Patent Application
20250011720
The present disclosure relates, in part, to a method of stem cell generation, e.g., using an enzyme capable of targeted genomic integration, such as a mobile element enzyme.
The contents of the text file submitted electronically herewith are incorporated herein by reference in their entirety: A computer readable format copy of the Sequence Listing (filename: “Sequence_Listing_SAL-008PC/126933-5008.xml”; date recorded: Nov. 4, 2022; file size: 962,560 bytes).
Stem cells are the precursor cells from which all cell types emerge. Recent advances in controlling cellular differentiation processes allow a stem cell to be converted into specialized cells such as nerve cells, blood vessel cells and cardiac muscle, or cells such as fibroblasts or PBMCs can be reprogrammed to stem cells (iPSCs). Stem-cell replacement therapy offers the ability to replace damaged or mutant cells but combining it with gene therapy has the potential to correct pathological genetic mutations and incorporate those corrections into new cell populations in the body.
Hematopoietic stem cell transplantation (HSCT) is a globally accepted practice for the treatment of malignant and non-malignant disorders of the blood and immune systems. Almost 90% of HSCTs worldwide are done for the treatment of hematological malignancies, including leukemia, lymphoma and myeloma. For these cases, patients initially receive a chemotherapy regimen to destroy tumor cells, but since the treatment targets all rapidly dividing cells in the body, it also depletes the HSC compartment in the bone marrow. The HSCT is thus aimed at replenishing the bone marrow with stem cells, which engraft and reconstitute the immune system with functional hematopoietic lineages. For non-malignant conditions, primarily rare inherited diseases of the blood and immune systems, the rationale for HSCT is to provide the patient with a hematopoietic lineage that replaces or compensates for the underlying genetic deficiency. Allogeneic HSCT, i.e., transplantation of HSCs harvested from a healthy donor, is essentially the only option for cure of these disorders. This, however, comes with notable limitations and safety concerns, including the need for a genetically-matched donor (which may not be available for up to 70% of cases), graft rejection, delayed immune reconstitution, graft-versus-host disease and a significant rate of mortality. Autologous transplantation of the patient's own HSCs, which have been gene-modified to correct for the underlying genetic cause of the disease, would thus be the preferred form of treatment for these patients.
Accordingly, there is a need for improved approach to development of stem cells.
In aspects, there is provided a method of making an engineered stem cell, the method comprising: obtaining a stem cell from a biological sample; and transfecting the stem cell with a first nucleic acid encoding an enzyme capable of targeted genomic integration, wherein the first nucleic acid is RNA, and a second, non-viral nucleic acid encoding a donor DNA comprising a transgene and flanked by ends recognized by the enzyme, to thereby create a transfected stem cell comprising the transgene in a certain genomic locus and/or site and being able to express the transgene.
In aspects, there is provided a method of making an engineered stem cell, the method comprising: obtaining a somatic cell from a biological sample; transfecting the somatic cell with a first nucleic acid encoding an enzyme capable of targeted genomic integration, wherein the first nucleic acid is RNA, and a second, non-viral nucleic acid encoding a donor DNA comprising a transgene and flanked by ends recognized by the enzyme, to thereby create a transfected somatic cell; and reprogramming the transfected somatic cell to produce a pluripotent stem cell comprising the transgene in a certain genomic locus and/or site.
In embodiments, the enzyme capable of performing targeted genomic integration is a recombinase, e.g., an integrase or a mobile element enzyme. In embodiments, the enzyme is a mobile element enzyme, e.g., derived from, or an engineered version of a mobile element enzyme of Bombyx mori, Xenopus tropicalis, Trichoplusia ni, Myotis lucifugus, Rhinolophus ferrumequinum, Rousettus aegyptiacus, Phyllostomus discolor, Myotis myotis, Pteropus vampyrus, Pipistrellus kuhlii, Molossus molossus, Pan troglodytes, or Homo sapiens, e.g., one or more of the Tn1, Tn2, Tn3, Tn5, Tn7, Tn9, Tn10, Tn552, Tn903, Tn1000/Gamma-delta, Tn/O, tnsA, tnsB, tnsC, tniQ, IS10, ISS, IS911, Minos, Sleeping beauty, piggyBac, Tol2, Mos1, Himar1, Hermes, Tol2, Minos, Tel, P-element, MuA, Ty1, Chapaev, transib, Tc1/mariner, or Tc3 donor DNA system, or biologically active fragments variants thereof, inclusive of hyperactive variants. In embodiments, the mobile element enzyme has the amino acid sequence of SEQ ID NO: 1, or a variant thereof, e.g., having an amino acid other than serine at the position corresponding to position 2 of SEQ ID NO: 1 (e.g., selected from G, A, V, L, I and P, optionally A), not having additional residues at the C terminus relative to SEQ ID NO: 1, and/or having one or more mutations which confer hyperactivity (e.g., of TABLE 1) and/or having one or more mutations which modulation integration (e.g., of TABLE 2A or TABLE 2B). In embodiments, the mobile element enzyme having at least about 90% identity to the amino acid sequence of SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11 or SEQ ID NO: 430, or a variant thereof, e.g. having one or more mutations which confer hyperactivity (e.g., of TABLE 1) and/or having one or more mutations which modulation integration (e.g., of TABLE 2A or TABLE 2B). In embodiments, the mobile element enzyme has gene cleavage activity (Exc+) and/or gene integration activity (Int+). In embodiments, the mobile element enzyme has gene cleavage activity (Exc+) and/or a lack of gene integration activity (Int−).
In embodiments, the enzyme comprises a targeting element, and an enzyme that is capable of inserting the donor DNA comprising a transgene, optionally at a TA dinucleotide site or a TTAA (SEQ ID NO: 440) tetranucleotide site in a genomic safe harbor site (GSHS). In embodiments, the mobile element enzyme is a chimeric mobile element enzyme. In embodiments, the targeting element comprises one or more of a gRNA, optionally associated with a Cas enzyme, which is optionally catalytically inactive, transcription activator-like effector (TALE), catalytically inactive Zinc finger, catalytically inactive transcription factor, nickase, a transcriptional activator, a transcriptional repressor, a recombinase, a DNA methyltransferase, a histone methyltransferase, a paternally expressed gene 10 (PEG10), and a TnsD.
In embodiments, the GSHS is in an open chromatin location in a chromosome. In embodiments, the GSHS is selected from adeno-associated virus site 1 (AAVS1), chemokine (C—C motif) receptor 5 (CCR5) gene, HIV-1 coreceptor, and human Rosa26 locus. In embodiments, the GSHS is located on human chromosome 2, 4, 6, 10, 11, 17, 22, or X. In embodiments, the GSHS is selected from TALC1, TALC2, TALC3, TALC4, TALC5, TALC7, TALC8, AVS1, AVS2, AVS3, ROSA1, ROSA2, TALER1, TALER2, TALER3, TALER4, TALER5, SHCHR2-1, SHCHR2-2, SHCHR2-3, SHCHR2-4, SHCHR4-1, SHCHR4-2, SHCHR4-3, SHCHR6-1, SHCHR6-2, SHCHR6-3, SHCHR6-4, SHCHR10-1, SHCHR10-2, SHCHR10-3, SHCHR10-4, SHCHR10-5, SHCHR11-1, SHCHR11-2, SHCHR11-3, SHCHR17-1, SHCHR17-2, SHCHR17-3, and SHCHR17-4.
In embodiments, the disclosure provides a stem cell generated by a method described herein.
In embodiments, the disclosure provides a method of delivering a stem cell therapy, comprising administering to a patient in need thereof the stem cell generated by a method described herein.
In embodiments, the disclosure provides a method of treating a disease or condition using a stem cell therapy, comprising administering to a patient in need thereof the stem cell generated by a method described herein.
In embodiments, a stem cell for gene therapy is provided, wherein the transfected cell is generated using a stem cell generated by a method described herein.
In embodiments, a method of delivering a cell therapy is provided, comprising administering to a patient in need thereof the stem cell generated using a method in accordance with embodiments of the present disclosure.
The details of the invention are set forth in the accompanying description below. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, illustrative methods and materials are now described. Other features, objects, and advantages of the invention will be apparent from the description and from the claims. In the specification and the appended claims, the singular forms also include the plural unless the context clearly dictates otherwise. Unless defined otherwise, 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 invention belongs.
The present disclosure is based, in part, on the discovery that stem cell generation can be made more efficient with the use of enzymatic transposition.
The disclosure provides, in aspects or embodiments, use of a donor DNA and helper RNA system to generate genetically modified human stem cells (HSCs) for either allogeneic or autologous transplantation. The system, in aspects or embodiments, uses site- and locus-specific genomic targeting to efficiently establish stem cells with a transgene integrated in the same genomic location. These stem cells are stable and durable throughout, e.g., a patient's lifetime. The system is highly efficient compared to current methods, e.g., using lentivirus. The disclosure provides, in aspects or embodiments, uses of a mammal-derived, helper RNA mobile element enzyme and donor DNA system to transfect autologous stem cells or express genes of interest in allogeneic stem cells (e.g., CD34+) to treat human disorders. It also describes transfecting somatic cells such as fibroblasts or peripheral blood mononuclear cells (PBMCs) before reprogramming to produce corrected individual pluripotent stem cells (iPSCs).
In aspects, there is provided a method of making an engineered stem cell, the method comprising: obtaining a stem cell from a biological sample; and transfecting the stem cell with a first nucleic acid encoding an enzyme capable of performing targeted genomic integration, wherein the first nucleic acid is RNA, and a second, non-viral nucleic acid encoding a donor DNA comprising a transgene and flanked by ends recognized by the enzyme, to thereby create a transfected stem cell comprising the transgene in a certain genomic locus and/or site and being able to express the transgene.
In aspects, there is provided a method of making an engineered stem cell, the method comprising: obtaining a somatic cell from a biological sample; transfecting the somatic cell with a first nucleic acid encoding an enzyme capable of performing targeted genomic integration, wherein the first nucleic acid is RNA, and a second, non-viral nucleic acid encoding a donor DNA comprising a transgene and flanked by ends recognized by the enzyme, to thereby create a transfected somatic cell; and reprogramming the transfected somatic cell to produce a pluripotent stem cell comprising the transgene in a certain genomic locus and/or site.
In embodiments, the transfected stem cell or engineered stem cell is an autologous stem cell. In embodiments, the transfected stem cell or engineered stem cell is an allogeneic stem cell. In embodiments, the transfected stem cell or engineered stem cell is a CD34+ cell. In embodiments, the transfected stem cell or engineered stem cell is an induced pluripotent stem cell (iPSC).
In embodiments, the somatic cell is a skin cell, optionally a fibroblast or a keratinocyte. In embodiments, the somatic cell is a peripheral blood mononuclear cell (PBMC).
In embodiments, the transfected stem cell or engineered stem cell is a mesenchymal stem cell.
In embodiments, the biological sample comprises a blood sample or biopsy.
In embodiments, the obtaining of a stem cell from the biological sample comprises administering to the subject a stem cell mobilization agent, optionally a granulocyte colony stimulating factor (G-CSF), recombinant G-CSF, an G-CSF analogue having the function of G-CSF, and/or plerixafor.
In embodiments, the method comprises culturing the transfected stem cell or engineered stem cell in a medium that selectively enhances proliferation of stem cells.
In embodiments, the engineered stem cell is created in about 1 day or about 2 days. In embodiments, the engineered stem cell is created in less than about 2 days, or less than about 3 days, or less than about 7 days, or less than about 14 days. In embodiments, the engineered stem cell is created in about 2 to about 14 days, or about 2 to about 10 days, or about 2 to about 7 days, or about 7 to about 14 days, or about 10 to about 14 days.
In embodiments, the method obviates a use of ex vivo expansion of stem cells.
In embodiments, the method obviates a use of clonal selection of stem cells.
In embodiments, the reprogramming of the transfected somatic cell is performed using one or more reprogramming factors. In embodiments, the one or more reprogramming factors are selected from Oct4, Sox2, Klf4, c-Myc, I-Myc, Tert, Nanog, Lin28, Utf1, Aicda, miR200 micro-RNA, miR302 micro-RNA, miR367 micro-RNA, miR369 micro-RNA and biologically active fragments, analogues, variants and family-members thereof. In embodiments, the one or more reprogramming factors are selected from Sox2 protein, Klf4 protein, c-Myc protein, and Lin28 protein. In embodiments, the reprogramming factor is a fusion protein. In embodiments, the reprogramming the transfected somatic cell comprises contacting the cell with a surface that is contacted with one or more cell-adhesion molecules, wherein the one or more cell-adhesion molecules optionally include at least one element comprising: poly-L-lysine, poly-L-ornithine, RGD peptide, fibronectin, vitronectin, collagen, and laminin or a biologically active fragment, analogue, variant or family-member thereof. In embodiments, the transfected somatic cell is reprogrammed in a low-oxygen environment. In embodiments, the reprogramming the transfected somatic cell is carried out via a series of transfections.
In embodiments, the method comprises culturing the cells in a medium that supports the reprogramming. In embodiments, the method comprises culturing the cells in a medium that does not include feeders.
In embodiments, the method comprises culturing the cells in a medium that does not include an immunosuppressant.
In embodiments, the method comprises culturing the cells in a medium that includes an immunosuppressant, optionally B18R or dexamethasone.
In embodiments, the one or more cell-adhesion molecules is fibronectin or a biologically active fragment thereof, wherein the fibronectin is optionally recombinant. In embodiments, the one or more cell-adhesion molecules is a mixture of fibronectin and vitronectin or biologically active fragments thereof, wherein both the fibronectin and vitronectin are optionally recombinant.
In embodiments, the transfecting of the cell is carried out using electroporation, or calcium phosphate precipitation.
In embodiments, the transfecting of the cell is carried out using a lipid vehicle, optionally N-[1-(2,3-dioleoyloxy)propyl]-N,N,N-trimethylammonium chloride (DOTMA), 1,2-bis(oleoyloxy)-3-3-(trimethylammonia) propane (DOTAP), or 1,2-dioleoyl-3-dimethylammonium-propane (DODAP), dioleoylphosphatidylethanolamine (DOPE), cholesterol, LIPOFECTIN (cationic liposome formulation), LIPOFECTAMINE (cationic liposome formulation), LIPOFECTAMINE 2000 (cationic liposome formulation), LIPOFECTAMINE 3000 (cationic liposome formulation), TRANSFECTAM (cationic liposome formulation), a lipid nanoparticle, or a liposome and combinations thereof.
In embodiments, the transfecting of the cell is carried out using a lipid selected from one or more of the following categories: cationic lipids; anionic lipids; neutral lipids; multi-valent charged lipids; and zwitterionic lipids. In embodiments, a cationic lipid may be used to facilitate a charge-charge interaction with nucleic acids. In embodiments, the lipid is a neutral lipid. In embodiments, the neutral lipid is dioleoylphosphatidylethanolamine (DOPE), 1,2-Dioleoyl-sn-glycero-3-phosphocholine (DOPC), or cholesterol. In embodiments, cholesterol is derived from plant sources. In other embodiments, cholesterol is derived from animal, fungal, bacterial or archaeal sources. In embodiments, the lipid is a cationic lipid. In embodiments, the cationic lipid is N-[1-(2,3-dioleoyloxy)propyl]-N,N,N-trimethylammonium chloride (DOTMA), 1,2-bis(oleoyloxy)-3-3-(trimethylammonia) propane (DOTAP), or 1,2-dioleoyl-3-dimethylammonium-propane (DODAP). In embodiments, one or more of the phospholipids 18:0 PC, 18:1 PC, 18:2 PC, DMPC, DSPE, DOPE, 18:2 PE, DMPE, or a combination thereof are used as lipids. In embodiments, the lipid is DOTMA and DOPE, optionally in a ratio of about 1:1. In embodiments, the lipid is DHDOS and DOPE, optionally in a ratio of about 1:1. In embodiments, the lipid is a commercially available product (e.g., LIPOFECTIN (cationic liposome formulation), LIPOFECTAMINE (cationic liposome formulation), LIPOFECTAMINE 2000 (cationic liposome formulation), LIPOFECTAMINE 3000 (cationic liposome formulation) (Life Technologies)).
In embodiments, the transfecting of the cell is carried out using a cationic vehicle, optionally LIPOFECTIN or TRANSFECTAM.
In embodiments, the transfecting of the cell is carried out using a lipid nanoparticle, or a liposome.
In embodiments, the method is helper virus-free.
In embodiments, the second nucleic acid is included in an expression vector. In embodiments, the expression vector comprises a plasmid. In embodiments, the expression vector includes a neomycin phosphotransferase gene.
In embodiments, the second nucleic acid is DNA, optionally cDNA.
In embodiments, the second nucleic acid has at least one chromatin element, wherein the at least one chromatin element is optionally a Matrix Attachment Region (MAR) element.
