SUPPRESSION OF CYTOKINE RELEASE SYNDROME IN CHIMERIC ANTIGEN RECEPTOR CELL THERAPY

Information

  • Patent Application
  • 20230074186
  • Publication Number
    20230074186
  • Date Filed
    May 17, 2022
    2 years ago
  • Date Published
    March 09, 2023
    a year ago
Abstract
Disclosed herein are methods of gene editing, or endogenous suppression, of cytokines/chemokines/transcription factors secreted from chimeric antigen receptor (CAR)-bearing immune effector cell such as CAR-T cells for the mitigation of cytokine release syndrome and/or CAR-T associated neuropathy. These methods involve insertion of the CAR into a locus of a cytokine gene, blocking its expression. Also disclosed herein are (CAR)-bearing immune effector cells with CARs inserted into a locus of a cytokine gene, and methods of treatment of diseases with immunotherapy with a reduced incidence of cytokine release syndrome and/or CAR-T associated neuropathy.
Description
SEQUENCE LISTING

The instant application contains a Sequence Listing which has been submitted electronically in ASCII format and is hereby incorporated by reference in its entirety. Said ASCII copy, created on Aug. 6, 2019, is named WGN0003-401-PC_SL.txt and is 956,834 bytes in size.


CAR-T cells have emerged as a promising therapy for the treatment of hematological malignancies. Despite remarkable clinical efficacy against B cell malignancies, the success of CAR-T therapy has been limited by severe, life-threatening toxicities, observed in over 50% of patients. These toxicities have resulted in several deaths leading to early termination of clinical trial. Toxicities primarily manifest as cytokine release syndrome (CRS, also referred to as “cytokine storm”) characterized by high elevations of cytokines including INFγ, granulocyte-macrophage colony-stimulating factor, IL-10, and IL-6. These cytokine elevations result in a plethora of clinical symptoms including fever, hypotension, organ dysfunction, respiratory failure and coagulopathy. CRS can be fatal. Additionally, neurotoxicity often presents even after the initial symptoms of CRS have subsided. The pathogenesis of CRS and associated neurotoxicity is poorly understood and further understanding of the mechanism would be useful for the successful translation of CAR-T therapy. In the meanwhile, disrupting the pathogenesis of CRS by reducing the level of cytokine genes available for expression is one way to mitigate the condition.


Disclosed herein are methods of gene deletion and endogenous suppression, of cytokines/chemokines/transcription factors secreted from chimeric antigen receptor (CAR)-bearing immune effector cell, such as CAR-T cell, for the mitigation of cytokine release syndrome and/or CAR-T associated neuropathy. These gene deletion methods may include, but are not limited to, insertion of the CAR into a locus of a cytokine/chemokine/transcription factor gene, blocking its expression; gene editing with Transcription Activator-like Effector Nucleases (TALENs), Zinc Finger Nucleases (ZFNs), or CRISPR; expression of an scFv with an endoplasmic reticulum (ER) binding tether to bind the cytokine in the ER and prevent secretion; and transfection of small hairpin RNAs (shRNAs) or small interfering RNAs (siRNAs). Also disclosed herein are CAR-bearing immune effector cells modified with these described cytokine/chemokine/transcription factor gene deletion methods, and methods of treatment of diseases with immunotherapy with a reduced incidence of cytokine release syndrome (CRS) and/or CAR-T associated neuropathy (CAN).





BRIEF DESCRIPTION OF THE DRAWINGS


FIG. 1—Illustrates the concept of inserting a CAR into the gene for a cytokine in order to block its translation, thereby deleting it or reducing its level, thus preventing or reducing cytokine release syndrome and/or CAR-bearing immune effector cell associated neuropathy (CAR-T associated neuropathy).



FIG. 2—Shows a timeline for a method of treatment of hematologic malignancies using the CAR-T cells disclosed herein. Those of skill in the art will appreciate that some flexibility is possible in the time frames shown.



FIG. 3—Shows the AAV donor construct for insertion of CD34 into the GM-CSF locus.



FIG. 4—Shows the WC40 plasmid vector for use in inserting GFP into the CD38 locus.



FIG. 5—Shows the AAV donor construct for insertion of GFP into the CD38 locus.



FIG. 6—Shows a CAR19-GM-CSF PEBL-trCD34 construct.



