, BsmI, BspMI, BsrBI, BsrBI, BsrDI, BtgZI, BtsI, EarI, EciI, MmeI, NmeAIII, BbvCI, Bpu10I, BspQI, SapI, BaeI, BsaXI, CspCI, BfiI, MboII, Acc36I, FokI or Clo051. In certain embodiments of the disclosure, the type IIS endonuclease may comprise, consist essentially of or consist of Clo051 (SEQ ID NO: 17010).

In certain embodiments of the disclosure, the nuclease domain of the genomic editing composition or construct may comprise, consist essentially of or consist of a nuclease domain isolated, derived or recombined from a zinc finger nuclease (ZFN) and a type I1S endonuclease. In certain embodiments of the disclosure, the type IIS endonuclease may comprise, consist essentially of or consist of AciI, Mn1I, AlwI, BbvI, BccI, BceAI, BsmAI, BsmFI, BspCNI, BsrI, BtsCI, HgaI, HphI, HpyAV, Mbo1I, My1I, PleI, SfaNI, AcuI, BciVI, BfuAI, BmgBI, BmrI, BpmI, BpuEI, BsaI, BseRI, BsgI, BsmI, BspMI, BsrBI, BsrBI, BsrDI, BtgZI, BtsI, EarI, EciI, MmeI, NmeAIII, BbvCI, Bpu10I, BspQI, SapI, BaeI, BsaXI, CspCI, BfiI, Mbo1I, Acc36I, FokI or Clo051. In certain embodiments of the disclosure, the type IIS endonuclease may comprise, consist essentially of or consist of Clo051 (SEQ ID NO: 17010).

In certain embodiments of the genomic editing compositions or constructs of the disclosure, the DNA binding domain and the nuclease domain may be covalently linked. For example, a fusion protein may comprise the DNA binding domain and the nuclease domain. In certain embodiments of the genomic editing compositions or constructs of the disclosure, the DNA binding domain and the nuclease domain may be operably linked through a non-covalent linkage.

Non-Transposition Based Methods of Modification

In some embodiments of the methods of the disclosure, a modified HSC or modified HSC descendent cell of the disclosure may be produced by introducing a transgene into an HSC or an HSC descendent cell of the disclosure. The introducing step may comprise delivery of a nucleic acid sequence and/or a genomic editing construct via a non-transposition delivery system.

In some embodiments of the methods of the disclosure, introducing a nucleic acid sequence and/or a genomic editing construct into an HSC or HSC descendent cell ex vivo, in vivo, in vitro or in situ comprises one or more of topical delivery, adsorption, absorption, electroporation, spin-fection, co-culture, transfection, mechanical delivery, sonic delivery, vibrational delivery, magnetofection or by nanoparticle-mediated delivery. In some embodiments of the methods of the disclosure, introducing a nucleic acid sequence and/or a genomic editing construct into an HSC or HSC descendent cell ex vivo, in vivo, in vitro or in situ comprises liposomal transfection, calcium phosphate transfection, fugene transfection, and dendrimer-mediated transfection. In some embodiments of the methods of the disclosure, introducing a nucleic acid sequence and/or a genomic editing construct into an HSC or HSC descendent cell ex vivo, in vivo, in vitro or in situ by mechanical transfection comprises cell squeezing, cell bombardment, or gene gun techniques. In some embodiments of the methods of the disclosure, introducing a nucleic acid sequence and/or a genomic editing construct into an HSC or HSC descendent cell ex vivo, in vivo, in vitro or in situ by nanoparticle-mediated transfection comprises liposomal delivery, delivery by micelles, and delivery by polymerosomes.

In some embodiments of the methods of the disclosure, introducing a nucleic acid sequence and/or a genomic editing construct into an HSC or HSC descendent cell ex vivo, in vivo, in vitro or in situ comprises a non-viral vector. In some embodiments, the non-viral vector comprises a nucleic acid. In some embodiments, the non-viral vector comprises plasmid DNA, linear double-stranded DNA (dsDNA), linear single-stranded DNA (ssDNA), DoggyBone™ DNA, nanoplasmids, minicircle DNA, single-stranded oligodeoxynucleotides (ssODN), DDNA oligonucleotides, single-stranded mRNA (ssRNA), and double-stranded mRNA (dsRNA). In some embodiments, the non-viral vector comprises a transposon of the disclosure.