Epigenetic regulatory elements can be used to protect a transgene from unwanted epigenetic effects when placed near the transgene on a vector including the transgene. See Ley et al., PloS One vol. 8,4 e62784. 30 Apr. 2013, doi:10.1371/journal.pone.0062784. For example, MARs were shown to increase genomic integration and integration of a transgene while preventing heterochromatin silencing, as exemplified by the human MAR 1-68. See id.; see also Grandjean et al., Nucleic Acids Res. 2011 August; 39(15):e104. MARs can also act as insulators and thereby prevent the activation of neighboring cellular genes. Gaussin et al., Gene Ther. 2012 January; 19(1):15-24. It has been shown that a piggyBac donor DNA containing human MARs in CHO cells mediated efficient and sustained expression from a few transgene copies, using cell populations generated without an antibiotic selection procedure. See Ley et al. (2013).
In embodiments, the cell is further transfected with a third nucleic acid having at least one chromatin element, wherein the at least one chromatin element is optionally a Matrix Attachment Region (MAR) element. MARs are expression enhancing, epigenetic regulator elements which are used to enhance and/or facilitate transgene expression, as described, for example, in PCT/IB2010/002337 (WO2011033375) which is incorporated by reference herein in its entirety. A MAR element can be located in cis or trans to the transgene.
In embodiments, the transgene has a size of 100,000 bases or less, e.g., about 100,000 bases, or about 50,000 bases, or about 30,000 bases, or about 10,000 bases, or about 5,000 bases, or about 10,000 to about 100,000 bases, or about 30,000 to about 100,000 bases, or about 50,000 to about 100,000 bases, or about 10,000 to about 50,000 bases, or about 10,000 to about 30,000 bases, or about 30,000 to about 50,000 bases.
In embodiments, the transgene has a size of about 200,000 bases or less, e.g., about 200,000 bases, or about 10,000 to about 200,000 bases, or about 30,000 to about 200,000 bases, or about 50,000 to about 200,000 bases, or about 100,000 to about 200,000 bases, or about 150,000 to about 200,000 bases.
In embodiments, an enzyme capable of performing targeted genomic integration is any type of an enzyme that cause a transgene to be inserted from one location (e.g., without limitation, donor DNA) to a specific site and/or locus in a subject's genome.
In embodiments, the enzyme capable of performing targeted genomic integration is a recombinase.
In embodiments, the recombinase is an integrase. In embodiments, the enzyme is a mobile element enzyme. In embodiments, the recombinase is an integrase or a mobile element enzyme.
In embodiments, the mobile element enzyme is an engineered mammalian mobile element enzyme. In embodiments, the mobile element enzyme is a mammal-derived, helper RNA mobile element enzyme. Messenger RNA (mRNA) is an effective alternative to DNA as a source of a mobile element enzyme for targeting somatic cells and tissues, given that RNA is a safer alternative to DNA as a source of a mobile element enzyme for somatic gene therapy applications. See, e.g., Wilber et al., Mol. Ther. 13, 625-630 (2006). Successful use of in vitro-transcribed mRNA as a transient source of mobile element enzyme and subsequent transposition in cultured human cells and in live mice was previously reported for Sleeping Beauty mobile element enzyme. See id. It was demonstrated that in vitro-transcribed, UTR-stabilized mobile element enzyme-encoding mRNA can be used as a source of mobile element enzyme for Sleeping Beauty-mediated transposition in cultured somatic cells. Id. Also, Hoerr et al. reported that a specific cytotoxic T cell response and circulating antigen-specific antibodies were detected after administration of in vitro-transcribed, UTR-stabilized, and protamine-condensed bacterial lacZ mRNA into the ear pinna of Balb/C mice. Hoerr et al., Eur. J. Immunol. 2000; 30: 1-7; see also Wilber et al. (2006).
In embodiments, the mobile element enzyme is a mammal-derived, DNA mobile element enzyme. In embodiments, the mobile element enzyme is a chimeric mobile element enzyme.
In embodiments, the enzyme capable of performing targeted genomic integration is a mobile element enzyme, and the mobile element enzyme comprises (a) a targeting element which is or comprises a gene-editing system, and (b) a mobile element enzyme that is capable of inserting the donor DNA comprising a transgene at a TA dinucleotide site or a TTAA (SEQ ID NO: 440) tetranucleotide site in a genomic safe harbor site (GSHS), as described elsewhere herein.
In embodiments, the enzyme is derived from Bombyx mori, Xenopus tropicalis, Trichoplusia ni, Myotis lucifugus, Rhinolophus ferrumequinum, Rousettus aegyptiacus, Phyllostomus discolor, Myotis myotis, Pteropus vampyrus, Pipistrellus kuhlii, Molossus molossus, Pan troglodytes, or Homo sapiens. In embodiments, the enzyme is an engineered version, including but not limited to hyperactive forms, of an enzyme derived from Bombyx mori, Xenopus tropicalis, Trichoplusia ni, Myotis lucifugus, Rhinolophus ferrumequinum, Rousettus aegyptiacus, Phyllostomus discolor, Myotis myotis, Pteropus vampyrus, Pipistrellus kuhlii, Molossus molossus, Pan troglodytes, or Homo sapiens.
In embodiments, the enzyme is a mobile element enzyme derived from Bombyx mori, Xenopus tropicalis, Trichoplusia ni, Myotis lucifugus, Rhinolophus ferrumequinum, Rousettus aegyptiacus, Phyllostomus discolor, Myotis myotis, Pteropus vampyrus, Pipistrellus kuhlii, Molossus molossus, Pan troglodytes, or Homo sapiens. In embodiments, the enzyme is an engineered version, including but not limited to hyperactive forms, of a mobile element enzyme derived from Bombyx mori, Xenopus tropicalis, Trichoplusia ni, Myotis lucifugus, Rhinolophus ferrumequinum, Rousettus aegyptiacus, Phyllostomus discolor, Myotis myotis, Pteropus vampyrus, Pipistrellus kuhlii, Molossus molossus, Pan troglodytes, or Homo sapiens.
In embodiments, the mobile element enzyme is from one or more of the Tn1, Tn2, Tn3, Tn5, Tn7, Tn9, Tn10, Tn552, Tn903, Tn1000/Gamma-delta, Tn/O, tnsA, tnsB, tnsC, tniQ, IS10, ISS, IS911, Minos, Sleeping beauty, piggyBac, Tol2, Mos1, Himar1, Hermes, Tol2, Minos, Tel, P-element, MuA, Ty1, Chapaev, transib, Tc1/mariner, or Tc3 donor DNA system, or biologically active fragments variants thereof, inclusive of hyperactive mutants (e.g., without limitation selected from TABLE 1, or equivalents thereof).
In embodiments, the mobile element enzyme is from a MLT donor DNA system that is based on a cut-and-paste MLT element obtained from the little brown bat (Myotis lucifugus) or other bat mobile element enzymes, such as Rhinolophus ferrumequinum, Rousettus aegyptiacus, Phyllostomus discolor, Myotis myotis, Pipistrellus kuhlii, and Molossus molossus. See Mitra et al., Proc Natl Acad Sci USA. 2013 Jan. 2; 110(1):234-9; Jebb et al., Nature, volume 583, pages 578-584 (2020), which are incorporated by reference herein in their entireties. In embodiments, hyperactive forms of a bat mobile element enzyme are used. The MLT mobile element enzyme has been shown to be capable of transposition in bat, human, and yeast cells. The hyperactive forms of the MLT mobile element enzyme enhance the transposition process. In addition, chimeric MLT mobile element enzymes are capable of site-specific excision without genomic integration.
In embodiments, the mobile element enzyme is a Myotis lucifugus mobile element enzyme (MLT), which is either the wild type, monomer, dimer, tetramer (or another multimer), hyperactive, an Int-mutant, or of any other form.
In embodiments, the MLT mobile element enzyme has an amino acid sequence of SEQ ID NO: 1, or a variant having at least about 80%, at least about 90%, or at least about 93%, or at least about 95%, or at least about 97%, or at least about 98%, or at least about 99% identity thereto, and one or more mutations selected from L573X, E574X, and S2X, wherein X is any amino acid or no amino acid, optionally X is A, G, or a deletion, optionally the mutations are L573del E574del, and S2A). In embodiments, the MLT mobile element enzyme has the nucleotide sequence of SEQ ID NO: 2 (which is a codon-optimized form of MLT), or a nucleotide sequence having at least about 90%, or at least about 93%, or at least about 95%, or at least about 97%, or at least about 98%, or at least about 99% identity thereto.
In embodiments, the MLT mobile element enzyme has an amino acid sequence of SEQ ID NO: 1 or a variant having at least about 80%, at least about 90%, or at least about 93%, or at least about 95%, or at least about 97%, or at least about 98%, or at least about 99% identity thereto and comprises an amino acid other than serine at the position corresponding to position 2 of SEQ ID NO: 1. In embodiments, the amino acid is a non-polar aliphatic amino acid, optionally a non-polar aliphatic amino acid optionally selected from G, A, V, L, I and P, optionally A. In embodiments, the mobile element enzyme does not have additional residues at the C terminus relative to SEQ ID NO: 1.
In embodiments, the MLT mobile element enzyme has a nucleotide sequence of SEQ ID NO: 2 (which is codon-optimized) and an amino acid sequence SEQ ID NO: 1, respectively. In embodiments, the MLT mobile element enzyme has a nucleotide sequence of SEQ ID NO: 2, or a nucleotide sequence having at least about 90%, or at least about 93%, or at least about 95%, or at least about 97%, or at least about 98%, or at least about 99% identity thereto, or a codon-optimized form thereof. In embodiments, the MLT mobile element enzyme has an amino acid sequence SEQ ID NO: 1, or an amino acid sequence having at least about 90%, or at least about 93%, or at least about 95%, or at least about 97%, or at least about 98%, or at least about 99% identity thereto.
In embodiments, the mobile element enzyme can act on an MLT left terminal end, or a sequence having at least about 90%, or at least about 93%, or at least about 95%, or at least about 97%, or at least about 98%, or at least about 99% identity thereto, wherein the nucleotide sequence of the MLT left terminal end (5′ to 3′) is as follows:
In embodiments, the mobile element enzyme can act on an MLT right terminal end, or a sequence having at least about 90%, or at least about 93%, or at least about 95%, or at least about 97%, or at least about 98%, or at least about 99% identity thereto, wherein the nucleotide sequence of the MLT right terminal end (5′ to 3′) is as follows:
In embodiments, the donor DNA is flanked by one or more terminal ends. In embodiments, the donor DNA is or comprises a gene encoding a compete polypeptide. In embodiments, the donor DNA is or comprises a gene which is defective or substantially absent in a disease state.
In embodiments, the enzyme (e.g., without limitation, a mobile element enzyme, e.g., without limitation, MLT mobile element enzyme), inclusive of any described herein has one or more mutations which confer hyperactivity.
In embodiments, the enzyme (e.g., without limitation, a mobile element enzyme, e.g., without limitation, MLT mobile element enzyme) has gene cleavage activity (Exc+) and/or gene integration activity (Int+). In embodiments, the enzyme (e.g., without limitation, a mobile element enzyme, e.g., without limitation, MLT mobile element enzyme) has gene cleavage activity (Exc+) and/or a lack of gene integration activity (Int−).
In embodiments, the mobile element enzyme, e.g., without limitation, MLT mobile element enzyme includes a hyperactive mutation, e.g., about 1, or about 2, or about 3, or about 4, or about 5 hyperactive mutations or combinations thereof. In embodiments, the mobile element enzyme can include any number of any of the hyperactive mutations, or equivalents thereof, described herein.
In embodiments, the MLT mobile element enzyme includes a hyperactive mutation, e.g., about 1, or about 2, or about 3, or about 4, or about 5 hyperactive mutations, or combinations thereof. In embodiments, the mobile element enzyme can include any number of any of the hyperactive mutations, or equivalents thereof, described herein.
In embodiments, the enzyme comprises one or more mutations corresponding to TABLE 1, or positions corresponding thereto, which, without being bound by theory, provides hyperactive mutations. Numbering relative to the amino acid sequence of protein of SEQ ID NO: 1, and nucleic acid sequence of SEQ ID NO: 2.
In embodiments, the MLT mobile element enzyme has one or more amino acid substitutions selected from S8X1, C13X2 and/or N125X3, or positions corresponding thereto, relative to SEQ ID NO: 1, wherein X1 is selected from G, A, V, L, I and P, X2 is selected from K, R, and H, and X3 is selected from K, R, and H, or wherein: X1 is P, X2 is R, and/or X3 is K.
In embodiments, the MLT mobile element enzyme has S8X1, C13X2 and N125X3 substitutions, at positions corresponding to SEQ ID NO: 1, wherein X1 is selected from G, A, V, L, I and P, X2 is selected from K, R, and H, and X3 is selected from K, R, and H, or wherein: X1 is P, X2 is R, and/or X3 is K.
In embodiments, the MLT mobile element enzyme has S8X1 and C13X2 substitutions, at positions corresponding to SEQ ID NO: 1, wherein X1 is selected from G, A, V, L, I and P, X2 is selected from K, R, and H, and X3 is selected from K, R, and H, or wherein: X1 is P, X2 is R, and/or X3 is K.
In embodiments, the MLT mobile element enzyme has S8X1 and N125X3 substitutions, at positions corresponding to SEQ ID NO: 1, wherein X1 is selected from G, A, V, L, I and P, X2 is selected from K, R, and H, and X3 is selected from K, R, and H, or wherein: X1 is P, X2 is R, and/or X3 is K.
In embodiments, the MLT mobile element enzyme has C13X2 and N125X3 substitutions, at positions corresponding to SEQ ID NO: 1, wherein X1 is selected from G, A, V, L, I and P, X2 is selected from K, R, and H, and X3 is selected from K, R, and H, or wherein: X1 is P, X2 is R, and/or X3 is K.
In embodiments, the MLT mobile element enzyme has an amino acid sequence of SEQ ID NO: 1, or a variant thereof, and S8P and C13R mutations (SEQ ID NO: 11). In embodiments, the MLT mobile element enzyme has an amino acid sequence having mutations at positions which correspond to at least one of S8P and C13R mutations relative to the amino acid of SEQ ID NO: 1 or a functional equivalent thereof. In embodiments, the MLT mobile element enzyme has an amino acid sequence having mutations at positions which correspond to S8P and C13R mutations relative to the amino acid of SEQ ID NO: 1 or a functional equivalent thereof.
In embodiments, the MLT mobile element enzyme has an amino acid sequence of SEQ ID NO: 1, or a variant thereof, and S8P, C13R, and N125K mutations (SEQ ID NO: 10).
In embodiments, a MLT mobile element enzyme comprising the amino acid sequence of SEQ ID NO: 1, or a variant thereof, and includes one or more hyperactive mutations selected from a substitution or deletion at one or more of positions S5, S8, D9, D10, E11, C13, A14, S36, S54, N125, K130, G239, T294, T300, 1345, R427, D475, M481, P491, A520, and A561, or positions corresponding thereto.
In embodiments, a MLT mobile element enzyme comprising the amino acid sequence of SEQ ID NO: 1, or a variant thereof, and includes one or more hyperactive mutations selected from S5P, S8P, S8P/C13R, D9G, D10G, E11G, C13R, A14V, S36G, S54N, N125K, K130T, G239S, T294A, T300A, 1345V, R427H, D475G, M481V, P491Q, A520T, and A561T, or positions corresponding thereto.
In embodiments, the MLT mobile element enzyme comprises one or more of hyperactive mutants selected from S8X1, C13X2 and/or N125X3 (e.g., all of S8X1, C13X2 and N125X3, S8X1 and C13X2, S8X1 and N125X3, and C13X2 and N125X3), where X1, X2, and X3 is each independently any amino acid, or X1 is a non-polar aliphatic amino acid, selected from G, A, V, L, I and P, X2 is a positively charged amino acid selected from K, R, and H, and/or X3 is a positively charged amino acid selected from K, R, and H. In embodiments, X1 is P, X2 is R, and/or X3 is K.
In embodiments, the enzyme (e.g., without limitation, a mobile element enzyme, e.g., without limitation, MLT mobile element enzyme) has gene cleavage activity (Exc+) and/or gene integration activity (Int+). In embodiments, the enzyme (e.g., without limitation, a mobile element enzyme) has gene cleavage activity (Exc+) and/or a lack of gene integration activity (Int−). In embodiments, the MLT mobile element enzyme has gene cleavage activity (Exc+) and/or gene integration activity (Int+). In embodiments, the MLT mobile element enzyme has gene cleavage activity (Exc+) and/or a lack of gene integration activity (Int−).
In embodiments, the mobile element enzyme, e.g., without limitation, MLT mobile element enzyme includes an integration reduced or deficient mutation, e.g., about 1, or about 2, or about 3, or about 4, or about 5 integration reduced or deficient mutations or combinations thereof. In embodiments, the mobile element enzyme can include any number of any of the integration reduced or deficient mutations, or equivalents thereof, described herein.
In embodiments, the MLT mobile element enzyme includes an integration reduced or deficient mutations, e.g., about 1, or about 2, or about 3, or about 4, or about 5 integration reduced or deficient mutations, or combinations thereof. In embodiments, the mobile element enzyme can include any number of any of the integration reduced or deficient mutations, or equivalents thereof, described herein.