FIG. 7—Shows IL-6 expression in T cells, iDC cells, T cells+iDC, T cells+beads, and iDC+beads+ T cells for GM-CSF knock-out, wild-type, and controls at 24 hours.



FIG. 8—Shows IL-6 expression in CAR19, iDC only, Act MQ only, MC only, RAMOS only, CAR19+iDC, CAR19+ActMQ, CAR19+MQ, CAR19+RAMOS, iDC+RAMOS+CAR-T, ActMQ+RAMOS+CAR-T, and MQ+RAMOS+CAR-T for CAR19 GM-CSF knock-out, CAR19, and controls at 24 hours.



FIG. 9—Shows IL-6 expression in T cells, iDC cells, T cells+iDC, T cells+beads, and iDC+beads+ T cells for GM-CSF knock-out, wild-type, and controls at 48 hours.



FIG. 10—Shows IL-6 expression in CAR19, iDC only, Act MQ only, MC only, RAMOS only, CAR19+iDC, CAR19+ActMQ, CAR19+MQ, CAR19+RAMOS, iDC+RAMOS+CAR-T, ActMQ+RAMOS+CAR-T, and MQ+RAMOS+CAR-T for CAR19 GM-CSF knock-out, CAR19, and controls at 48 hours.



FIG. 11—Shows a set-up of an ELISA plate for detecting specific markers of CAR-T cells.



FIG. 12a and FIG. 12b—Shows the results for an ELISA assay detecting specific markers of the CAR-T cells. FIG. 12a shows the top half of the ELISA plate, and FIG. 12b shows the bottom half of the plate. In each of rows A-H, from top to bottom, the sub-rows indicate [450] test, [540] ref, Pathlength, 450, 540, Corrected [450], and Corrected [540].





DETAILED DESCRIPTION

Accordingly, disclosed herein as Embodiment 1 is a chimeric antigen receptor (CAR)-bearing immune effector cell that is deficient in a cytokine or in a chemokine or in a transcription factor that is involved in cytokine release syndrome.


The following disclosure will detail embodiments, alternatives, and uses of the cytokine-deficient cells, as well as the use of such cells in, for example, immunotherapy and adoptive cell transfer for the treatment of diseases. Accordingly, provided herein are the following additional embodiments.


Embodiment 2—The cell as recited in embodiment 1, wherein the cytokine or chemokine or transcription factor deficiency is effected by deletion or suppression of a gene encoding the cytokine or chemokine or transcription factor.


Embodiment 3—The cell as recited in any of embodiments 1 or 2, wherein the deletion or suppression is effected by inserting the CAR into a locus of the cytokine or chemokine or transcription factor gene.


Embodiment 4—The cell as recited in any of embodiments 1 to 3, wherein the CAR is part of a construct that also includes a selectable marker.


Embodiment 5—The cell as recited in any of embodiments 1 to 4, wherein the selectable marker comprises a green fluorescence (GFP) gene, a yellow fluorescent (YFP) gene, a truncated CD34 (tCD34) gene, or a truncated EGFR (tEGFR) gene.


Embodiment 6—The cell as recited in any of embodiments 1 to 5, wherein the cytokine or chemokine or transcription factor deficiency is effected by deletion or suppression of the cytokine or chemokine gene, by Transcription Activator-like Effector Nucleases (TALENs), Zinc Finger Nucleases (ZFNs), or Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) editing.


Embodiment 7—The cell as recited in any of embodiments 1 to 6, wherein deletion or suppression is effected using CRISPR.


Embodiment 8—The cell as recited in any of embodiments 1 to 7, wherein deletion or suppression is effected using Cas9-CRISPR.


Embodiment 9—The cell as recited in any of embodiments 1 to 8, wherein the Cas9 is delivered into the cell as mRNA or protein.


Embodiment 10—The cell as recited in any of embodiments 1 to 9, wherein the Cas9 is delivered into the cell as mRNA.


Embodiment 11—The cell as recited in any of embodiments 1 to 10, wherein the Cas9 is delivered into the cell as protein.


Embodiment 12—The cell as recited in any of embodiments 1 to 11, wherein a guide RNA (gRNA) targeting the gene to be deleted or suppressed is delivered contemporaneously with the Cas9.