In some embodiments of the methods of the disclosure, introducing a nucleic acid sequence and/or a genomic editing construct into an HSC or HSC descendent cell ex vivo, in vivo, in vitro or in situ comprises a viral vector. In some embodiments, the viral vector is a non-integrating non-chromosomal vector. Exemplary non-integrating non-chromosomal vectors include, but are not limited to, adeno-associated virus (AAV), adenovirus, and herpes viruses. In some embodiments, the viral vector is an integrating chromosomal vector. Integrating chromosomal vectors include, but are not limited to, adeno-associated vectors (AAV). Lentiviruses, and gamma-retroviruses.

In some embodiments of the methods of the disclosure, introducing a nucleic acid sequence and/or a genomic editing construct into an HSC or HSC descendent cell ex vivo, in vivo, in vitro or in situ comprises a combination of vectors. Exemplary, non-limiting vector combinations include: viral and non-viral vectors, a plurality of non-viral vectors, or a plurality of viral vectors. Exemplary but non-limiting vectors combinations include: a combination of a DNA-derived and an RNA-derived vector, a combination of an RNA and a reverse transcriptase, a combination of a transposon and a transposase, a combination of a non-viral vector and an endonuclease, and a combination of a viral vector and an endonuclease.

In some embodiments of the methods of the disclosure, genome modification comprising introducing a nucleic acid sequence and/or a genomic editing construct into an HSC or HSC descendent cell ex vivo, in vivo, in vitro or in situ stably integrates a nucleic acid sequence. In some embodiments, the stable chromosomal integration can be a random integration, a site-specific integration, or a biased integration. In some embodiments, the site-specific integration can be non-assisted or assisted. In some embodiments, the assisted site-specific integration is co-delivered with a site-directed nuclease. In some embodiments, the site-directed nuclease comprises a transgene with 5′ and 3′ nucleotide sequence extensions that contain a percentage homology to upstream and downstream regions of the site of genomic integration. In some embodiments, the transgene with homologous nucleotide extensions enable genomic integration by homologous recombination, microhomology-mediated end joining, or nonhomologous end-joining. In some embodiments the site-specific integration occurs at a safe harbor site. Genomic safe harbor sites are able to accommodate the integration of new genetic material in a manner that ensures that the newly inserted genetic elements function reliably (for example, are expressed at a therapeutically effective level of expression) and do not cause deleterious alterations to the host genome that cause a risk to the host organism. Potential genomic safe harbors include, but are not limited to, intronic sequences of the human albumin gene, the adeno-associated virus site 1 (AAVS1), a naturally occurring site of integration of AAV virus on chromosome 19, the site of the chemokine (C-C motif) receptor 5 (CCR5) gene and the site of the human ortholog of the mouse Rosa26 locus.

In some embodiments, the site-specific transgene integration occurs at a site that disrupts expression of a target gene. In some embodiments, disruption of target gene expression occurs by site-specific integration at introns, exons, promoters, genetic elements, enhancers, suppressors, start codons, stop codons, and response elements. In some embodiments, exemplary target genes targeted by site-specific integration include but are not limited to TRAC, TRAB, PDI, any immunosuppressive gene, and genes involved in allo-rejection.

In some embodiments, the site-specific transgene integration occurs at a site that results in enhanced expression of a target gene. In some embodiments, enhancement of target gene expression occurs by site-specific integration at introns, exons, promoters, genetic elements, enhancers, suppressors, start codons, stop codons, and response elements.

In some embodiments of the methods of the disclosure, enzymes may be used to create strand breaks in the host genome to facilitate delivery or integration of the transgene. In some embodiments, enzymes create single-strand breaks. In some embodiments, enzymes create double-strand breaks. In some embodiments, examples of break-inducing enzymes include but are not limited to: transposases, integrases, endonucleases, CRISPR-Cas9, transcription activator-like effector nucleases (TALEN), zinc finger nucleases (ZFN), Cas-CLOVER™, and CPF1. In some embodiments, break-inducing enzymes can be delivered to the cell encoded in DNA, encoded in mRNA, as a protein, as a nucleoprotein complex with a guide RNA (gRNA).

In some embodiments of the methods of the disclosure, the site-specific transgene integration is controlled by a vector-mediated integration site bias. In some embodiments vector-mediated integration site bias is controlled by the chosen lentiviral vector. In some embodiments vector-mediated integration site bias is controlled by the chosen gamma-retroviral vector.