In embodiments, the enzyme comprises one or more mutations corresponding to TABLE 2A, or positions corresponding thereto, which, without being bound by theory, provides integration reduced or deficient mutations. Numbering relative to the amino acid sequence of protein of SEQ ID NO: 1, and nucleic acid sequence of SEQ ID NO: 2.
In embodiments, the enzyme comprises one or more mutations corresponding to TABLE 2B, or positions corresponding thereto, which, without being bound by theory, provides excision positive and integration deficient mutations. Numbering relative to the amino acid sequence of protein of SEQ ID NO: 1, and nucleic acid sequence of SEQ ID NO: 2.
In embodiments, a MLT mobile element enzyme comprising the amino acid sequence of SEQ ID NO: 1, or a variant thereof, and includes one or more mutations selected from S8P and/or C13R and one of R164N, W168V, M278A, K286A, R287A, R333A, K334A, N335A, K349A, K350A, K368A, K369A, and D416N.
In embodiments, a MLT mobile element enzyme comprising the amino acid sequence of SEQ ID NO: 1, or a variant thereof, and includes one or more mutations selected from S8P and/or C13R and one of R164N, W168V, M278A, K286A, R287A, R333A, K334A, N335A, K349A, K350A, K368A, K369A, and D416N and/or one or more of E284A, K286A, R287A, N310A, R333A, K334A, R336A, K349A, K350A, K368A, and K369A.
In embodiments, a MLT mobile element enzyme comprising the amino acid sequence of SEQ ID NO: 1, or a variant thereof, and includes one or more mutations selected from S8P and/or C13R and one of R164N, W168V, M278A, K286A, R287A, R333A, K334A, N335A, K349A, K350A, K368A, K369A, and D416N and/or one or more of E284A, K286A, R287A, N31 OA, R333A, K334A, R336A, K349A, K350A, K368A, and K369A and/or one R336A.
In embodiments, the mobile element enzyme is or is derived from any of Bombyx mori, Xenopus tropicalis, Trichoplusia ni, Rhinolophus ferrumequinum, Rousettus aegyptiacus, Phyllostomus discolor, Myotis myotis, Myotis lucifugus, Pipistrellus kuhlii, Pteropus vampyrus, and Molossus molossus. In embodiments, the mobile element enzyme is or is derived from any of Trichoplusia ni (SEQ ID NO: 433), Myotis myotis (SEQ ID NO: 435, SEQ ID NO: 436, SEQ ID NO: 438, or SEQ ID NO: 439), or Pteropus vampyrus (SEQ ID NO: 434). In embodiments, the mobile element enzymes have one or more hyperactive and/or integration deficient mutations selected from TABLE 1, TABLE 2A, and/or TABLE 2B, or equivalents thereof. One skilled in the art can correspond such mutants to mobile element enzymes from any of Trichoplusia ni (SEQ ID NO: 433), Myotis lucifugus (SEQ ID NO: 437), Myotis myotis (SEQ ID NO: 435, SEQ ID NO: 436, SEQ ID NO: 438, or SEQ ID NO: 439), or Pteropus vampyrus (SEQ ID NO: 434), e.g.:
Trichnoplusia ni
Pteropus vampyrus
Myotis myotis (“2a”)
Myotis myotis (“1”)
Myotis lucifugus (“2”)
Myotis myotis (“2”)
Myotis myotis (“2b”)
In embodiments, the mobile element enzyme is derived from Bombyx mori, Xenopus tropicalis, or Trichoplusia ni. In embodiments, the mobile element enzyme is an engineered version of a mobile element enzyme, including but not limited to monomers, dimers, tetramers, hyperactive, or Int-forms, derived from Bombyx mori, Xenopus tropicalis, or Trichoplusia ni.
In embodiments, the mobile element enzyme is derived from Bombyx mori, Xenopus tropicalis, Trichoplusia ni, or Myotis lucifugus. In embodiments, the mobile element enzyme is an engineered version, including but not limited to a mobile element enzyme that is a monomer, dimer, tetramer (or another multimer), hyperactive, or has a reduced interaction with non-TTAA (SEQ ID NO: 440) recognitions sites (Int−), derived from Bombyx mori, Xenopus tropicalis, Trichoplusia ni or Myotis lucifugus. In embodiments, the mobile element enzymes have one or more hyperactive and/or integration deficient mutations selected from TABLE 1, TABLE 2A, and TABLE 2B, or equivalents thereof.
In embodiments, one skilled in the art can correspond such mutants to mobile element enzymes from any of Bombyx mori, Xenopus tropicalis, Trichoplusia ni, Rhinolophus ferrumequinum, Rousettus aegyptiacus, Phyllostomus discolor, Myotis myotis, Myotis lucifugus, Pipistrellus kuhlii, Pteropus vampyrus, Pan troglodytes, and Molossus molossus.
In embodiments, the mobile element enzyme has a nucleotide sequence having at least about 90%, or at least about 93%, or at least about 95%, or at least about 97%, or at least about 98%, or at least about 99% identity to a nucleotide sequence of any of Rhinolophus ferrumequinum, Rousettus aegyptiacus, Phyllostomus discolor, Myotis myotis, Myotis lucifugus, Pteropus vampyrus, Pipistrellus kuhliim, Pan troglodytes, and Molossus molossus. In embodiments, the mobile element enzyme has an amino acid sequence having at least about 90%, or at least about 93%, or at least about 95%, or at least about 97%, or at least about 98%, or at least about 99% identity to an amino acid sequence of any of Rhinolophus ferrumequinum, Rousettus aegyptiacus, Phyllostomus discolor, Myotis myotis, Myotis lucifugus, Pteropus vampyrus, Pipistrellus kuhlii, and Molossus molossus. See Jebb, et al. (2020).
In embodiments, the enzyme (e.g., without limitation, a mobile element enzyme) is derived from Bombyx mori, Xenopus tropicalis, Trichoplusia ni, Myotis lucifugus, Rhinolophus ferrumequinum, Rousettus aegyptiacus, Phyllostomus discolor, Myotis myotis, Pteropus vampyrus, Pipistrellus kuhlii, Molossus molossus, Pan troglodytes, or Homo sapiens. In embodiments, the enzyme (e.g., without limitation, a mobile element enzyme) is an engineered version, including but not limited to hyperactive forms, of a mobile element enzyme derived from Bombyx mori, Xenopus tropicalis, Trichoplusia ni, Myotis lucifugus, Rhinolophus ferrumequinum, Rousettus aegyptiacus, Phyllostomus discolor, Myotis myotis, Pteropus vampyrus, Pipistrellus kuhlii, Molossus molossus, Pan troglodytes, or Homo sapiens. The enzyme is either the wild type, monomer, dimer, tetramer, hyperactive, or an Int-mutant. In embodiments, the mobile element enzymes have one or more hyperactive and/or integration deficient mutations selected from TABLE 1, TABLE 2A, and/or TABLE 2B, or equivalents thereof.
In embodiments, the mobile element enzyme has a nucleotide sequence having at least about 90%, or at least about 93%, or at least about 95%, or at least about 97%, or at least about 98%, or at least about 99% identity to a nucleotide sequence of any of Rhinolophus ferrumequinum, Rousettus aegyptiacus, Phyllostomus discolor, Myotis myotis, Pteropus vampyrus, Pipistrellus kuhlii, Molossus molossus, and Pan troglodytes. In embodiments, the mobile element enzyme has an amino acid sequence having at least about 90%, or at least about 93%, or at least about 95%, or at least about 97%, or at least about 98%, or at least about 99% identity to an amino acid sequence of any of Rhinolophus ferrumequinum, Rousettus aegyptiacus, Phyllostomus discolor, Myotis myotis, Pteropus vampyrus, Pipistrellus kuhlii, Molossus molossus, Pan troglodytes, and Homo sapiens.
In embodiments, the mobile element enzyme is an engineered version, including but not limited to a mobile element enzyme that is a monomer, dimer, tetramer, hyperactive, or has a reduced interaction with non-TTAA (SEQ ID NO: 440) recognitions sites (Int−), derived from any of Bombyx mori, Xenopus tropicalis, Trichoplusia ni, Rhinolophus ferrumequinum, Rousettus aegyptiacus, Phyllostomus discolor, Myotis myotis, Myotis lucifugus, Pipistrellus kuhlii, Pteropus vampyrus, and Molossus molossus Bombyx mori, Xenopus tropicalis, Trichoplusia ni, Pan troglodytes, Myotis lucifugus, and Homo sapiens. The mobile element enzyme is either the wild type, monomer, dimer, tetramer or another multimer, hyperactive, or a an Int-mutant.
In embodiments, the mobile element enzyme is from a Tc1/mariner donor DNA system. See, e.g., Plasterk et al. Trends in Genetics. 1999; 15(8):326-32.
In embodiments, the mobile element enzyme is from a Sleeping Beauty donor DNA system (see, e.g., Cell. 1997; 91:501-510), e.g., a hyperactive form of Sleeping Beauty (hypSB), e.g., SB100X (see Gene Therapy volume 18, pages 849-856(2011), or a piggyBac (PB) donor DNA system (see, e.g., Trends Biotechnol. 2015 September; 33(9):525-33, which is incorporated herein by reference in its entirety), e.g., a hyperactive form of PB mobile element enzyme (hypPB), e.g., with seven amino acid substitutions (e.g., I30V, S103P, G165S, M282V, S509G, N570S, N538K on mPB, or functional equivalents in non-mPB, see Mol Ther Nucleic Acids. 2012 October; 1(10): e50, which is incorporated herein by reference in its entirety); see also Yusa et al., PNAS Jan. 25, 2011 108 (4) 1531-1536; Voigt et al., Nature Communications volume 7, Article number: 11126 (2016).
The piggyBac mobile element enzymes belong to the IS4 mobile element enzyme family. De Palmenaer et al., BMC Evolutionary Biology. 2008; 8:18. doi: 10.1186/1471-2148-8-18. The piggyBac family includes a large diversity of donor DNAs, and any of these donor DNAs can be used in embodiments of the present disclosure. See, e.g., Bouallègue et al., Genome Biol Evol. 2017; 9(2):323-339. The founding member of the piggyBac (super)family, insect piggyBac, was originally identified in the cabbage looper moth (Trichoplusiani ni) and studied both in vivo and in vitro. Insect piggyBac is known to transpose by a canonical cut-and-paste mechanism promoted by an element-encoded mobile element enzyme with a catalytic site resembling the RNase H fold shared by many recombinases. The insect piggyBac donor DNA system has been shown to be highly active in a wide range of animals, including Drosophila and mice, where it has been developed as a powerful tool for gene tagging and genome engineering. Other donor DNAs affiliated to the piggyBac superfamily are common in arthropods and vertebrates including Xenopus and Bombyx. Mammalian piggyBac donor DNAs and mobile element enzymes, including hyperactive mammalian piggyBac variants, which can be used in embodiments of the present disclosure, are described, e.g., in International Application WO2010085699, which is incorporated herein by reference in its entirety.
In embodiments, the mobile element enzyme is from a LEAP-IN 1 type or LEAP-IN donor DNA system (Biotechnol J. 2018 October; 13(10):e1700748. doi: 10.1002/biot.201700748. Epub 2018 Jun. 11). The LEAPIN mobile element enzyme system includes a mobile element enzyme (e.g., without limitation, a mobile element enzyme mRNA) and a vector containing one or more genes of interest (donor DNAs), selection markers, regulatory elements, insulators, etc., flanked by the donor DNA cognate inverted terminal ends and the transposition recognition motif (TTAT). Upon co-transfection of vector DNA and mobile element enzyme mRNA, the transiently expressed enzyme catalyzes high-efficiency and precise integration of a single copy of the donor DNA cassette (all sequences between the terminal ends) at one or more sites across the genome of the host cell. Hottentot et al. In Genotyping: Methods and Protocols. White S J, Cantsilieris S, eds: 185-196. (New York, NY: Springer): 2017. pp. 185-196. The LEAPIN mobile element enzyme generates stable transgene integrants with various advantageous characteristics, including single copy integrations at multiple genomic loci, primarily in open chromatin segments; no payload limit, so multiple independent transcriptional units may be expressed from a single construct; the integrated transgenes maintain their structural and functional integrity; and maintenance of transgene integrity ensures the desired chain ratio in every recombinant cell.
In embodiments, the mobile element enzyme is an engineered form of a mobile element enzyme reconstructed from Homo sapiens or a predecessor thereof.
Donor DNAs in Humans have 5 inactive elements, designated piggyBac domain (PGBD)1, PGBD2, PGBD3, PGBD4, and PGBD5. PGBD1, PGBD2, and PGBD3 have multiple coding exons, but in each case the mobile element enzyme-related sequence is encoded by a single uninterrupted 3′ terminal exon. Thus, PGBD1 and PGBD2 may resemble the PGBD3 donor DNA in which the mobile element enzyme ORF is flanked upstream by a 3′ splice site and downstream by a polyadenylation site. See Newman et al., PLoS Genet 2008; 4:e1000031. PLoS Genet 4(3): e1000031. https://doi.org/10.1371/journal.pgen.1000031; Gray et al., PLoS Genet 8(9): e1002972. https://doi.org/10.1371/journal.pgen.1002972.
The PGBD5 inactive mobile element enzyme sequence belongs to the RNase H clan of Pfam structures, while PGBD3 has sustained only a single D to N mutation in the essential catalytic triad DDD(D) and retains the ability to bind the upstream piggyBac terminal inverted repeat. Bailey et al., DNA Repair (Amst) 2012; 11:488-501. The PGBD5 mobile element enzyme does not retain the catalytic DDD (D) motif found in active elements, and the mobile element enzyme is not only inactive but fails to associate with either DNA or chromatin in vivo. Pavelitz et al., Mob DNA 2013; 4:23. However, in vitro studies showed that it is transpositionally active in HEK293 cells. See Henssen et al., Elife 2015; 4. PGBD1 and PGBD2 are thought to be present in the common ancestor of mammals, while PGBD3 and PGBD4 are restricted to primates. See Sarkar et al., Mol Genet Genomics 2003; 270:173-80. The Pteropus vampyrus mobile element enzyme is closely related to PGBD4 and shares DDD catalytic domain and the C-terminal region that are involved in excision mechanisms. See Mitra et al., EMBO J 2008; 27:1097-109.
A mammalian mobile element enzyme, which has gene cleavage and/or gene integration activity, can be constructed based on alignment of the amino acid sequence of Pteropus vampyrus mobile element enzyme to PGBD1, PGBD2, PGBD3, PGBD4, and PGBD5 sequences. Also, in embodiments, the mammalian mobile element enzyme has mutations that confers hyperactivity to a recombinant mammalian mobile element enzyme. Accordingly, in embodiments, the mobile element enzyme has gene cleavage activity (Exc+) and/or gene integration activity (Int+). In embodiments, the mobile element enzyme has gene cleavage activity (Exc+) and/or lacks gene integration activity (Int−).
In some aspects, an enzyme capable of performing targeted genomic integration is a recombinant mammalian mobile element enzyme that was derived by, in part, aligning several inactive mobile element enzyme sequences from a human genome to Pteropus vampyrus mobile element enzyme sequence. In embodiments, the Pteropus vampyrus mobile element enzyme has an amino acid sequence having at least 90% identity (e.g., a sequence having at least about 90%, or at least about 93%, or at least about 95%, or at least about 97%, or at least about 98%, or at least about 99% identity) to SEQ ID NO: 430 (or a functional equivalent thereof. In embodiments, the Pteropus vampyrus mobile element enzyme has an amino acid sequence of SEQ ID NO: 430, or a functional equivalent thereof. In embodiments, the Pteropus vampyrus mobile element enzyme has a nucleotide sequence having at least 90% identity (e.g., a sequence having at least about 90%, or at least about 93%, or at least about 95%, or at least about 97%, or at least about 98%, or at least about 99% identity) to SEQ ID NO: 429 or a codon-optimized variant thereof.
In embodiments, the mobile element enzyme is a mammalian mobile element enzyme, such as a mobile element enzyme from a bat, e.g., without limitation, Pteropus vampyrus.
In embodiments, the mobile element enzyme is an engineered form that is based on a mobile element enzyme reconstructed from Homo sapiens or a predecessor thereof. In embodiments, the mobile element enzyme includes but is not limited to an engineered version that is a monomer, dimer, tetramer (or another multimer), hyperactive, or has a reduced interaction with non-TTAA (SEQ ID NO: 440) recognitions sites (Int−), of an engineered version of a mobile element enzyme reconstructed from Homo sapiens or a predecessor thereof.
In embodiments, the mobile element enzyme is an engineered form that is based on a mobile element enzyme reconstructed from mammalian species. In embodiments, the mobile element enzyme includes but is not limited to an engineered that is a monomer, dimer, tetramer (or another multimer), hyperactive, or has a reduced interaction with non-TTAA (SEQ ID NO: 440) recognitions sites (Int−), of a mobile element enzyme reconstructed from mammalian species.
In embodiments, the donor DNA is included in a vector comprising left and right end sequences recognized by the mobile element enzyme.