Embodiment 13—The cell as recited in any of embodiments 1 to 12, wherein the delivery is by electroporation.


Embodiment 14—The cell as recited in any of embodiments 1 to 13, wherein the cytokine or chemokine or transcription factor deficiency is effected by suppression of the cytokine or chemokine or transcription factor gene transcript by transfection of one or more types of small interfering RNAs (siRNA).


Embodiment 15—The cell as recited in any of embodiments 1 to 14, wherein the cytokine or chemokine or transcription factor deficiency is effected by suppression of the cytokine or chemokine or transcription factor gene transcript by transduction of one or more types of short hairpin RNAs (shRNA).


Embodiment 16—A chimeric antigen receptor (CAR)-bearing immune effector cell expressing at least one CAR, wherein:


the at least one CAR is inserted into a locus of a cytokine or chemokine or transcription factor gene or transcription factor gene;


the cytokine or chemokine or transcription factor gene is deleted or suppressed by a method chosen from Transcription Activator-like Effector Nucleases (TALENs), Zinc Finger Nucleases (ZFNs), and Clustered Regularly Interspaces Short Palindromic Repeats (CRISPR) editing;


the cytokine or chemokine or transcription factor is suppressed by expression of an scFv with an endoplasmic reticulum (ER) binding tether to bind the cytokine or chemokine in the ER and prevent secretion;


the cytokine or chemokine or transcription factor gene transcript is suppressed by transfection of small interfering RNAs (siRNAs); or


the cytokine or chemokine or transcription factor gene transcript is suppressed by transduction of short hairpin RNAs (shRNAs).


Embodiment 17—The cell as recited in any of embodiments 1 to 16, wherein the cell is chosen from a chimeric antigen receptor T cell (CAR-T), a CAR-bearing iNKT cell (iNKT-CAR), and a CAR-bearing natural killer (NK) cell (NK-CAR), or a CAR-bearing macrophage.


Embodiment 18—The cell as recited in any of embodiments 1 to 17, wherein the cell is a CAR-T.


Embodiment 19—The cell as recited in any of embodiments 1 to 18, wherein the cell is a dual or tandem CAR-T.


Embodiment 20—The cell as recited in any of embodiments 1 to 17, wherein the cell is an iNKT-CAR.


Embodiment 21—The cell as recited in any of embodiments 1 to 20, wherein the cell is a dual or tandem iNKT-CAR.


Embodiment 22—The cell as recited in any of embodiments 1 to 17, wherein the cell is a CAR-macrophage.


Embodiment 23—The cell as recited in any of embodiments 1 to 22, wherein the cell is a dual or tandem CAR-macrophage.


Embodiment 24—The cell as recited in any of embodiments 1 to 23, wherein the cytokine or chemokine or transcription factor contributes to the development of cytokine release syndrome.


Embodiment 25—The cell as recited in any of embodiments 1 to 24, wherein the cytokine or chemokine or transcription factor is selected from among those recited in Table 10.


Embodiment 26—The cell as recited in any of embodiments 1 to 25, wherein the cytokine or chemokine or transcription factor is produced by T cells that activate or localize myeloid cells.


Embodiment 27—The cell as recited in any of embodiments 1 to 26, wherein the cytokine or chemokine or transcription factor is a T cell surface receptor gene that activates myeloid or CAR-T cells.


Embodiment 28—The cell as recited in any of embodiments 1 to 27, wherein the gene that is deleted or suppressed is a T cell surface receptor that is integrated into CAR-T cell signaling.


Embodiment 29—The cell as recited in any of embodiments 1 to 28, wherein the cytokine or chemokine or transcription factor drives T cell/CAR-T cell differentiation.


Embodiment 30—The cell as recited in any of embodiments 1 to 29, wherein the cytokine or chemokine is a transcription factor that drives T cell/CAR-T cell differentiation.


Embodiment 31—The cell as recited in any of embodiments 1 to 30, wherein the cytokine or chemokine or transcription factor is chosen from MCP1 (CCL2), MCP-2, GM-CSF, G-CSF, M-CSF, Il-4, and IFNγ.


Embodiment 32—The cell as recited in any of embodiments 1 to 31, wherein the cytokine or chemokine or transcription factor is GM-CSF.