In some embodiments of the methods of the disclosure, the site-specific transgene integration site is a non-stable chromosomal insertion. In some embodiments, the integrated transgene may become silenced, removed, excised, or further modified.

In some embodiments of the methods of the disclosure, the genome modification is a non-stable integration of a transgene. In some embodiments, the non-stable integration can be a transient non-chromosomal integration, a semi-stable non chromosomal integration, a semi-persistent non-chromosomal insertion, or a non-stable chromosomal insertion. In some embodiments, the transient non-chromosomal insertion can be epi-chromosomal or cytoplasmic.

In some embodiments, the transient non-chromosomal insertion of a transgene does not integrate into a chromosome and the modified genetic material is not replicated during cell division.

In some embodiments of the methods of the disclosure, the genome modification is a semi-stable or persistent non-chromosomal integration of a transgene. In some embodiments, a DNA vector encodes a Scaffold/matrix attachment region (S-MAR) module that binds to nuclear matrix proteins for episomal retention of a non-viral vector allowing for autonomous replication in the nucleus of dividing cells.

In some embodiments of the methods of the disclosure, the genome modification is a non-stable chromosomal integration of a transgene. In some embodiments, the integrated transgene may become silenced, removed, excised, or further modified.

In some embodiments of the methods of the disclosure, the modification to the genome by transgene insertion can occur via host cell-directed double-strand breakage repair (homology-directed repair) by homologous recombination (HR), microhomology-mediated end joining (MMEJ), nonhomologous end joining (NHEJ), transposase enzyme-mediated modification, integrase enzyme-mediated modification, endonuclease enzyme-mediated modification, or recombinant enzyme-mediated modification. In some embodiments, the modification to the genome by transgene insertion can occur via CRISPR-Cas9, TALEN, ZFNs, Cas-CLOVER, and cpf1.

Nanoparticle Delivery

Poly(histidine) (i.e., poly(L-histidine)), is a pH-sensitive polymer due to the imidazole ring providing an electron lone pair on the unsaturated nitrogen. That is, poly(histidine) has amphoteric properties through protonation-deprotonation. The various embodiments enable intracellular delivery of gene editing tools by complexing with poly(histidine)-based micelles. In particular, the various embodiments provide triblock copolymers made of a hydrophilic block, a hydrophobic block, and a charged block. In some embodiments, the hydrophilic block may be poly(ethylene oxide) (PEO), and the charged block may be poly(L-histidine). An example tri-block copolymer that may be used in various embodiments is a PEO-b-PLA-b-PHIS, with variable numbers of repeating units in each block varying by design. The gene editing tools may be various molecules that are recognized as capable of modifying, repairing, adding and/or silencing genes in various cells. The correct and efficient repair of double-strand breaks (DSBs) in DNA is critical to maintaining genome stability in cells. Structural damage to DNA may occur randomly and unpredictably in the genome due to any of a number of intracellular factors (e.g., nucleases, reactive oxygen species, etc.) as well as external forces (e.g., ionizing radiation, ultraviolet (UV) radiation, etc.). In particular, correct and efficient repair of double-strand breaks (DSBs) in DNA is critical to maintaining genome stability. Accordingly, cells naturally possess a number of DNA repair mechanisms, which can be leveraged to alter DNA sequences through controlled DSBs at specific sites. Genetic modification tools may therefore be composed of programmable, sequence-specific DNA-binding modules associated with a nonspecific DNA nuclease, introducing DSBs into the genome. For example CRISPR, mostly found in bacteria, are loci containing short direct repeats, and are part of the acquired prokaryotic immune system, conferring resistance to exogenous sequences such as plasmids and phages. RNA-guided endonucleases are programmable genetic engineering tools that are adapted from the CRISPR/CRISPR-associated protein 9 (Cas9) system, which is a component of prokaryotic innate immunity.