In embodiments, the end sequences are selected from MER, MER75A, MER75B, and MER85.
In embodiments, the end sequences are selected from nucleotide sequences of SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 441, and SEQ ID NO: 22, or a nucleotide sequence having at least about 90% identity (e.g., a sequence having at least about 90%, or at least about 93%, or at least about 95%, or at least about 97%, or at least about 98%, or at least about 99% identity) thereto. In embodiments, one or more of the end sequences are optionally flanked by a TTAA (SEQ ID NO: 440) sequence.
In embodiments, the end sequences include at least one repeat from a nucleotide sequence having at least about 90% (e.g., a sequence having at least about 90%, or at least about 93%, or at least about 95%, or at least about 97%, or at least about 98%, or at least about 99% identity) identity to the nucleotide sequence of SEQ ID NO: 12, and wherein the at least one repeat from the nucleotide sequence having at least about 90% identity (e.g., a sequence having at least about 90%, or at least about 93%, or at least about 95%, or at least about 97%, or at least about 98%, or at least about 99% identity) to the nucleotide sequence of SEQ ID NO: 12 is positioned at the 5′ end of the donor DNA. The end sequences can further include at least one repeat from a nucleotide sequence having at least about 90% identity (e.g., a sequence having at least about 90%, or at least about 93%, or at least about 95%, or at least about 97%, or at least about 98%, or at least about 99% identity to the nucleotide sequence of SEQ ID NO: 17, and wherein the at least one repeat from the nucleotide sequence having at least about 90% identity (e.g., a sequence having at least about 90%, or at least about 93%, or at least about 95%, or at least about 97%, or at least about 98%, or at least about 99% identity) to the nucleotide sequence of SEQ ID NO: 17 is positioned at the 3′ end of the donor DNA. The end sequences, which can be from, e.g., Pteropus vampyrus, are optionally flanked by a TTAA (SEQ ID NO: 440) sequence.
In embodiments, the end sequences include at least one repeat from a nucleotide sequence having at least about 90% identity (e.g., a sequence having at least about 90%, or at least about 93%, or at least about 95%, or at least about 97%, or at least about 98%, or at least about 99% identity) to the nucleotide sequence of SEQ ID NO: 13, and wherein the at least one repeat from the nucleotide sequence having at least about 90% identity (e.g., a sequence having at least about 90%, or at least about 93%, or at least about 95%, or at least about 97%, or at least about 98%, or at least about 99% identity) to the nucleotide sequence of SEQ ID NO: 13 is positioned at the 5′ end of the donor DNA. The end sequences can further include at least one repeat from a nucleotide sequence having at least about 90% identity (e.g., a sequence having at least about 90%, or at least about 93%, or at least about 95%, or at least about 97%, or at least about 98%, or at least about 99% identity) to the nucleotide sequence of SEQ ID NO: 18, and wherein the at least one repeat from the nucleotide sequence having at least about 90% identity (e.g., a sequence having at least about 90%, or at least about 93%, or at least about 95%, or at least about 97%, or at least about 98%, or at least about 99% identity) to the nucleotide sequence of SEQ ID NO: 18 is positioned at the 3′ end of the donor DNA. The end sequences, which can be, e.g., PGBD4, are optionally flanked by a TTAA (SEQ ID NO: 440) sequence.
In embodiments, the end sequences include at least one repeat from a nucleotide sequence having at least about 90% identity (e.g., a sequence having at least about 90%, or at least about 93%, or at least about 95%, or at least about 97%, or at least about 98%, or at least about 99% identity) to the nucleotide sequence of SEQ ID NO: 14, wherein the at least one repeat from the nucleotide sequence having at least about 90% identity (e.g., a sequence having at least about 90%, or at least about 93%, or at least about 95%, or at least about 97%, or at least about 98%, or at least about 99% identity) to the nucleotide sequence of SEQ ID NO: 14 is positioned at the 5′ end of the donor DNA. The end sequences include at least one repeat from a nucleotide sequence having at least about 90% identity (e.g. a sequence having at least about 90%, or at least about 93%, or at least about 95%, or at least about 97%, or at least about 98%, or at least about 99% identity) to the nucleotide sequence of SEQ ID NO: 18, wherein the at least one repeat from the nucleotide sequence having at least about 90% identity (e.g., a sequence having at least about 90%, or at least about 93%, or at least about 95%, or at least about 97%, or at least about 98%, or at least about 99% identity) to the nucleotide sequence of SEQ ID NO: 19 is positioned at the 3′ end of the donor DNA. The end sequences, which can be, e.g., MER75, are optionally flanked by a TTAA (SEQ ID NO: 440) sequence.
In embodiments, the end sequences include at least one repeat from a nucleotide sequence having at least about 90% identity (e.g., a sequence having at least about 90%, or at least about 93%, or at least about 95%, or at least about 97%, or at least about 98%, or at least about 99% identity) to the nucleotide sequence of SEQ ID NO: 15, wherein the at least one repeat from the nucleotide sequence having at least about 90% identity (e.g., a sequence having at least about 90%, or at least about 93%, or at least about 95%, or at least about 97%, or at least about 98%, or at least about 99% identity) to the nucleotide sequence of SEQ ID NO: 15 is positioned at the 5′ end of the donor DNA. The end sequences include at least one repeat from a nucleotide sequence having at least about 90% identity (e.g., a sequence having at least about 90%, or at least about 93%, or at least about 95%, or at least about 97%, or at least about 98%, or at least about 99% identity) to the nucleotide sequence of SEQ ID NO: 20, wherein the at least one repeat from the nucleotide sequence having at least about 90% identity (e.g., a sequence having at least about 90%, or at least about 93%, or at least about 95%, or at least about 97%, or at least about 98%, or at least about 99% identity) to the nucleotide sequence of SEQ ID NO: 20 is positioned at the 3′ end of the donor DNA. The end sequences, which can be, e.g., MER75B, are optionally flanked by a TTAA (SEQ ID NO: 440) sequence.
In embodiments, the end sequences include at least one repeat from a nucleotide sequence having at least about 90% (e.g., a sequence having at least about 90%, or at least about 93%, or at least about 95%, or at least about 97%, or at least about 98%, or at least about 99% identity) identity to the nucleotide sequence of SEQ ID NO: 16, wherein the at least one repeat from the nucleotide sequence having at least about 90% identity (e.g., a sequence having at least about 90%, or at least about 93%, or at least about 95%, or at least about 97%, or at least about 98%, or at least about 99% identity) to the nucleotide sequence of SEQ ID NO: 16 is positioned at the 5′ end of the donor DNA. The end sequences include at least one repeat from a nucleotide sequence having at least about 90% identity (e.g., a sequence having at least about 90%, or at least about 93%, or at least about 95%, or at least about 97%, or at least about 98%, or at least about 99% identity) to the nucleotide sequence of SEQ ID NO: 21 or SEQ ID NO: 441, wherein the at least one repeat from the nucleotide sequence having at least about 90% identity (e.g., a sequence having at least about 90%, or at least about 93%, or at least about 95%, or at least about 97%, or at least about 98%, or at least about 99% identity) to the nucleotide sequence of SEQ ID NO: 21 or SEQ ID NO: 441 is positioned at the 3′ end of the donor DNA. The end sequences, which can be, e.g., MER75A, are optionally flanked by a TTAA (SEQ ID NO: 440) sequence.
In embodiments, a donor DNA is or comprises a vector comprising a donor DNA comprising one or more end sequences recognized by an enzyme such as, for example a mobile element enzyme. In embodiments, the end sequences are selected from Pteropus vampyrus, MER75, MER75A, and MER75B. MERs contain end sequences with similarity to piggyBac-like mobile elements and exhibit duplications of their presumed TTAA (SEQ ID NO: 440) target sites. In embodiments, the end sequences are selected from nucleotide sequences of SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 441, and SEQ ID NO: 22, or a nucleotide sequence having at least about 90% identity (e.g., a sequence having at least about 90%, or at least about 93%, or at least about 95%, or at least about 97%, or at least about 98%, or at least about 99% identity) thereto.
In embodiments, the mobile element enzyme has an amino acid sequence of SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, or SEQ ID NO: 9, or a variant sequence having at least about 90%, or at least about 93%, or at least about 95%, or at least about 97%, or at least about 98%, or at least about 99% identity thereto, or at least about 10 mutations, or at least about 9 mutations, or at least about 8 mutations, or at least about 7 mutations, or at least about 6 mutations, or at least about 5 mutations, or at least about 4 mutations, or at least about 3 mutations, or at least about 2 mutations, or at least about 1 mutation.
In embodiments, the mobile element enzyme has an amino acid sequence having S8P, G17R, and/or K134K mutation relative to the amino acid sequence of SEQ ID NO: 4 or a functional equivalent thereof.
In embodiments, the mobile element enzyme has an amino acid sequence having S8P, G17R, and/or K134K mutation relative to the amino acid sequence of SEQ ID NO: 5 or a functional equivalent thereof.
In embodiments, the mobile element enzyme has an amino acid sequence having 183P and/or V118R mutation relative to the amino acid sequence of SEQ ID NO: 6 or a functional equivalent thereof.
In embodiments, the mobile element enzyme has an amino acid sequence having S20P and/or A29R mutation relative to the amino acid sequence of SEQ ID NO: 7 or a functional equivalent thereof.
In embodiments, the mobile element enzyme has an amino acid sequence having T4P and/or L13R mutation relative to the amino acid sequence of SEQ ID NO: 8 or a functional equivalent thereof.
In embodiments, the mobile element enzyme has an amino acid sequence having A12P and/or 128R mutation and/or R152K mutation relative to the amino acid sequence of SEQ ID NO: 9 or a functional equivalent thereof.
In embodiments, the enzyme capable of performing targeted genomic integration (e.g., without limitations, a mobile element enzyme) is in a monomeric or dimeric form. In embodiments, the enzyme capable of performing targeted genomic integration (e.g., without limitations, a mobile element enzyme) is in a multimeric form.
In embodiments, the enzyme (e.g., without limitation, a mobile element enzyme) is an engineered version, including but not limited to a mobile element enzyme that is a monomer, dimer, tetramer, hyperactive, or has a reduced interaction with non-TTAA (SEQ ID NO: 440) recognitions sites (Int−), and is derived from any of Bombyx mori, Xenopus tropicalis, Trichoplusia ni, Myotis lucifugus, Rhinolophus ferrumequinum, Rousettus aegyptiacus, Phyllostomus discolor, Myotis myotis, Pteropus vampyrus, Pipistrellus kuhlii, Pan troglodytes, Molossus molossus, or Homo sapiens. In embodiments, the mobile element enzyme is either the wild type, monomer, dimer, tetramer or another multimer, hyperactive, or an Int-mutant.
In aspects, the present disclosure provides for targeted chimeras, e.g., in embodiments, the enzyme, without limitation, a mobile element enzyme, comprises a targeting element.
in embodiments, the enzyme, without limitation, a mobile element enzyme, associated with the targeting element, is capable of inserting the donor DNA comprising a transgene, optionally at a TA dinucleotide site or a TTAA (SEQ ID NO: 440) tetranucleotide site in a genomic safe harbor site (GSHS).
In embodiments, the enzyme, without limitation, a mobile element enzyme, associated with the targeting element has one or more mutations which confer hyperactivity.
In embodiments, the enzyme, without limitation, a mobile element enzyme, associated with the targeting element has gene cleavage activity (Exc+) and/or gene integration activity (Int+).
In embodiments, the enzyme, without limitation, a mobile element enzyme, associated with the targeting element has gene cleavage activity (Exc+) and/or a lack of gene integration activity (Int−).
In embodiments, the targeting element comprises one or more proteins or nucleic acids that are capable of binding to a nucleic acid.
In embodiments, the targeting element comprises one or more of a of a gRNA, optionally associated with a Cas enzyme, which is optionally catalytically inactive, transcription activator-like effector (TALE), catalytically inactive Zinc finger, catalytically inactive transcription factor, nickase, a transcriptional activator, a transcriptional repressor, a recombinase, a DNA methyltransferase, a histone methyltransferase, paternally expressed gene 10 (PEG10), and TnsD.
In embodiments, the targeting element comprises a transcription activator-like effector (TALE) DNA binding domain (DBD).
In embodiments, the TALE DBD comprises one or more repeat sequences. In embodiments, the TALE DBD comprises about 14, or about 15, or about, 16, or about 17, or about 18, or about 18.5 repeat sequences. In embodiments, the TALE DBD repeat sequences comprise 33 or 34 amino acids. In embodiments, the TALE DBD repeat sequences comprise a repeat variable di-residue (RVD) at residue 12 or 13 of the 33 or 34 amino acids. In embodiments, the RVD recognizes one base pair in the nucleic acid molecule. In embodiments, the RVD recognizes a C residue in the nucleic acid molecule and is selected from HD, N(gap), HA, ND, and HI. In embodiments, the RVD recognizes a G residue in the nucleic acid molecule and is selected from NN, NH, NK, HN, and NA. In embodiments, the RVD recognizes an A residue in the nucleic acid molecule and is selected from NI and NS. In embodiments, the RVD recognizes a T residue in the nucleic acid molecule and is selected from NG, HG, H(gap), and IG. In embodiments, the GSHS is in an open chromatin location in a chromosome. In embodiments, the GSHS is selected from adeno-associated virus site 1 (AAVS1), chemokine (C—C motif) receptor 5 (CCR5) gene, HIV-1 coreceptor, and human Rosa26 locus. In embodiments, the GSHS is located on human chromosome 2, 4, 6, 10, 11, 17, 22, or X. In embodiments, the GSHS is selected from TALC1, TALC2, TALC3, TALC4, TALC5, TALC7, TALC8, AVS1, AVS2, AVS3, ROSA1, ROSA2, TALER1, TALER2, TALER3, TALER4, TALER5, SHCHR2-1, SHCHR2-2, SHCHR2-3, SHCHR2-4, SHCHR4-1, SHCHR4-2, SHCHR4-3, SHCHR6-1, SHCHR6-2, SHCHR6-3, SHCHR6-4, SHCHR10-1, SHCHR10-2, SHCHR10-3, SHCHR10-4, SHCHR10-5, SHCHR11-1, SHCHR11-2, SHCHR11-3, SHCHR17-1, SHCHR17-2, SHCHR17-3, and SHCHR17-4.
In embodiments, the targeting element comprises a Cas9 enzyme guide RNA complex. In embodiments, the Cas9 enzyme guide RNA complex comprises a nuclease-deficient dCas9 guide RNA complex. In embodiments, the targeting element comprises a Cas12 enzyme guide RNA complex. In embodiments, the targeting element comprises a nuclease-deficient dCas12 guide RNA complex, optionally dCas12j guide RNA complex or dCas12a guide RNA complex. In embodiments, the targeting element comprises a Cas12k enzyme guide RNA complex. In embodiments, the targeting element comprises a nuclease-deficient dCas12 guide RNA complex, optionally dCas12k guide RNA complex.
In embodiments, a targeting chimeric system or construct, having a DBD fused to a mobile element enzyme, directs binding of an enzyme capable of performing targeted genomic integration (e.g., without limitation, a mobile element enzyme) to a specific sequence (e.g., transcription activator-like effector proteins (TALE) repeat variable di-residues (RVD) or gRNA) near an enzyme recognition site. The enzyme is thus prevented from binding to random recognition sites. In embodiments, the targeting chimeric construct binds to human GSHS. In embodiments, dCas9 (i.e., deficient for nuclease activity) is programmed with gRNAs directed to bind at a desired sequence of DNA in GSHS.
In embodiments, TALEs described herein can physically sequester the enzyme such as, e.g., a mobile element enzyme, to GSHS and promote transposition to nearby TTAA (SEQ ID NO: 440) sequences in close proximity to the RVD TALE nucleotide sequences. GSHS in open chromatin sites are specifically targeted based on the predilection for mobile element enzymes to insert into open chromatin.
In embodiments, an enzyme capable of performing targeted genomic integration (e.g., without limitation, a recombinase, integrase, or a mobile element enzyme such as, without limitation, a mammalian mobile element enzyme) is linked to or fused with a TALE DNA binding domain (DBD) or a Cas-based gene-editing system, such as, e.g., Cas9 or a variant thereof.
In embodiments, the targeting element targets the enzyme to a locus of interest. In embodiments, the targeting element comprises CRISPR (Clustered Regularly Interspaced Short Palindromic Repeat) associated protein 9 (Cas9), or a variant thereof. A CRISPR/Cas9 tool only requires Cas9 nuclease for DNA cleavage and a single-guide RNA (sgRNA) for target specificity. See Jinek et al. (2012) Science 337, 816-821; Chylinski et al. (2014) Nucleic Acids Res 42, 6091-6105. The inactivated form of Cas9, which is a nuclease-deficient (or inactive, or “catalytically dead” Cas9, is typically denoted as “dCas9,” has no substantial nuclease activity. Qi, L. S. et al. (2013). Cell 152, 1173-1183. CRISPR/dCas9 binds precisely to specific genomic sequences through targeting of guide RNA (gRNA) sequences. See Dominguez et al., Nat Rev Mol Cell Biol. 2016; 17:5-15; Wang et al., Annu Rev Biochem. 2016; 85:227-64. dCas9 is utilized to edit gene expression when applied to the transcription binding site of a desired site and/or locus in a genome. When the dCas9 protein is coupled to guide RNA (gRNA) to create dCas9 guide RNA complex, dCas9 prevents the proliferation of repeating codons and DNA sequences that might be harmful to an organism's genome. Essentially, when multiple repeat codons are produced, it elicits a response, or recruits an abundance of dCas9 to combat the overproduction of those codons and results in the shut-down of transcription. Thus, dCas9 works synergistically with gRNA and directly affects the DNA polymerase II from continuing transcription.