Embodiment 33—The cell as recited in any of embodiments 1 to 32, wherein the cell is a GM-CSF deficient CAR-T cell.


Embodiment 34—The cell as recited in any of embodiments 1 to 33, wherein the cell is a GM-CSF deficient iNKT-CAR cell.


Embodiment 35—The cell as recited in any of embodiments 1 to 34, wherein the immune effector cells to be used are harvested from a healthy donor.


Embodiment 36—The cell as recited in any of embodiments 1 to 35, wherein the donor is a human.


Embodiment 37—The cell as recited in any of embodiments 1 to 36, wherein the chimeric antigen receptor(s) specifically binds at least one antigen expressed on a malignant cell.


Embodiment 38—The cell as recited in any of embodiments 1 to 37, wherein the one or more antigens expressed on a malignant cell is chosen from BCMA, CS1, CD38, CD138, CD19, CD33, CD123, CD371, CD117, CD135, Tim-3, CD5, CD7, CD2, CD4, CD3, CD79A, CD79B, APRIL, CD56, and CD1a.


Embodiment 39—The cell as recited in any of embodiments 1 to 38, wherein the chimeric antigen receptor specifically binds at least one antigen expressed on a malignant T cell.


Embodiment 40—The cell as recited in any of embodiments 1 to 39, wherein the antigen is selected from CD2, CD38, CD4, CD5, CD7, TCRA, and TCRβ.


Embodiment 41—The cell as recited in any of embodiments 1 to 38, wherein the chimeric antigen receptor specifically binds at least one antigen expressed on a malignant B cell.


Embodiment 42—The cell as recited in any of embodiments 1 to 41, wherein the antigen is selected from CD19, CD20, CD21, CD22, CD23, CD24, CD25, CD27, CD38, and CD45.


Embodiment 43—The cell as recited in any of embodiments 1 to 42, wherein the antigen is selected from CD19 and CD20.


Embodiment 44—The cell as recited in any of embodiments 1 to 38, wherein the chimeric antigen receptor specifically binds at least one antigen expressed on a malignant mesothelial cell.


Embodiment 45—The cell as recited in any of embodiments 1 to 44, wherein the antigen is mesothelin.


Embodiment 46—The cell as recited in any of embodiments 1 to 38, wherein the chimeric antigen receptor specifically binds at least one antigen expressed on a malignant plasma cell.


Embodiment 47—The cell as recited in any of embodiments 1 to 46, wherein the antigen is selected from BCMA, CS1, CD38, and CD19.


Embodiment 48—The cell as recited in any of embodiments 1 to 47, wherein the chimeric antigen receptor expresses the extracellular portion of the APRIL protein, the ligand for BCMA and TACI, effectively co-targeting both BCMA and TACI.


Embodiment 49—The cell as recited in any of embodiments 1 to 48, wherein the CAR-T cell further comprises a suicide gene.


Embodiment 50—The cell as recited in any of embodiments 1 to 49, wherein endogenous T cell receptor mediated signaling is negligible in the cell.


Embodiment 51—The cell as recited in any of embodiments 1 to 50, wherein the cell does not induce alloreactivity or graft-versus-host disease.


Embodiment 52—The cell as recited in any of embodiments 1 to 51, wherein the cell does not induce fratricide.


Embodiment 53—A method of treatment of cancer in a patient, which has a reduced incidence of cytokine release syndrome and/or CAR-T associated neuropathy, comprising the administration of cells as recited in any of embodiments 1 to 52.


Embodiment 54—The method as recited in embodiment 53, wherein the cancer is a hematologic malignancy.


Embodiment 55—The method as recited in any of embodiments 53 to 54, wherein the hematologic malignancy is a T-cell malignancy.


Embodiment 56—The method as recited in any of embodiments 53 to 55, wherein the T cell malignancy is T-cell acute lymphoblastic leukemia (T-ALL).


Embodiment 57—The method as recited in any of embodiments 53 to 56, wherein the T cell malignancy is non-Hodgkin's lymphoma.


Embodiment 58—The method as recited in any of embodiments 53 to 57, wherein the hematologic malignancy is multiple myeloma.


Embodiment 59—The method as recited in any of embodiments 53 to 58, wherein the hematologic malignancy is AML.