Diblock copolymers that may be used as intermediates for making triblock copolymers of the embodiment micelles may have hydrophilic biocompatible poly(ethylene oxide) (PEO), which is chemically synonymous with PEG, coupled to various hydrophobic aliphatic poly(anhydrides), poly(nucleic acids), poly(esters), poly(ortho esters), poly(peptides), poly(phosphazenes) and poly(saccharides), including but not limited by poly(lactide) (PLA), poly(glycolide) (PLGA), poly(lactic-co-glycolic acid) (PLGA), poly(ε-caprolactone) (PCL), and poly (trimethylene carbonate) (PTMC). Polymeric micelles comprised of 100% PEGylated surfaces possess improved in vitro chemical stability, augmented in vivo bioavailablity, and prolonged blood circulatory half-lives. For example, aliphatic polyesters, constituting the polymeric micelle's membrane portions, are degraded by hydrolysis of their ester linkages in physiological conditions such as in the human body. Because of their biodegradable nature, aliphatic polyesters have received a great deal of attention for use as implantable biomaterials in drug delivery devices, bioresorbable sutures, adhesion barriers, and as scaffolds for injury repair via tissue engineering.

In various embodiments, molecules required for gene editing (i.e., gene editing tools) may be delivered to cells using one or more micelle formed from self-assembled triblock copolymers containing poly(histidine). The term “gene editing” as used herein refers to the insertion, deletion or replacement of nucleic acids in genomic DNA so as to add, disrupt or modify the function of the product that is encoded by a gene. Various gene editing systems require, at a minimum, the introduction of a cutting enzyme (e.g., a nuclease or recombinase) that cuts genomic DNA to disrupt or activate gene function.

Further, in gene editing systems that involve inserting new or existing nucleotides/nucleic acids, insertion tools (e.g. DNA template vectors, transposable elements (transposons or retrotransposons) must be delivered to the cell in addition to the cutting enzyme (e.g. a nuclease, recombinase, integrase or transposase). Examples of such insertion tools for a recombinase may include a DNA vector. Other gene editing systems require the delivery of an integrase along with an insertion vector, a transposase along with a transposon/retrotransposon, etc. In some embodiments, an example recombinase that may be used as a cutting enzyme is the CRE recombinase. In various embodiments, example integrases that may be used in insertion tools include viral based enzymes taken from any of a number of viruses including, but not limited to, AAV, gamma retrovirus, and lentivirus. Example transposons/retrotransposons that may be used in insertion tools include, but are not limited to, the piggyBac® transposon, Sleeping Beauty transposon, and the L1 retrotransposon.

In certain embodiments of the methods of the disclosure, the transgene is delivered in vivo. In certain embodiments of the methods of the disclosure, in vivo transgene delivery can occur by: topical delivery, adsorption, absorption, electroporation, spin-fection, co-culture, transfection, mechanical delivery, sonic delivery, vibrational delivery, magnetofection or by nanoparticle-mediated delivery. In certain embodiments of the methods of the disclosure, in vivo transgene delivery by transfection can occur by liposomal transfection, calcium phosphate transfection, fugene transfection, and dendrimer-mediated transfection. In certain embodiments of the methods of the disclosure, in vivo mechanical transgene delivery can occur by cell squeezing, bombardment, and gene gun. In certain embodiments of the methods of the disclosure, in vivo nanoparticle-mediated transgene delivery can occur by liposomal delivery, delivery by micelles, and delivery by polymerosomes. In various embodiments, nucleases that may be used as cutting enzymes include, but are not limited to. Cas9, transcription activator-like effector nucleases (TALENs) and zinc finger nucleases.

In various embodiments, the gene editing systems described herein, particularly proteins and/or nucleic acids, may be complexed with nanoparticles that are poly(histidine)-based micelles. In particular, at certain pHs, poly(histidine)-containing triblock copolymers may assemble into a micelle with positively charged poly(histidine) units on the surface, thereby enabling complexing with the negatively-charged gene editing molecule(s). Using these nanoparticles to bind and release proteins and/or nucleic acids in a pH-dependent manner may provide an efficient and selective mechanism to perform a desired gene modification. In particular, this micelle-based delivery system provides substantial flexibility with respect to the charged materials, as well as a large payload capacity, and targeted release of the nanoparticle payload. In one example, site-specific cleavage of the double stranded DNA may be enabled by delivery of a nuclease using the poly(histidine)-based micelles.