In embodiments, the targeting element comprises a nuclease-deficient Cas enzyme guide RNA complex. In embodiments, the targeting element comprises a nuclease-deficient (or inactive, or “catalytically dead” Cas, e.g., Cas9, typically denoted as “dCas” or “dCas9”) guide RNA complex.
In embodiments, guide RNAs (gRNAs) for targeting human genomic safe harbor sites using any of the gRNA-based targeting elements, e.g., without limitation dCas, in areas of open chromatin are as shown in TABLE 3A-3F.
In embodiments, guide RNAs (gRNAs) for targeting human genomic safe harbor sites using any of the gRNA-based targeting elements, e.g., without limitation dCas, in areas of open chromatin are as shown in TABLE 3A:
In embodiments, gRNAs for targeting human genomic safe harbor sites using any of the gRNA-based targeting elements, e.g., without limitation dCas, to the TTAA site in hROSA26 (e.g., hg38 chr3:9,396,133-9,396,305) are shown in TABLE 3B:
In embodiments, gRNAs for targeting human genomic safe harbor sites using any of the gRNA-based targeting elements, e.g., without limitation dCas, to the AAVS1 (e.g., hg38 chr19:55,112,851-55,113,324) are shown in TABLE 3C:
In embodiments, gRNAs for targeting human genomic safe harbor sites using any of the gRNA-based targeting elements, e.g., without limitation dCas, to Chromosome 4 (e.g., hg38 chr4:30,793,534-30,875,476 or hg38 chr4:30,793,533-30,793,537 (9677); chr4:30,875,472-30,875,476 (8948)) are shown in TABLE 3D:
In embodiments, gRNAs for targeting human genomic safe harbor sites using any of the gRNA-based targeting elements, e.g., without limitation dCas, to Chromosome 22 (e.g., hg38 chr22:35,370,000-35,380,000 or hg38 chr22:35,373,912-35,373,916 (861); chr22:35,377,843-35,377,847 (1153)) are shown in TABLE 3E:
In embodiments, gRNAs for targeting human genomic safe harbor sites using any of the gRNA-based targeting elements, e.g., without limitation dCas, to Chromosome X (e.g., hg38 chrX:134,419,661-134,541,172 or hg38 chrX:134,476,304-134,476,307 (85); chrX:134,476,337-134,476,340 (51)) are shown in TABLE 3F:
In embodiments, the gRNA comprises one or more of the sequences outlined herein or a variant sequence having at least about 10 mutations, or at least about 9 mutations, or at least about 8 mutations, or at least about 7 mutations, or at least about 6 mutations, or at least about 5 mutations, or at least about 4 mutations, or at least about 3 mutations, or at least about 2 mutations, or at least about 1 mutation.
In embodiments, a Cas-based targeting element comprises Cas12 or a variant thereof, e.g., without limitation, Cas12a (e.g., dCas12a), or Cas12j (e.g., dCas12j), or Cas12k (e.g., dCas12k). In embodiments, the targeting element comprises a Cas12 enzyme guide RNA complex. In embodiments, comprises a nuclease-deficient dCas12 guide RNA complex, optionally dCas12j guide RNA complex or dCas12a guide RNA complex.
In embodiments, the targeting element is selected from a zinc finger (ZF), transcription activator-like effector (TALE), meganuclease, and clustered regularly interspaced short palindromic repeat (CRISPR)-associated protein, any of which are, in embodiments, catalytically inactive. In embodiments, the CRISPR-associated protein is selected from Cas9, CasX, CasY, Cas12a (Cpf1), and gRNA complexes thereof. In embodiments, the CRISPR-associated protein is selected from Cas9, xCas9, Cas 6, Cas7, Cas8, Cas12a (Cpf1), Cas13a, Cas14, CasX, CasY, a Class 1 Cas protein, a Class 2 Cas protein, MAD7, MG1 nuclease, MG2 nuclease, MG3 nuclease, or catalytically inactive forms thereof, and gRNA complexes thereof.
In embodiments, the mobile element enzyme is capable of inserting a donor DNA at a TA dinucleotide site or a TTAA tetranucleotide site in a genomic safe harbor site (GSHS) of a nucleic acid molecule. The mobile element enzyme is suitable for causing insertion of the donor DNA in a GSHS when contacted with a biological cell.
In embodiments, the targeting element is suitable for directing the mobile element enzyme to the GSHS sequence.
In embodiments, the targeting element comprises transcription activator-like effector (TALE) DNA binding domain (DBD). The TALE DBD comprises one or more repeat sequences. For example, in embodiments, the TALE DBD comprises about 14, or about 15, or about, 16, or about 17, or about 18, or about 18.5 repeat sequences. In embodiments, the TALE DBD repeat sequences comprise 33 or 34 amino acids.
In embodiments, the one or more of the TALE DBD repeat sequences comprise a repeat variable di-residue (RVD) at residue 12 or 13 of the 33 or 34 amino acids.
In embodiments, the targeting element (e.g., TALE or Cas (e.g., Cas9 or Cas12, or variants thereof) DBDs cause the mammalian mobile element enzyme to bind specifically to human GSHS. In embodiments, the TALEs or Cas DBDs sequester the mobile element enzyme to GSHS and promote transposition to nearby TA dinucleotide or a TTAA tetranucleotide sites which can be located in proximity to the repeat variable di-residues (RVD) TALE or gRNA nucleotide sequences. The GSHS regions are located in open chromatin sites that are susceptible to mobile element enzyme activity. Accordingly, the mammalian mobile element enzyme does not only operate based on its ability to recognize TA or TTAA sites, but it also directs a donor DNA (having a transgene) to specific locations in proximity to a TALE or Cas DBD. The chimeric mobile element enzyme in accordance with embodiments of the present disclosure has negligible risk of genotoxicity and exhibits superior features as compared to existing gene therapies.
In embodiments, a chimeric mobile element enzyme is mutated to be characterized by reduced or inhibited binding of off-target sequences and consequently reliant on a DBD fused thereto, such as a TALE or Cas DBD, for transposition.
The described cells, compositions, and methods allow reducing vector and transgene insertions that increase a mutagenic risk. The described cells and methods make use of a gene transfer system that reduces genotoxicity compared to viral- and nuclease-mediated gene therapies. The dual system is designed to avoid the persistence of an active mobile element enzyme and efficiently transfect human cell lines without significant cytotoxicity.
In embodiments, TALE or Cas DBDs are customizable, such as a TALE or Cas DBDs is selected for targeting a specific genomic location. In embodiments, the genomic location is in proximity to a TA dinucleotide site or a TTAA (SEQ ID NO: 440) tetranucleotide site.
Embodiments of the present disclosure make use of the ability of TALE or Cas or dCas9/gRNA DBDs to target specific sites in a host genome. The DNA targeting ability of a TALE or Cas DBD or dCas9/gRNA DBD is provided by TALE repeat sequences (e.g., modular arrays) or gRNA which are linked together to recognize flanking DNA sequences.
Each TALE or gRNA can recognize certain base pair(s) or residue(s).
TALE nucleases (TALENs) are a known tool for genome editing and introducing targeted double-stranded breaks.
TALENs comprise endonucleases, such as Fokl nuclease domain, fused to a customizable DBD. This DBD is composed of highly conserved repeats from TALEs, which are proteins secreted by Xanthomonas bacteria to alter transcription of genes in host plant cells. The DBD includes a repeated highly conserved 33-34 amino acid sequence with divergent 12th and 13th amino acids. These two positions, referred to as the RVD, are highly variable and show a strong correlation with specific base pair or nucleotide recognition. This straightforward relationship between amino acid sequence and DNA recognition has allowed for the engineering of specific DBDs by selecting a combination of repeat segments containing the appropriate RVDs. Boch et al. Nature Biotechnology. 2011; 29 (2): 135-6.
Accordingly, TALENs can be readily designed using a “protein-DNA code” that relates modular DNA-binding TALE repeat domains to individual bases in a target-binding site. See Joung et al. Nat Rev Mol Cell Biol. 2013; 14(1):49-55. doi:10.1038/nrm3486. The following table, for example, shows such code:
It has been demonstrated that TALENs can be used to target essentially any DNA sequence of interest in human cell. Miller et al. Nat Biotechnol. 2011; 29:143-148. Guidelines for selection of potential target sites and for use of particular TALE repeat domains (harboring NH residues at the hypervariable positions) for recognition of G bases have been proposed. See Streubel et al. Nat Biotechnol. 2012; 30:593-595.
Accordingly, in embodiments, the TALE DBD comprises one or more repeat sequences. In embodiments, the TALE DBD comprises about 15, or about, 16, or about 17, or about 18, or about 18.5 repeat sequences. In embodiments, the TALE DBD repeat sequences comprise 33 or 34 amino acids.
In embodiments, the one or more of the TALE DBD repeat sequences comprise an RVD at residue 12 or 13 of the 33 or 34 amino acids. The RVD can recognize certain base pair(s) or residue(s). In embodiments, the RVD recognizes one base pair in the nucleic acid molecule. In embodiments, the RVD recognizes a C residue in the nucleic acid molecule and is selected from HD, N(gap), HA, ND, and HI. In embodiments, the RVD recognizes a G residue in the nucleic acid molecule and is selected from NN, NH, NK, HN, and NA. In embodiments, the RVD recognizes an A residue in the nucleic acid molecule and is selected from NI and NS. In embodiments, the RVD recognizes a T residue in the nucleic acid molecule and is selected from NG, HG, H(gap), and IG.
In embodiments, the GSHS is in an open chromatin location in a chromosome. In embodiments, the GSHS is selected from adeno-associated virus site 1 (AAVS1), chemokine (C—C motif) receptor 5 (CCR5) gene, HIV-1 coreceptor; and human Rosa26 locus. In embodiments, the GSHS is located on human chromosome 2, 4, 6, 10, 11, 17, 22, or X.
In embodiments, the GSHS is selected from TALC1, TALC2, TALC3, TALC4, TALC5, TALC7, TALC8, AVS1, AVS2, AVS3, ROSA1, ROSA2, TALER1, TALER2, TALER3, TALER4, TALER5, SHCHR2-1, SHCHR2-2, SHCHR2-3, SHCHR2-4, SHCHR4-1, SHCHR4-2, SHCHR4-3, SHCHR6-1, SHCHR6-2, SHCHR6-3, SHCHR6-4, SHCHR10-1, SHCHR10-2, SHCHR10-3, SHCHR10-4, SHCHR10-5, SHCHR11-1, SHCHR11-2, SHCHR11-3, SHCHR17-1, SHCHR17-2, SHCHR17-3, and SHCHR17-4.
In embodiments, the TALE DBD comprises one or more of the sequences outlined herein or a variant sequence having at least about 90%, or at least about 93%, or at least about 95%, or at least about 97%, or at least about 98%, or at least about 99% identity thereto, or at least about 10 mutations, or at least about 9 mutations, or at least about 8 mutations, or at least about 7 mutations, or at least about 6 mutations, or at least about 5 mutations, or at least about 4 mutations, or at least about 3 mutations, or at least about 2 mutations, or at least about 1 mutation.
In embodiments, the GSHS is within about 25, or about 50, or about 100, or about 150, or about 200, or about 300, or about 500 nucleotides of the TA dinucleotide site or TTAA (SEQ ID NO: 440) tetranucleotide site.
Illustrative DNA binding codes for human genomic safe harbor in areas of open chromatin via TALEs, encompassed by various embodiments are provided in TABLE 4A-4F. In embodiments, there is provided a variant of the TALEs, encompassed by various embodiments are provided in TABLE 4A-4F, e.g., having a sequence having at least about 90%, or at least about 93%, or at least about 95%, or at least about 97%, or at least about 98%, or at least about 99% identity to any of the sequences in TABLE 4A-4F.
Illustrative DNA binding codes for human genomic safe harbor in areas of open chromatin via TALEs, encompassed by various embodiments are provided in TABLE 4A:
In embodiments, TALEs for targeting human genomic safe harbor sites using any of the TALE-based targeting elements to the TTAA site in hROSA26 (e.g., hg38 chr3:9,396,133-9,396,305) are shown in TABLE 4B:
In embodiments, TALEs for targeting human genomic safe harbor sites using any of the TALE-based targeting elements to the AAVS1 (e.g., hg38 chr19:55,112,851-55,113,324) are shown in TABLE 4C:
In embodiments, TALEs for targeting human genomic safe harbor sites using any of the TALE-based targeting elements to Chromosome 4 (e.g., hg38 chr4:30,793,534-30,875,476 or hg38 chr4:30,793,533-30,793,537 (9677); chr4:30,875,472-30,875,476 (8948)) are shown in TABLE 4D:
In embodiments, TALEs for targeting human genomic safe harbor sites using any of the TALE-based targeting elements to Chromosome 22 (e.g., hg38 chr22:35,370,000-35,380,000 or hg38 chr22:35,373,912-35,373,916 (861); chr22:35,377,843-35,377,847 (1153)) are shown in TABLE 4E:
In embodiments, TALEs for targeting human genomic safe harbor sites using any of the TALE-based targeting elements to Chromosome X (e.g., hg38 chrX:134,419,661-134,541,172 or hg38 chrX:134,476,304-134,476,307 (85); chrX:134,476,337-134,476,340 (51)) are shown in TABLE 4F:
In embodiments, the mobile element enzyme is capable of inserting a donor DNA at a TA dinucleotide site. In embodiments, the mobile element enzyme is capable of inserting a donor DNA at a TTAA (SEQ ID NO: 440) tetranucleotide site.
Illustrative DNA binding codes for human genomic safe harbor in areas of open chromatin via ZNFs, encompassed by various embodiments are provided in TABLE 5A-5E. In embodiments, there is provided a variant of the ZNFs, encompassed by various embodiments are provided in TABLE 5A-5E, e.g., having a sequence having at least about 90%, or at least about 93%, or at least about 95%, or at least about 97%, or at least about 98%, or at least about 99% identity to any of the sequences in TABLE 5A-5E.
In embodiments, ZNFs for targeting human genomic safe harbor sites using any of the ZNF-based targeting elements to the TTAA site in hROSA26 (e.g., hg38 chr3:9,396,133-9,396,305) are shown in TABLE 5A:
In embodiments, ZNFs for targeting human genomic safe harbor sites using any of the ZNF-based targeting elements to the AAVS1 (e.g., hg38 chr19:55,112,851-55,113,324) are shown in TABLE 56:
In embodiments, ZNFs for targeting human genomic safe harbor sites using any of the ZNF-based targeting elements to Chromosome 4 (e.g., hg38 chr4:30,793,534-30,875,476 or hg38 chr4:30,793,533-30,793,537 (9677); chr4:30,875,472-30,875,476 (8948)) are shown in TABLE 5C:
In embodiments, ZNFs for targeting human genomic safe harbor sites using any of the ZNF-based targeting elements to Chromosome 22 (e.g., hg38 chr22:35,370,000-35,380,000 or hg38 chr22:35,373,912-35,373,916 (861); chr22:35,377,843-35,377,847 (1153)) are shown in TABLE 50:
In embodiments, ZNFs for targeting human genomic safe harbor sites using any of the ZNF-based targeting elements to Chromosome X (e.g., hg38 chrX:134,419,661-134,541,172 or hg38 chrX:134,476,304-134,476,307 (85); chrX:134,476,337-134,476,340 (51)) are shown in TABLE 5E:
In embodiments, the mobile element enzyme is capable of inserting a donor DNA at a TA dinucleotide site. In embodiments, the mobile element enzyme is capable of inserting a donor DNA at a TTAA (SEQ ID NO: 440) tetranucleotide site.
In embodiments, the present disclosure relates to a system having nucleic acids encoding the enzyme and the donor DNA, respectively.
In embodiments, the transgene is an exogenous wild-type gene that, e.g., corrects a defective function of one or more mutations in a recipient. For instance, in embodiments, the recipient may have a mutation that provides a disease phenotype (e.g., a defective or absent gene product). In embodiments, the present stem cell, i.e., produced using the present methods, provides a correction that restores the gene product and diminishes the disease phenotype.
In embodiments, the transgene is a gene that replaces, inactivates, or provides suicide or helper functions.
In embodiments, the transgene is flanked by insulators, optionally HS4 and D4Z4.
In embodiments, the transgene and/or or disease to be treated is one or more of:
In embodiments, the transgene and/or or disease to be treated is one or more of:
In embodiments, the targeting element comprises a nucleic acid binding component of the gene-editing system. In embodiments, the enzyme capable of performing targeted genomic integration (e.g., without limitation, a chimeric mobile element enzyme) and the targeting element, e.g., nucleic acid binding component of the gene-editing system are fused or linked to one another. For example, in embodiments, the mobile element enzyme and the targeting element, e.g., nucleic acid binding component of the gene-editing system are fused or linked to one another. In embodiments, the mobile element enzyme and the targeting element, e.g., nucleic acid binding component of the gene-editing system are connected via a linker.
In embodiments, the linker is a flexible linker. In embodiments, the flexible linker is substantially comprised of glycine and serine residues, optionally wherein the flexible linker comprises (Gly4Ser)n, where n is from about 1 to about 12. In embodiments, the flexible linker is of about 20, or about 30, or about 40, or about 50, or about 60 amino acid residues.
In embodiments, the flexible linker is about 50, or about 100, or about 150, or about 200 amino acid residues in length. In embodiments, the flexible linker comprises at least about 150 nucleotides (nt), or at least about 200 nt, or at least about 250 nt, or at least about 300 nt, or at least about 350 nt, or at least about 400 nt, or at least about 450 nt, or at least about 500 nt, or at least about 500 nt, or at least about 600 nt. In embodiments, the flexible linker comprises from about 450 nt to about 500 nt.
In embodiments, the mobile element enzyme and the targeting element, e.g., nucleic acid binding component of the gene-editing system are encoded on a single polypeptide.
In embodiments, the donor DNA comprises a gene encoding a complete polypeptide. In embodiments, the donor DNA comprises a gene which is defective or substantially absent in a disease state.
Inteins (INTervening protEINS) are mobile genetic elements that are protein domains, found in nature, with the capability to carry out the process of protein splicing. See Sarmiento & Camarero (2019) Current Protein & Peptide Science, 20(5), 408-424, which is incorporated by reference herein in its entirety. Protein spicing is a post-translation biochemical modification which results in the cleavage and formation of peptide bonds between precursor polypeptide segments flanking the intein. Id. Inteins apply standard enzymatic strategies to excise themselves post-translationally from a precursor protein via protein splicing. Nanda et al., Microorganisms vol. 8,12 2004. 16 Dec. 2020, doi:10.3390/microorganisms8122004. An intein can splice its flanking N- and C-terminal domains to become a mature protein and excise itself from a sequence. For example, split inteins have been used to control the delivery of heterologous genes into transgenic organisms. See Wood & Camarero (2014) J Biol Chem. 289(21):14512-14519. This approach relies on splitting the target protein into two segments, which are then post-translationally reconstituted in vivo by protein trans-splicing (PTS). See Aboye & Camarero (2012) J. Biol. Chem. 287, 27026-27032. More recently, an intein-mediated split-Cas9 system has been developed to incorporate Cas9 into cells and reconstitute nuclease activity efficiently. Truong et al., Nucleic Acids Res. 2015, 43 (13), 6450-6458. The protein splicing excises the internal region of the precursor protein, which is then followed by the ligation of the N-extein and C-extein fragments, resulting in two polypeptides—the excised intein and the new polypeptide produced by joining the C- and N-exteins. Sarmiento & Camarero (2019).
In embodiments, intein-mediated incorporation of DNA binders such as, without limitation, dCas9, dCas12j, or TALEs, allows creation of a split-enzyme system such as, without limitation, split-MLT mobile element enzyme system, that permits reconstitution of the full-length enzyme, e.g., MLT mobile element enzyme, from two smaller fragments. This allows avoiding the need to express DNA binders at the N- or C-terminus of an enzyme, e.g., MLT mobile element enzyme. In this approach, the two portions of an enzyme, e.g., MLT mobile element enzyme, are fused to the intein and, after co-expression, the intein allows producing a full-length enzyme, e.g., MLT mobile element enzyme, by post-translation modification. Thus, in embodiments, a nucleic acid encoding the enzyme capable of performing targeted genomic integration comprises an intein. In embodiments, the nucleic acid encodes the enzyme in the form of first and second portions with the intein encoded between the first and second portions, such that the first and second portions are fused into a functional enzyme upon post-translational excision of the intein from the enzyme.
In embodiments, an intein is a suitable ligand-dependent intein, for example, an intein selected from those described in U.S. Pat. No. 9,200,045; Mootz et al., J. Am. Chem. Soc. 2002; 124, 9044-9045; Mootz et al., J. Am. Chem. Soc. 2003; 125,10561-10569; Buskirk et al., Proc. Natl. Acad. Sci. USA. 2004; 101, 10505-10510; Skretas & Wood. Protein Sci. 2005; 14, 523-532; Schwartz, et al., Nat. Chem. Biol. 2007; 3, 50-54; Peck et al., Chem. Biol. 2011; 18 (5), 619-630; the entire contents of each of which are hereby incorporated by reference herein.
In embodiments the intein is NpuN (Intein-N) (SEQ ID NO: 423) and/or NpuC (Intein-C) (SEQ ID NO: 424), or a variant thereof, e.g., a sequence having at least about 90%, or at least about 93%, or at least about 95%, or at least about 97%, or at least about 98%, or at least about 99% identity thereto.
In embodiments, a nucleic acid encoding the enzyme is RNA. In embodiments, a nucleic acid encoding the transgene is DNA.
In embodiments, the enzyme (e.g., without limitation, the mobile element enzyme) is encoded by a recombinant or synthetic nucleic acid. In embodiments, the nucleic acid is RNA, optionally a helper RNA. In embodiments, the nucleic acid is RNA that has a 5′-m7G cap (cap0, or cap1, or cap2), optionally with pseudouridine substitution (e.g., without limitation N1-methyl-pseudouridine) or a 5-methoxy substitution (e.g., without limitation, 5-methoxy-uridine), and optionally a poly-A tail of about 30, or about 34, or about 50, or about 55, or about 80, or about 100, of about 150 nucleotides in length (e.g. about 30 to about 70 nucleotides in length). In embodiments, the poly-A tail is of about 30 nucleotides in length, optionally about 34 nucleotides in length. In embodiments, a nuclear localization signal is placed before the enzyme start codon at the N-terminus, optionally at the C-terminus.
In embodiments, the nucleic acid that is RNA has a 5′-m7G cap (cap 0, or cap 1, or cap 2).
In embodiments, the nucleic acid comprises a 5′ cap structure, a 5′-UTR comprising a Kozak consensus sequence, a 5′-UTR comprising a sequence that increases RNA stability in vivo, a 3′-UTR comprising a sequence that increases RNA stability in vivo, and/or a 3′ poly(A) tail.
In embodiments, the enzyme (e.g., without limitation, a mobile element enzyme) is incorporated into a vector or a vector-like particle. In embodiments, the vector is a non-viral vector.
In embodiments, a nucleic acid encoding the enzyme in accordance with embodiments of the present disclosure, is DNA.
In various embodiments, a construct comprising a donor DNA is any suitable genetic construct, such as a nucleic acid construct, a plasmid, or a vector. In various embodiments, the construct is DNA, which is referred to herein as a donor DNA. In embodiments, sequences of a nucleic acid encoding the donor DNA is codon optimized to provide improved mRNA stability and protein expression in mammalian systems.
In embodiments, the enzyme and the donor DNA are included in different vectors. In embodiments, the enzyme and the donor DNA are included in the same vector.
In various embodiments, a nucleic acid encoding the enzyme capable of performing targeted genomic integration (e.g., without limitation, a mobile element enzyme which is a chimeric mobile element enzyme) is RNA (e.g., helper RNA), and a nucleic acid encoding a donor DNA is DNA.
As would be appreciated in the art, a donor DNA often includes an open reading frame that encodes a transgene at the middle of donor DNA and terminal repeat sequences at the 5′ and 3′ end of the donor DNA. The translated mobile element enzyme binds to the 5′ and 3′ sequence of the donor DNA and carries out the transposition function.
In embodiments, a donor DNA is used interchangeably with mobile elements, which are used to refer to polynucleotides capable of inserting copies of themselves into other polynucleotides. The term donor DNA is well known to those skilled in the art and includes classes of donor DNAs that can be distinguished on the basis of sequence organization, for example inverted terminal sequences at each end, and/or directly repeated long terminal repeats (LTRs) at the ends. In embodiments, the donor DNA as described herein may be described as a piggyBac like element, e.g., a donor DNA element that is characterized by its traceless excision, which recognizes TTAA (SEQ ID NO: 440) sequence and restores the sequence at the insert site back to the original TTAA (SEQ ID NO: 440) sequence after removal of the donor DNA.
In embodiments, donor DNA or transgene are used interchangeably with mobile elements.
In embodiments, the donor DNA is flanked by one or more end sequences or terminal ends. In embodiments, the donor DNA is or comprises a gene encoding a complete polypeptide. In embodiments, the donor DNA is or comprises a gene which is defective or substantially absent in a disease state.
In embodiments, the donor DNA includes a MLT mobile element enzyme (e.g., without limitation, a MLT mobile element enzyme having at least about 90% identity to the amino acid sequence of SEQ ID NO: 1, SEQ ID NO: 10, or SEQ ID NO: 11). For example, the mobile element enzyme can act on a left terminal end having a nucleotide sequence of SEQ ID NO: 431 or a sequence having at least about 90%, or at least about 93%, or at least about 95%, or at least about 97%, or at least about 98%, or at least about 99% identity thereto. In embodiments, the donor DNA can act on a right terminal end having a nucleotide sequence of SEQ ID NO: 432 or a sequence having at least about 90%, or at least about 93%, or at least about 95%, or at least about 97%, or at least about 98%, or at least about 99% identity thereto. In embodiments, the donor DNA acts on both MLT left donor DNA end and MLT right donor DNA end, having nucleotide sequences of SEQ ID NO: 431 and of SEQ ID NO: 432 respectively, or a sequence having at least about 90%, or at least about 93%, or at least about 95%, or at least about 97%, or at least about 98%, or at least about 99% identity thereto.
In embodiments, a transgene is associated with various regulatory elements that are selected to ensure stable expression of a construct with the transgene. Thus, in embodiments, a transgene is encoded by a non-viral vector (e.g., without limitation, a DNA plasmid) that can comprise one or more insulator sequences that prevent or mitigate activation or inactivation of nearby genes. The insulators flank the donor DNA (transgene cassette) to reduce transcriptional silencing and position effects imparted by chromosomal sequences. As an additional effect, the insulators can eliminate functional interactions of the transgene enhancer and promoter sequences with neighboring chromosomal sequences. In embodiments, the one or more insulator sequences comprise an HS4 insulator (1.2-kb 5′-HS4 chicken β-globin (cHS4) insulator element) and an D4Z4 insulator (tandem macrosatellite repeats linked to Facio-Scapulo-Humeral Dystrophy (FSHD). In embodiments, the sequences of the HS4 insulator and the D4Z4 insulator are as described in Rival-Gervier et al. Mol Ther. 2013 August; 21(8):1536-50, which is incorporated herein by reference in its entirety.
In embodiments, the transgene is inserted into a GSHS location in a host genome. GSHSs is defined as loci well-suited for gene transfer, as integrations within these sites are not associated with adverse effects such as proto-oncogene activation, tumor suppressor inactivation, or insertional mutagenesis. GSHSs can defined by the following criteria: 1) distance of at least 50 kb from the 5′ end of any gene, (2) distance of at least 300 kb from any cancer-related gene, (3) distance of at least 300 kb from any microRNA (miRNA), (4) location outside a transcription unit, and (5) location outside ultra-conserved regions (UCRs) of the human genome. See Papapetrou et al. Nat Biotechnol 2011; 29:73-8; Bejerano et al. Science 2004; 304:1321-5.
Furthermore, the use of GSHS locations can allow stable transgene expression across multiple cell types. One such site, chemokine C—C motif receptor 5 (CCR5) has been identified and used for integrative gene transfer. CCR5 is a member of the beta chemokine receptor family and is required for the entry of R5 tropic viral strains involved in primary infections. A homozygous 32 bp deletion in the CCR5 gene confers resistance to HIV-1 virus infections in humans. Disrupted CCR5 expression, naturally occurring in about 1% of the Caucasian population, does not appear to result in any reduction in immunity. Lobritz at al., Viruses 2010; 2:1069-105. A clinical trial has demonstrated safety and efficacy of disrupting CCR5 via targetable nucleases. Tebas at al., HIV. N Engl J Med 2014; 370:901-10.
In embodiments, the donor DNA is under control of a tissue-specific promoter. The tissue-specific promoter is, e.g., without limitation, a liver-specific promoter. In embodiments, the liver-specific promoter is an LP1 promoter that, in embodiments, is a human LP1 promoter. The LP1 promoter is described, e.g., in Nathwani et al. Blood vol. 2006; 107(7):2653-61, and it is constructed, without limitation, as described in Nathawani et al.
It should be appreciated however that a variety of promoters can be used, including other tissue-specific promoters, inducible promoters, constitutive promoters, etc.
In embodiments, the present nucleic acids include polymeric form of nucleotides of any length, either ribonucleotides or deoxyribonucleotides, or analogs or derivatives thereof. In embodiments, there is provided double- and single-stranded DNA, as well as double- and single-stranded RNA, and RNA-DNA hybrids. In embodiments, transcriptionally-activated polynucleotides such as methylated or capped polynucleotides are provided. In embodiments, the present compositions are mRNA or DNA.
In embodiments, the present non-viral vectors are linear or circular DNA molecules that comprise a polynucleotide encoding a polypeptide and is operably linked to control sequences, wherein the control sequences provide for expression of the polynucleotide encoding the polypeptide. In embodiments, the non-viral vector comprises a promoter sequence, and transcriptional and translational stop signal sequences. Such vectors may include, among others, chromosomal and episomal vectors, e.g., vectors bacterial plasmids, from donor DNAs, from yeast episomes, from insertion elements, from yeast chromosomal elements, and vectors from combinations thereof. The present constructs may contain control regions that regulate as well as engender expression.
In embodiments, the construct comprising the enzyme and/or transgene is codon optimized. Transgene codon optimization is used to optimize therapeutic potential of the transgene and its expression in the host organism. Codon optimization is performed to match the codon usage in the transgene with the abundance of transfer RNA (tRNA) for each codon in a host organism or cell. Codon optimization methods are known in the art and described in, for example, WO 2007/142954, which is incorporated by reference herein in its entirety. Optimization strategies can include, for example, the modification of translation initiation regions, alteration of mRNA structural elements, and the use of different codon biases.
In embodiments, the construct comprising the enzyme and/or transgene includes several other regulatory elements that are selected to ensure stable expression of the construct. Thus, in embodiments, the non-viral vector is a DNA plasmid that can comprise one or more insulator sequences that prevent or mitigate activation or inactivation of nearby genes. In embodiments, the one or more insulator sequences comprise an HS4 insulator (1.2-kb 5′-HS4 chicken β-globin (cHS4) insulator element) and an D4Z4 insulator (tandem macrosatellite repeats linked to Facio-Scapulo-Humeral Dystrophy (FSHD). In embodiments, the sequences of the HS4 insulator and the D4Z4 insulator are as described in Rival-Gervier et al. Mol Ther. 2013 August; 21(8):1536-50, which is incorporated herein by reference in its entirety. In embodiments, the gene of the construct comprising the enzyme and/or transgene is capable of transposition in the presence of a mobile element enzyme. In embodiments, the non-viral vector in accordance with embodiments of the present disclosure comprises a nucleic acid construct encoding a mobile element enzyme. The mobile element enzyme is an RNA mobile element enzyme plasmid. In embodiments, the non-viral vector further comprises a nucleic acid construct encoding a DNA donor plasmid. In embodiments, the mobile element enzyme is an in vitro-transcribed mRNA mobile element enzyme. The mobile element enzyme is capable of excising and/or transposing the gene from the construct comprising the enzyme and/or transgene to site- or locus-specific genomic regions.
In embodiments, the enzyme and the donor DNA are included in the same vector.
In embodiments, the enzyme is disposed on the same (cis) or different vector (trans) than a donor DNA with a transgene. Accordingly, in embodiments, the enzyme and the donor DNA encompassing a transgene are in cis configuration such that they are included in the same vector. In embodiments, the enzyme and the donor DNA encompassing a transgene are in trans configuration such that they are included in different vectors. The vector is any non-viral vector in accordance with the present disclosure.
In some aspects, a nucleic acid encoding the enzyme capable of performing targeted genomic integration (e.g., a mobile element enzyme or a chimeric mobile element enzyme) in accordance with embodiments of the present disclosure is provided. The nucleic acid is or comprises DNA or RNA. In embodiments, the nucleic acid encoding the enzyme is DNA. In embodiments, the nucleic acid encoding the enzyme capable of performing targeted genomic integration (e.g., a chimeric mobile element enzyme) is RNA such as, e.g., helper RNA. In embodiments, the chimeric mobile element enzyme is incorporated into a vector. In embodiments, the vector is a non-viral vector.