The various embodiments enable intracellular delivery of gene editing tools by complexing with poly(histidine)-based micelles. In particular, the various embodiments provide triblock copolymers made of a hydrophilic block, a hydrophobic block, and a charged block. In some embodiments, the hydrophilic block may be poly(ethylene oxide) (PEO), and the charged block may be poly(L-histidine). An example tri-block copolymer that may be used in various embodiments is a PEO-b-PLA-b-PHIS, with variable numbers of repeating units in each block varying by design. Without wishing to be bound by a particular theory, it is believed that believed that in the micelles that are formed by the various embodiment triblock copolymers, the hydrophobic blocks aggregate to form a core, leaving the hydrophilic blocks and poly(histidine) blocks on the ends to form one or more surrounding layer.

In certain embodiments of the methods of the disclosure, non-viral vectors are used for transgene delivery. In certain embodiments, the non-viral vector is a nucleic acid. In certain embodiments, the nucleic acid non-viral vector is plasmid DNA, linear double-stranded DNA (dsDNA), linear single-stranded DNA (ssDNA), DoggyBone™ DNA, nanoplasmids, minicircle DNA, single-stranded oligodeoxynucleotides (ssODN), DDNA oligonucleotides, single-stranded mRNA (ssRNA), and double-stranded mRNA (dsRNA). In certain embodiments, the non-viral vector is a transposon. In certain embodiments, the transposon is piggyBac®.

In certain embodiments of the methods of the disclosure, transgene delivery can occur via viral vector. In certain embodiments, the viral vector is a non-integrating non-chromosomal vectors. Non-integrating non-chromosomal vectors can include adeno-associated virus (AAV), adenovirus, and herpes viruses. In certain embodiments, the viral vector is an integrating chromosomal vectors. Integrating chromosomal vectors can include adeno-associated vectors (AAV), Lentiviruses, and gamma-retroviruses.

In certain embodiments of the methods of the disclosure, transgene delivery can occur by a combination of vectors. Exemplary but non-limiting vector combinations can include: viral plus non-viral vectors, more than one non-viral vector, or more than one viral vector. Exemplary but non-limiting vectors combinations can include: DNA-derived plus RNA-derived vectors, RNA plus reverse transcriptase, a transposon and a transposase, a non-viral vectors plus an endonuclease, and a viral vector plus an endonuclease.

In certain embodiments of the methods of the disclosure, the genome modification can be a stable integration of a transgene, a transient integration of a transgene, a site-specific integration of a transgene, or a biased integration of a transgene.

In certain embodiments of the methods of the disclosure, the genome modification can be a stable chromosomal integration of a transgene. In certain embodiments, the stable chromosomal integration can be a random integration, a site-specific integration, or a biased integration. In certain embodiments, the site-specific integration can be non-assisted or assisted. In certain embodiments, the assisted site-specific integration is co-delivered with a site-directed nuclease. In certain embodiments, the site-directed nuclease comprises a transgene with 5′ and 3′ nucleotide sequence extensions that contain homology to upstream and downstream regions of the site of genomic integration. In certain embodiments, the transgene with homologous nucleotide extensions enable genomic integration by homologous recombination, microhomology-mediated end joining, or nonhomologous end-joining. In certain embodiments the site-specific integration occurs at a safe harbor site. Genomic safe harbor sites are able to accommodate the integration of new genetic material in a manner that ensures that the newly inserted genetic elements function reliably (for example, are expressed at a therapeutically effective level of expression) and do not cause deleterious alterations to the host genome that cause a risk to the host organism. Potential genomic safe harbors include, but are not limited to, intronic sequences of the human albumin gene, the adeno-associated virus site 1 (AAVS1), a naturally occurring site of integration of AAV virus on chromosome 19, the site of the chemokine (C-C motif) receptor 5 (CCR5) gene and the site of the human ortholog of the mouse Rosa26 locus.

In certain embodiments, the site-specific transgene integration occurs at a site that disrupts expression of a target gene. In certain embodiments, disruption of target gene expression occurs by site-specific integration at introns, exons, promoters, genetic elements, enhancers, suppressors, start codons, stop codons, and response elements. In certain embodiments, exemplary target genes targeted by site-specific integration include but are not limited to TRAC, TRAB, PDI, any immunosuppressive gene, and genes involved in allo-rejection.

In certain embodiments, the site-specific transgene integration occurs at a site that results in enhanced expression of a target gene. In certain embodiments, enhancement of target gene expression occurs by site-specific integration at introns, exons, promoters, genetic elements, enhancers, suppressors, start codons, stop codons, and response elements.