In embodiments, a nucleic acid encoding the transgene in accordance with embodiments of the present disclosure is provided. The nucleic acid is or comprises DNA or RNA. In embodiments, the nucleic acid encoding the transgene is DNA. In embodiments, the nucleic acid encoding the e transgene is RNA such as, e.g., helper RNA. In embodiments, the transgene is incorporated into a vector. In embodiments, the vector is a non-viral vector.
In embodiments, the present enzyme can be in the form or an RNA or DNA and have one or two N-terminus nuclear localization signal (NLS) to shuttle the protein more efficiently into the nucleus. For example, in embodiments, the present enzyme further comprises one, two, three, four, five, or more NLSs. Examples of NLS are provided in Kosugi et al. (J. Biol. Chem. (2009) 284:478-485; incorporated by reference herein). In a particular embodiment, the NLS comprises the consensus sequence K(K/R)X(K/R) (SEQ ID NO: 348). In an embodiment, the NLS comprises the consensus sequence (K/R)(K/R)X10-12(K/R)3/5(SEQ ID NO: 349), where (K/R)3/5 represents at least three of the five amino acids is either lysine or arginine. In an embodiment, the NLS comprises the c-myc NLS. In a particular embodiment, the c-myc NLS comprises the sequence PAAKRVKLD (SEQ ID NO: 350). In a particular embodiment, the NLS is the nucleoplasmin NLS. In embodiments, the nucleoplasmin NLS comprises the sequence KRPAATKKAGQAKKKK (SEQ ID NO: 351). In embodiments, the NLS comprises the SV40 Large T-antigen NLS. In embodiments, the SV40 Large T-antigen NLS comprises the sequence PKKKRKV (SEQ ID NO: 352). In a particular embodiment, the NLS comprises three SV40 Large T-antigen NLSs (e.g., DPKKKRKVDPKKKRKVDPKKKRKV (SEQ ID NO: 353). In embodiments, the NLS may comprise mutations/variations in the above sequences such that they contain 1 or more substitutions, additions or deletions (e.g., about 1, or about 2, or about 3, or about 4, or about 5, or about 10 substitutions, additions, or deletions).
In some aspects, a host cell comprising the nucleic acid in accordance with embodiments of the present disclosure is provided.
In some aspects, there is provided a transgenic animal comprising a host cell comprising the nucleic acid in accordance with embodiments of the present disclosure is provided.
In some aspects, there is provided a transgenic animal that is generated using one or more of the stem cell of the present disclosure. In embodiments, embryonic stem cells are generated using the present methods. In embodiments, such embryonic stems are used to generate one or more transgenic animals. In embodiments, the transgenic animals are used as disease models, e.g., to test the efficacy of one or more agents that are potentially useful in the treatment of the disease. In embodiments, the animal is a mammal, e.g., a human, mouse, rat, guinea pig, dog, cat, horse, cow, pig, rabbit, bear, sheep, or non-human primate, such as a monkey, chimpanzee, or baboon. In other embodiments, the subject and/or animal is a non-mammal, such, for example, a zebrafish.
In embodiments, at least one of the first nucleic acid and the second nucleic acid is in the form of a lipid nanoparticle (LNP). In embodiments, a composition comprising the first and second nucleic acids is in the form of an LNP.
In embodiments, a nucleic acid encoding the enzyme and a nucleic acid encoding the transgene are contained within the same lipid nanoparticle (LNP). In embodiments, the nucleic acid encoding the enzyme and the nucleic acid encoding the donor DNA are a mixture incorporated into or associated with the same LNP. In embodiments, the nucleic acid encoding the enzyme and the nucleic acid encoding the donor DNA are in the form of a co-formulation incorporated into or associated with the same LNP.
In embodiments, the LNP is selected from 1,2-dioleoyl-3-trimethylammonium propane (DOTAP), a cationic cholesterol derivative mixed with dimethylaminoethane-carbamoyl (DC—Chol), phosphatidylcholine (PC), triolein (glyceryl trioleate), and 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[carboxy(polyethylene glycol)-2000] (DSPE-PEG), 1,2-dimyristoyl-rac-glycero-3-methoxypolyethyleneglycol—2000 (DMG-PEG 2K), and 1,2 distearol-sn-glycerol-3phosphocholine (DSPC) and/or comprising of one or more molecules selected from polyethylenimine (PEI) and poly(lactic-co-glycolic acid) (PLGA), and N-Acetylgalactosamine (GalNAc).
In embodiments, an LNP is as described, e.g., in Patel et al., J Control Release 2019; 303:91-100. The LNP can comprise one or more of a structural lipid (e.g., DSPC), a PEG-conjugated lipid (CDM-PEG), a cationic lipid (MC3), cholesterol, and a targeting ligand (e.g., GalNAc).
In embodiments, a nanoparticle is a particle having a diameter of less than about 1000 nm. In embodiments, nanoparticles of the present disclosure have a greatest dimension (e.g., diameter) of about 500 nm or less, or about 400 nm or less, or about 300 nm or less, or about 200 nm or less, or about 100 nm or less. In embodiments, nanoparticles of the present invention have a greatest dimension ranging between about 50 nm and about 150 nm, or between about 70 nm and about 130 nm, or between about 80 nm and about 120 nm, or between about 90 nm and about 110 nm. In embodiments, the nanoparticles of the present disclosure have a greatest dimension (e.g., a diameter) of about 100 nm.
In some aspects, the cell in accordance with the present disclosure is prepared via an in vivo genetic modification method. In embodiments, a genetic modification in accordance with the present disclosure is performed via an ex vivo method.
In some aspects, the cell in accordance with the present disclosure is prepared by contacting a cell with an enzyme capable of performing targeted genomic integration (e.g., without limitation, a mammalian mobile element enzyme) in vivo. In embodiments, the cell is contacted with the enzyme ex vivo.
In embodiments, the present method provides reduced insertional mutagenesis or oncogenesis as compared to a method with a non-chimeric mobile element enzyme.
In embodiments, the transgene of interest in accordance with embodiments of the present disclosure can encode various genes.
In embodiments, the enzyme (e.g., without limitations, a mobile element enzyme), and the donor DNA are included in the same pharmaceutical composition.
In embodiments, the enzyme (e.g., without limitations, a mobile element enzyme) and the donor DNA are included in different pharmaceutical compositions.
In embodiments, the enzyme and the donor DNA are co-transfected.
In embodiments, the enzyme and the donor DNA are transfected separately.
In embodiments, the donor DNA and the enzyme are transfected at a donor DNA to enzyme ratio of about 1 to about 4, or about 1 to about 2, or about 1 to about 1.
In embodiments, the donor DNA and the enzyme RNA are transfected at a donor DNA to enzyme RNA ratio of about 1 to about 4, or about 1 to about 2, or about 1 to about 1.
In embodiments, the amount of donor DNA transfected is about 2 μg to about 10 μg, or about 2 μg to about 8 μg, or about 2 μg to about 6 μg, or about 2 μg to about 4 μg, or about 2 μg, or about 4 μg, or about 6 μg, or about 8 μg, or about 10 μg.
In embodiments, the amount of donor DNA transfected is about 2 μg.
In embodiments, the amount of donor DNA transfected is about 2 μg and the amount of an enzyme RNA transfected is about 8 μg.
In embodiments, the disclosure provides a stem cell generated by a method described herein.
In embodiments, the disclosure provides a method of delivering a stem cell therapy, comprising administering to a patient in need thereof the stem cell generated by a method described herein.
In embodiments, the disclosure provides a method of treating a disease or condition using a stem cell therapy, comprising administering to a patient in need thereof the stem cell generated by a method described herein.
In embodiments, a stem cell for gene therapy is provided, wherein the transfected cell is generated using a stem cell generated by a method described herein.
In embodiments, a method of delivering a cell therapy is provided, comprising administering to a patient in need thereof the stem cell generated using a method in accordance with embodiments of the present disclosure.
In embodiments, the disease or condition is or comprises cancer. In embodiments, the cancer is or comprises an adrenal cancer, a biliary track cancer, a bladder cancer, a bone/bone marrow cancer, a brain cancer, a breast cancer, a cervical cancer, a colorectal cancer, a cancer of the esophagus, a gastric cancer, a head/neck cancer, a hepatobiliary cancer, a kidney cancer, a liver cancer, a lung cancer, an ovarian cancer, a pancreatic cancer, a pelvis cancer, a pleura cancer, a prostate cancer, a renal cancer, a skin cancer, a stomach cancer, a testis cancer, a thymus cancer, a thyroid cancer, a uterine cancer, a lymphoma, a melanoma, a multiple myeloma, or a leukemia.
In embodiments, the cancer is selected from one or more of the basal cell carcinoma, biliary tract cancer; bladder cancer; bone cancer; brain and central nervous system cancer; breast cancer; cancer of the peritoneum; cervical cancer; choriocarcinoma; colon and rectum cancer; connective tissue cancer; cancer of the digestive system; endometrial cancer; esophageal cancer; eye cancer; cancer of the head and neck; gastric cancer; glioblastoma; hepatic carcinoma; hepatoma; intra-epithelial neoplasm; kidney or renal cancer; larynx cancer; leukemia; liver cancer; lung cancer; melanoma; myeloma; neuroblastoma; oral cavity cancer; ovarian cancer; pancreatic cancer; prostate cancer; retinoblastoma; rhabdomyosarcoma; rectal cancer; cancer of the respiratory system; salivary gland carcinoma; sarcoma; skin cancer; squamous cell cancer; stomach cancer; testicular cancer; thyroid cancer; uterine or endometrial cancer; cancer of the urinary system; vulval cancer; Hodgkin's lymphoma; non-Hodgkin's lymphoma; B-cell lymphoma; small lymphocytic (SL) NHL; intermediate grade/follicular NHL; intermediate grade diffuse NHL; high grade immunoblastic NHL; high grade lymphoblastic NHL; high grade small non-cleaved cell NHL; bulky disease NHL; mantle cell lymphoma; AIDS-related lymphoma; Waldenstrom's Macroglobulinemia; chronic lymphocytic leukemia (CLL); acute lymphoblastic leukemia (ALL); and Hairy cell leukemia.
In embodiments, the cancer is selected from one or more of basal cell carcinoma, biliary tract cancer; bladder cancer; bone cancer; brain and central nervous system cancer; breast cancer; cancer of the peritoneum; cervical cancer; choriocarcinoma; colon and rectum cancer; connective tissue cancer; cancer of the digestive system; endometrial cancer; esophageal cancer; eye cancer; cancer of the head and neck; gastric cancer (including gastrointestinal cancer); glioblastoma; hepatic carcinoma; hepatoma; intra-epithelial neoplasm; kidney or renal cancer; larynx cancer; leukemia; liver cancer; lung cancer (e.g., small-cell lung cancer, non-small cell lung cancer, adenocarcinoma of the lung, and squamous carcinoma of the lung); melanoma; myeloma; neuroblastoma; oral cavity cancer (lip, tongue, mouth, and pharynx); ovarian cancer; pancreatic cancer; prostate cancer; retinoblastoma; rhabdomyosarcoma; rectal cancer; cancer of the respiratory system; salivary gland carcinoma; sarcoma; skin cancer; squamous cell cancer; stomach cancer; testicular cancer; thyroid cancer; uterine or endometrial cancer; cancer of the urinary system; vulvar cancer; lymphoma including Hodgkin's and non-Hodgkin's lymphoma, as well as B-cell lymphoma (including low grade/follicular non-Hodgkin's lymphoma (NHL); small lymphocytic (SL) NHL; intermediate grade/follicular NHL; intermediate grade diffuse NHL; high grade immunoblastic NHL; high grade lymphoblastic NHL; high grade small non-cleaved cell NHL; bulky disease NHL; mantle cell lymphoma; AIDS-related lymphoma; and Waldenstrom's Macroglobulinemia; chronic lymphocytic leukemia (CLL); acute lymphoblastic leukemia (ALL); Hairy cell leukemia; chronic myeloblastic leukemia; as well as other carcinomas and sarcomas; and post-transplant lymphoproliferative disorder (PTLD), as well as abnormal vascular proliferation associated with phakomatoses, edema (e.g., that associated with brain tumors), and Meigs syndrome.
In embodiments, the disease or condition is or comprises an infectious disease. In embodiments, the infectious disease is a coronavirus infection, optionally selected from infection with SAR-CoV, MERS-CoV, and SARS-CoV-2, or variants thereof.
In embodiments, the infectious disease is or comprises a disease comprising a viral infection, a parasitic infection, or a bacterial infection. In embodiments, the viral infection is caused by a virus of family Flaviviridae, a virus of family Picornaviridae, a virus of family Orthomyxoviridae, a virus of family Coronaviridae, a virus of family Retroviridae, a virus of family Paramyxoviridae, a virus of family Bunyaviridae, or a virus of family Reoviridae.
In embodiments, the virus of family Coronaviridae comprises a betacoronavirus or an alphacoronavirus, optionally wherein the betacoronavirus is selected from SARS-CoV-2, SARS-CoV, MERS-CoV, HCoV-HKU1, and HCoV-OC43, or the alphacoronavirus is selected from a HCoV-NL63 and HCoV-229E. In embodiments, the infectious disease comprises a coronavirus infection 2019 (COVID-19).
In embodiments, the disease or condition is or comprises a genetic disease or disorder, optionally cystic fibrosis, sickle cell disease, lysosomal acid lipase (LAL) defect 1, Tay-Sachs disease, phenylketonuria, mucopolysaccharidosis, glycogenosis (GSD, optionally, GSD type I, II, III, IV, V, VI, VII, VIII, IX, X, XI, XII, XIII, and XIV), galactosemia, thalassaemia, muscular dystrophy (e.g., Duchenne muscular dystrophy), and hemophilia.
In embodiments, the disease or condition is or comprises a rare disease or disorder, optionally selected from Erythropoietic Protoporphyria, Hailey-Hailey Disease, Xeroderma Pigmentosum, Ehlers-Danlos Syndrome, Cutis Laxa, Protein C & Protein S Deficiency, Alport Syndrome, Striate Palmoplantar Keratoderma, Lethal Acantholytic EB, Pseudoxanthoma Elasticum (PXE), Ichthyosis Vulgaris, Pemphigus Vulgaris, and Basal Cell Nevus Syndrome.
In embodiments, the disease or condition is or comprises cancer, optionally selected from acute lymphoblastic leukemia, chronic lymphocytic leukemia, non-Hodgkin lymphoma (NHL), and/or multiple myeloma. In embodiments, the cancer is relapsed or refractory acute lymphoblastic leukemia (ALL), a chronic lymphocytic leukemia (CLL), a chronic myelogenous leukemia (CML), a multiple myeloma (MM), an acute myeloid leukemia (AML), diffuse large B-cell lymphoma, primary mediastinal B-cell lymphoma, high grade B-cell lymphoma, transformed follicular lymphoma, and/or Mantle cell lymphoma. In embodiments, the disease or condition is or comprises cancer, optionally a solid tumor, optionally selected from a small cell lung cancer (SCLC), large cell neuroendocrine carcinoma (LCNEC), a gastric cancer, a colon cancer, a renal cell carcinoma, a hepatocellular carcinoma, a bladder urothelial carcinoma, a metastatic melanoma, a breast cancer, an ovarian cancer, a cervical cancer, a head and neck cancer, a pancreatic cancer, a glioma, and/or a glioblastoma.
In embodiments, there is provided a method of delivering a hematopoietic stem cell transplant (HSCT), comprising administering to a patient in need thereof the stem cell generated using a method described herein. In embodiments, the HSCT is autologous. In embodiments, the transplant is not rejected by the patient. In embodiments, the patient does not develop graft-versus-host disease (GVHD).
In embodiments, the disease or condition is or comprises an autoimmune disease or disorder. In embodiments, the autoimmune disease is or comprises multiple sclerosis, diabetes mellitus, lupus, celiac disease, Crohn's disease, ulcerative colitis, Guillain-Barre syndrome, sclerodermas, Goodpasture's syndrome, Wegener's granulomatosis, autoimmune epilepsy, Rasmussen's encephalitis, Primary biliary sclerosis, Sclerosing cholangitis, Autoimmune hepatitis, Addison's disease, Hashimoto's thyroiditis, Fibromyalgia, Meniere's syndrome; transplantation rejection (e.g., prevention of allograft rejection) pernicious anemia, rheumatoid arthritis, systemic lupus erythematosus, dermatomyositis, Sjogren's syndrome, lupus erythematosus, multiple sclerosis, myasthenia gravis, Reiter's syndrome, Grave's disease, and other autoimmune diseases.
In embodiments, the disease or condition is or comprises a neurologic disease or disorder. In embodiments, the neurologic disease is or comprises Friedreich's ataxia, multiple sclerosis (including without limitation, benign multiple sclerosis; relapsing-remitting multiple sclerosis (RRMS); secondary progressive multiple sclerosis (SPMS); progressive relapsing multiple sclerosis (PRMS); and primary progressive multiple sclerosis (PPMS)), Alzheimer's. disease (including, without limitation, Early-onset Alzheimer's, Late-onset Alzheimer's, and Familial Alzheimer's disease (FAD), Parkinson's disease and parkinsonism (including, without limitation, Idiopathic Parkinson's disease, Vascular parkinsonism, Drug-induced parkinsonism, Dementia with Lewy bodies, Inherited Parkinson's, Juvenile Parkinson's), Huntington's disease, Amyotrophic lateral sclerosis (ALS, including, without limitation, Sporadic ALS, Familial ALS, Western Pacific ALS, Juvenile ALS, Hiramaya Disease).
In embodiments, the disease or condition is or comprises a cardiovascular disease or disorder. In embodiments, the cardiovascular disease or disorder is or comprises coronary heart disease (CHD), cerebrovascular disease (CVD), aortic stenosis, peripheral vascular disease, atherosclerosis, arteriosclerosis, myocardial infarction (heart attack), cerebrovascular diseases (stroke), transient ischemic attacks (TIA), angina (stable and unstable), atrial fibrillation, arrhythmia, valvular disease, and/or congestive heart failure.
In embodiments, the method does not cause general immunosuppression.
In embodiments, the method of delivering a stem cell therapy is non-immunogenic.
In embodiments, the method of delivering a stem cell therapy reduces or avoids off-target effects.
In embodiments, the transfected stem cell or engineered stem cell is administered by injection.
In embodiments, the method of delivering a stem cell therapy comprises delivery via two or more doses.
In embodiments, the method of delivering a stem cell therapy comprises creating a high copy number of the transfected stem cells in a subject.
In embodiments, the method requires a single administration. In embodiments, the method requires a plurality of administrations.
In some aspects of the present disclosure, an isolated cell is provided that comprises the transfected cell in accordance with embodiments of the present disclosure.
In some aspects, the present disclosure provides an ex vivo gene therapy approach. Accordingly, in embodiments, the method that is used to treat an inherited or acquired disease in a patient in need thereof comprises (a) contacting a cell obtained from a patient (autologous) or another individual (allogeneic) with a transfected cell in accordance with embodiments of the present disclosure; and (b) administering the cell to a patient in need thereof.
One of the advantages of ex vivo gene therapy is the ability to “sample” the transduced cells before patient administration. This facilitates efficacy and allows performing safety checks before introducing the cell(s) to the patient. For example, the transduction efficiency and/or the clonality of integration can be assessed before infusion of the product. The present disclosure provides transfected cells and methods that can be effectively used for ex vivo gene modification.
In embodiments, a composition comprising transfected cells in accordance with the present disclosure comprises a pharmaceutically acceptable carrier, excipient or diluent.
Methods of formulating suitable pharmaceutical compositions are known in the art, see, e.g., Remington: The Science and Practice of Pharmacy, 21st ed., 2005; and the books in the series Drugs and the Pharmaceutical Sciences: a Series of Textbooks and Monographs (Dekker, N.Y.). For example, pharmaceutical compositions suitable for injectable use can include sterile aqueous solutions (where water soluble) or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersion. For intravenous administration, suitable carriers include physiological saline, bacteriostatic water, Cremophor EL™ (BASF, Parsippany, N.J.) or phosphate buffered saline (PBS). In all cases, the composition must be sterile and the fluid should be easy to draw up by a syringe. It should be stable under the conditions of manufacture and storage and must be preserved against the contaminating action of microorganisms such as bacteria and fungi. The carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyethylene glycol, and the like), and suitable mixtures thereof. The proper fluidity can be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersion and by the use of surfactants. Prevention of the action of microorganisms can be achieved by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, ascorbic acid, thimerosal, and the like. In many cases, it will be preferable to include isotonic agents, for example, sugars, polyalcohols such as mannitol, sorbitol, and sodium chloride in the composition. Prolonged absorption of the injectable compositions can be brought about by including in the composition an agent that delays absorption, for example, aluminum monostearate and gelatin.
Sterile injectable solutions can be prepared by incorporating the active compound in the required amount in an appropriate solvent with one or a combination of ingredients enumerated above, as required, followed by filtered sterilization. Generally, dispersions are prepared by incorporating the active compound into a sterile vehicle, which contains a basic dispersion medium and the required other ingredients from those enumerated above. In the case of sterile powders for the preparation of sterile injectable solutions, the preferred methods of preparation are vacuum drying and freeze-drying, which yield a powder of the active ingredient plus any additional desired ingredient from a previously sterile-filtered solution thereof.
Therapeutic compounds can be prepared with carriers that will protect the therapeutic compounds against rapid elimination from the body, such as a controlled release formulation, including implants and microencapsulated delivery systems. Biodegradable, biocompatible polymers can be used, such as collagen, ethylene vinyl acetate, polyanhydrides (e.g., poly[1,3-bis(carboxyphenoxy)propane-co-sebacic-acid] (PCPP-SA) matrix, fatty acid dimer-sebacic acid (FAD-SA) copolymer, poly(lactide-co-glycolide)), polyglycolic acid, collagen, polyorthoesters, polyethyleneglycol-coated liposomes, and polylactic acid. Such formulations can be prepared using standard techniques, or obtained commercially, e.g., from Alza Corporation and Nova Pharmaceuticals, Inc. Liposomal suspensions can also be used as pharmaceutically acceptable carriers. These can be prepared according to methods known to those skilled in the art, for example, as described in U.S. Pat. No. 4,522,811. Semisolid, gelling, soft-gel, or other formulations (including controlled release) can be used, e.g., when administration to a surgical site is desired. Methods of making such formulations are known in the art and can include the use of biodegradable, biocompatible polymers. See, e.g., Sawyer et al., Yale J Biol Med. 2006; 79(3-4): 141-152.
In embodiments, there is provided a transgenic organism that may comprise cells which have been transformed by the methods of the present disclosure. In embodiments, the organism may be a mammal or an insect. When the organism is a mammal, the organism may include, but is not limited to, a mouse, a rat, a monkey, a dog, a rabbit, bear and the like. When the organism is an insect, the organism may include, but is not limited to, a fruit fly, a mosquito, a bollworm and the like.
In embodiments, the cells produced in accordance with embodiments of the present disclosure, and/or components for generating cells, is included in a container, kit, pack, or dispenser together with instructions for administration.
Also provided herein are kits comprising: one or more genetic constructs encoding the present enzyme and donor DNA and) instructions and/or reagents for the use of the same.
Also provided herein are kits comprising: i) a transfected cell in accordance with embodiments of the present disclosure, ii) instructions for the use of the transfected cell.
Furthermore, in embodiments, a kit is provided for creating a stem cell, and instructions for creating the same, and. optionally, reagents for the same (e.g., media, factors, and the like).
In embodiments, a kit is provided that comprises an enzyme (e.g., without limitation, a recombinant mammalian mobile element enzyme) or a nucleic acid in accordance with embodiments of the present disclosure, and instructions for introducing DNA and/or RNA into a cell using the enzyme.
The following definitions are used in connection with the disclosure disclosed herein. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood to one of skill in the art to which this invention belongs.
As used herein, “a,” “an,” or “the” can mean one or more than one.
Further, the term “about” when used in connection with a referenced numeric indication means the referenced numeric indication plus or minus up to 10% of that referenced numeric indication. For example, the language “about 50” covers the range of 45 to 55.
An “effective amount,” when used in connection with medical uses is an amount that is effective for providing a measurable treatment, prevention, or reduction in the rate of pathogenesis of a disease of interest.
The term “in vivo” refers to an event that takes place in a subject's body.
The term “ex vivo” refers to an event which involves treating or performing a procedure on a cell, tissue and/or organ which has been removed from a subject's body. Aptly, the cell, tissue and/or organ may be returned to the subject's body in a method of treatment or surgery.
As used herein, the term “variant” encompasses but is not limited to nucleic acids or proteins which comprise a nucleic acid or amino acid sequence which differs from the nucleic acid or amino acid sequence of a reference by way of one or more substitutions, deletions and/or additions at certain positions. The variant may comprise one or more conservative substitutions. Conservative substitutions may involve, e.g., the substitution of similarly charged or uncharged amino acids.
“Carrier” or “vehicle” as used herein refer to carrier materials suitable for drug administration. Carriers and vehicles useful herein include any such materials known in the art, e.g., any liquid, gel, solvent, liquid diluent, solubilizer, surfactant, lipid or the like, which is non-toxic and which does not interact with other components of the composition in a deleterious manner.
The phrase “pharmaceutically acceptable” refers to those compounds, materials, compositions, and/or dosage forms that are, within the scope of sound medical judgment, suitable for use in contact with the tissues of human beings and animals without excessive toxicity, irritation, allergic response, or other problems or complications commensurate with a reasonable benefit/risk ratio.
The terms “pharmaceutically acceptable carrier” or “pharmaceutically acceptable excipient” are intended to include any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and inert ingredients. The use of such pharmaceutically acceptable carriers or pharmaceutically acceptable excipients for active pharmaceutical ingredients is well known in the art. Except insofar as any conventional pharmaceutically acceptable carrier or pharmaceutically acceptable excipient is incompatible with the active pharmaceutical ingredient, its use in the therapeutic compositions of the disclosure is contemplated. Additional active pharmaceutical ingredients, such as other drugs, can also be incorporated into the described compositions and methods.
As referred to herein, all compositional percentages are by weight of the total composition, unless otherwise specified. As used herein, the word “include,” and its variants, is intended to be non-limiting, such that recitation of items in a list is not to the exclusion of other like items that may also be useful in the compositions and methods of this technology. Similarly, the terms “can” and “may” and their variants are intended to be non-limiting, such that recitation that an embodiment can or may comprise certain elements or features does not exclude other embodiments of the present technology that do not contain those elements or features.
Although the open-ended term “comprising,” as a synonym of terms such as including, containing, or having, is used herein to describe and claim the invention, the present invention, or embodiments thereof, may alternatively be described using alternative terms such as “consisting of” or “consisting essentially of.”
As used herein, the words “preferred” and “preferably” refer to embodiments of the technology that afford certain benefits, under certain circumstances. However, other embodiments may also be preferred, under the same or other circumstances. Furthermore, the recitation of one or more preferred embodiments does not imply that other embodiments are not useful, and is not intended to exclude other embodiments from the scope of the technology.
The amount of compositions described herein needed for achieving a therapeutic effect may be determined empirically in accordance with conventional procedures for the particular purpose. Generally, for administering therapeutic agents for therapeutic purposes, the therapeutic agents are given at a pharmacologically effective dose. A “pharmacologically effective amount,” “pharmacologically effective dose,” “therapeutically effective amount,” or “effective amount” refers to an amount sufficient to produce the desired physiological effect or amount capable of achieving the desired result, particularly for treating the disorder or disease. An effective amount as used herein would include an amount sufficient to, for example, delay the development of a symptom of the disorder or disease, alter the course of a symptom of the disorder or disease (e.g., slow the progression of a symptom of the disease), reduce or eliminate one or more symptoms or manifestations of the disorder or disease, and reverse a symptom of a disorder or disease. Therapeutic benefit also includes halting or slowing the progression of the underlying disease or disorder, regardless of whether improvement is realized.
Effective amounts, toxicity, and therapeutic efficacy can be determined by standard pharmaceutical procedures in cell cultures or experimental animals, e.g., for determining the LD50 (the dose lethal to about 50% of the population) and the ED50 (the dose therapeutically effective in about 50% of the population). The dosage can vary depending upon the dosage form employed and the route of administration utilized. The dose ratio between toxic and therapeutic effects is the therapeutic index and can be expressed as the ratio LD50/ED50. In embodiments, compositions and methods that exhibit large therapeutic indices are preferred. A therapeutically effective dose can be estimated initially from in vitro assays, including, for example, cell culture assays. Also, a dose can be formulated in animal models to achieve a circulating plasma concentration range that includes the IC50 as determined in cell culture, or in an appropriate animal model. Levels of the described compositions in plasma can be measured, for example, by high performance liquid chromatography. The effects of any particular dosage can be monitored by a suitable bioassay. The dosage can be determined by a physician and adjusted, as necessary, to suit observed effects of the treatment.
As used herein, “methods of treatment” are equally applicable to use of a composition for treating the diseases or disorders described herein and/or compositions for use and/or uses in the manufacture of a medicaments for treating the diseases or disorders described herein.
In embodiments, the present disclosure provides for any of the sequence provided herein, including the below, and a variant sequence having at least about 90%, or at least about 93%, or at least about 95%, or at least about 97%, or at least about 98%, or at least about 99% identity thereto, or at least about 10 mutations, or at least about 9 mutations, or at least about 8 mutations, or at least about 7 mutations, or at least about 6 mutations, or at least about 5 mutations, or at least about 4 mutations, or at least about 3 mutations, or at least about 2 mutations, or at least about 1 mutation.
Myotis lucifugus (Wild-type) Amino Acid Sequence with Hyperactive
Myotis lucifugus Corrected Amino Acid Sequence with Hyperactive
Pteropus vampyrus Left End Sequence
Pteropus vampyrus Right End Sequence
Trichnoplusia ni
Myotis myotis (“2a”)
Myotis myotis (“1”)
Myotis lucifugus (“2”)
Myotis myotis (“2”)
Myotis myotis (“2b”)
This invention is further illustrated by the following non-limiting examples.
Hereinafter, the present disclosure will be described in further detail with reference to examples. These examples are illustrative purposes only and are not to be construed to limit the scope of the present invention. In addition, various modifications and variations can be made without departing from the technical scope of the present invention.
Donor DNA and helper RNA system are used to generate stem cells to deliver therapeutic genes. Human induced pluripotent stem cells are derived from peripheral blood mononuclear cells (PBMC) or fibroblasts by standard methods. Alternatively, CD34+ cells are isolated from umbilical cord blood for human stem cell transplantation (HSCT). The purified or reprogrammed cells are transfected with a gene of interest using the DNA donor and RNA helper mobile element enzyme system as shown in
Illustrative advantages of the present methods to a standard process include:
Human peripheral blood CD34+ hematopoietic stem cells (HSCs) mobilized with G-SCM (Stemcell Technologies, #70060) were cultured in StemSpan-XF media (Stemcell Technologies, #100-0073) at a density of 1×105 cells/mL and expanded with a cytokine cocktail including rhlL-3 (CellGenix, #1402-050), rhlL-6 (CellGenix, #1404-050), SCF (CellGenix, #1418-050), and Flt3-L (CellGenix, #1415-050), each at 100 ng/mL final. After 2 days of culturing, the cells were transfected with the P3 Primary Cell 4D-Nucleofector™ X Kit S (Lonza, #V4XP-3032). Transfections were performed in 20 μL reactions with 5×105 cells per condition across a range of donor DNA [0.5-4 μg] and MLT transposase mRNA [0.5-16 μg] using program EO-100. The donor DNA nanoplasmid (Nature Technologies) contains the following features: MLT transposase ITRs flanking the 5′ and 3′ insertion cassette, 5′ dimer HS4 insulator, CAG promoter, EGFP reporter gene, rabbit beta-globin 3′UTR polyA, and 3′ D4Z4-c insulator (
After transfection, the cells were recovered in 1 mL of culture media. At 24 hours post transfection, 200 μL of cells were stained with zombie violet dye (Biolegend, #77477) and analyzed with flow cytometry (Beckman Coulter CytoFLEX S). Donor DNA amounts greater than 2 μg showed a drop-off in viability, while 2 μg or less showed >75% viability at 24 hours (
To identify a useful DNA amount, the viability and delivery efficiency of each test condition were compared (
The transfected HSCs were then monitored for ˜2 weeks with continual flow cytometry analysis and reseeding with fresh media to a density of 1×105 cells/mL every 2 to 3 days. Data is shown for the DNA amount of 2 μg. The viability of the transfected cells increased to >90% by day 8, with a minor decline in cell health near the day 15 endpoint (
Similar results were observed at three other amounts of DNA (0.5, 1, and 4 μg).
These experiments shows that MLT transposase successfully mediates genome editing of primary human HSCs.
While the invention has been described in connection with specific embodiments thereof, it will be understood that it is capable of further modifications and this application is intended to cover any variations, uses, or adaptations of the invention following, in general, the principles of the invention and including such departures from the present disclosure as come within known or customary practice within the art to which the invention pertains and as may be applied to the essential features herein set forth and as follows in the scope of the appended claims.
Those skilled in the art will recognize, or be able to ascertain, using no more than routine experimentation, numerous equivalents to the specific embodiments described specifically herein. Such equivalents are intended to be encompassed in the scope of the following claims.
All patents and publications referenced herein are hereby incorporated by reference in their entireties.
The publications discussed herein are provided solely for their disclosure prior to the filing date of the present application. Nothing herein is to be construed as an admission that the present invention is not entitled to antedate such publication by virtue of prior invention.
As used herein, all headings are simply for organization and are not intended to limit the disclosure in any manner. The content of any individual section may be equally applicable to all sections.
This application claims priority to and the benefit of U.S. Provisional Patent Application No. 63/275,776, filed on Nov. 4, 2021, the entire content of which is hereby incorporated herein by reference in its entirety.
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/US22/79293 | 11/4/2022 | WO |
| Number | Date | Country | |
|---|---|---|---|
| 63275776 | Nov 2021 | US |