In certain embodiments of the methods of the disclosure, enzymes may be used to create strand breaks in the host genome to facilitate delivery or integration of the transgene. In certain embodiments, enzymes create single-strand breaks. In certain embodiments, enzymes create double-strand breaks. In certain embodiments, examples of break-inducing enzymes include but are not limited to: transposases, integrases, endonucleases, CRISPR-Cas9, transcription activator-like effector nucleases (TALEN), zinc finger nucleases (ZFN), Cas-CLOVER™, and cpf1. In certain embodiments, break-inducing enzymes can be delivered to the cell encoded in DNA, encoded in mRNA, as a protein, as a nucleoprotein complex with a guide RNA (gRNA).

In certain embodiments of the methods of the disclosure, the site-specific transgene integration is controlled by a vector-mediated integration site bias. In certain embodiments vector-mediated integration site bias is controlled by the chosen lentiviral vector. In certain embodiments vector-mediated integration site bias is controlled by the chosen gamma-retroviral vector.

In certain embodiments of the methods of the disclosure, the site-specific transgene integration site is a non-stable chromosomal insertion. In certain embodiments, the integrated transgene may become silenced, removed, excised, or further modified. In certain embodiments of the methods of the disclosure, the genome modification is a non-stable integration of a transgene. In certain embodiments, the non-stable integration can be a transient non-chromosomal integration, a semi-stable non chromosomal integration, a semi-persistent non-chromosomal insertion, or a non-stable chromosomal insertion. In certain embodiments, the transient non-chromosomal insertion can be epi-chromosomal or cytoplasmic. In certain embodiments, the transient non-chromosomal insertion of a transgene does not integrate into a chromosome and the modified genetic material is not replicated during cell division.

In certain embodiments of the methods of the disclosure, the genome modification is a semi-stable or persistent non-chromosomal integration of a transgene. In certain embodiments, a DNA vector encodes a Scaffold/matrix attachment region (S-MAR) module that binds to nuclear matrix proteins for episomal retention of a non-viral vector allowing for autonomous replication in the nucleus of dividing cells.

In certain embodiments of the methods of the disclosure, the genome modification is a non-stable chromosomal integration of a transgene. In certain embodiments, the integrated transgene may become silenced, removed, excised, or further modified.

In certain embodiments of the methods of the disclosure, the modification to the genome by transgene insertion can occur via host cell-directed double-strand breakage repair (homology-directed repair) by homologous recombination (HR), microhomology-mediated end joining (MMEJ), nonhomologous end joining (NHEJ), transposase enzyme-mediated modification, integrase enzyme-mediated modification, endonuclease enzyme-mediated modification, or recombinant enzyme-mediated modification. In certain embodiments, the modification to the genome by transgene insertion can occur via CRISPR-Cas9, TALEN, ZFNs, Cas-CLOVER, and cpf1.

In certain embodiments of the methods of the disclosure, a cell with an in vivo or ex vivo genomic modification can be a germline cell or a somatic cell. In certain embodiments the modified cell can be a human, non-human, mammalian, rat, mouse, or dog cell. In certain embodiments, the modified cell can be differentiated, undifferentiated, or immortalized. In certain embodiments, the modified undifferentiated cell can be a stem cell. In certain embodiments, the modified cell can be differentiated, undifferentiated, or immortalized. In certain embodiments, the modified undifferentiated cell can be an induced pluripotent stem cell. In certain embodiments, the modified cell can be a T cell, a hematopoietic stem cell, a natural killer cell, a macrophage, a dendritic cell, a monocyte, a megakaryocyte, or an osteoclast. In certain embodiments, the modified cell can be modified while the cell is quiescent, in an activated state, resting, in interphase, in prophase, in metaphase, in anaphase, or in telophase. In certain embodiments, the modified cell can be fresh, cryopreserved, bulk, sorted into sub-populations, from whole blood, from leukapheresis, or from an immortalized cell line.

Other Embodiments

While particular embodiments of the disclosure have been illustrated and described, various other changes and modifications can be made without departing from the spirit and scope of the disclosure. The scope of the appended claims includes all such changes and modifications that are within the scope of this disclosure.

Number	Date	Country
62815880	Mar 2019	US
62815334	Mar 2019	US
62744073	Oct 2018	US
62727498	Sep 2018	US

ALLOGENEIC CELL COMPOSITIONS AND METHODS OF USE

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