Embodiment 60—The method as recited in any of embodiments 53 to 59, wherein the cancer is a solid tumor.


Embodiment 61—The method as recited any of embodiments 53 to 60, wherein the cancer is cervical cancer, pancreatic cancer, ovarian cancer, mesothelioma, and lung cancer.


Embodiment 62—A method of prevention or reduction of cytokine release syndrome or CAR-T associated neuropathy in a patient receiving chimeric antigen receptor T cell (CAR-T), CAR-bearing iNKT cell (iNKT-CAR), CAR-bearing natural killer (NK) cell (NK-CAR), or CAR-bearing macrophage (CAR-macrophage) immunotherapy, comprising the administration of cells as recited in any of embodiments 53 to 61 as the immunotherapy.


Embodiment 63—The method of any of embodiments 53 to 62, wherein the patient is being treated for cancer.


Embodiment 64—The method as recited in any of embodiments 53 to 63, wherein the cancer is a hematologic malignancy.


Embodiment 65—The method as recited in any of embodiments 53 to 64, wherein the hematologic malignancy is a T-cell malignancy.


Embodiment 66—The method as recited in any of embodiments 53 to 65, wherein the T cell malignancy is T-cell acute lymphoblastic leukemia (T-ALL).


Embodiment 67—The method as recited in any of embodiments 53 to 66, wherein the T cell malignancy is non-Hodgkin's lymphoma.


Embodiment 68—The method as recited in any of embodiments 53 to 67, wherein the hematologic malignancy is multiple myeloma.


Embodiment 69—The method as recited in any of embodiments 53 to 68, wherein the hematologic malignancy is AML.


Embodiment 70—The method as recited in any of embodiments 53 to 69, wherein the cancer is a solid tumor.


Embodiment 71—The method as recited in any of embodiments 53 to 70, wherein the cancer is cervical cancer, pancreatic cancer, ovarian cancer, mesothelioma, and lung cancer.


Embodiment 72—A method of blocking the expression of a cytokine gene or chemokine gene or transcription factor gene in a chimeric antigen receptor T cell (CAR-T), CAR-bearing iNKT cell (iNKT-CAR), CAR-bearing natural killer (NK) cell (NK-CAR), or CAR-bearing macrophage (CAR-macrophage) comprising the insertion of a CAR into a locus of the cytokine gene or chemokine gene or transcription factor gene.


Embodiment 73—The method of any of embodiments 53 to 72, wherein blocking the expression of the cytokine gene or chemokine gene or transcription factor gene does not reduce CAR-T cell-mediated killing.


Embodiment 74—A method of making a CAR-T (immune effector) cell that does not cause or contribute to CRS or CAR-T-associated neuropathy (CAN) comprising deleting or suppressing a cytokine or chemokine or transcription factor gene.


Embodiment 75—The method of any of embodiments 53 to 74, wherein deleting or suppressing the cytokine or chemokine or transcription factor gene does not reduce CAR-T cell-mediated killing.


Embodiment 76—The method as recited in any of embodiments 53 to 75, wherein the deletion or suppression is effected by inserting the CAR into a locus of the cytokine or chemokine or transcription factor gene.


Embodiment 77—The method as recited in any of embodiments 53 to 76, wherein the CAR is part of a construct that also includes a selectable marker.


Embodiment 78—The method as recited in any of embodiments 53 to 77, wherein the selectable marker comprises a green fluorescence (GFP) gene, a YFP gene, a tCD34 gene, or a tEGFR gene.


Embodiment 79—The method of any of embodiments 53 to 78, wherein the insertion of a CAR with a selectable marker into the cytokine or chemokine or transcription factor gene allows a single-step purification of TCR-negative cells.


Embodiment 80—The method of any of embodiments 53 to 79, wherein the insertion of a CAR with a selectable marker into the cytokine or chemokine or transcription factor gene allows a single step purification of CAR+ cytokine negative cells.


Embodiment 81—The method as recited in any of embodiments 53 to 80, wherein the deletion or suppression is effected using Transcription Activator-like Effector Nucleases (TALENs), Zinc Finger Nucleases (ZFNs), or Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) editing.


Embodiment 82—The method as recited in any of embodiments 53 to 81, wherein the deletion or suppression is effected using CRISPR.


Embodiment 83—The method as recited in any of embodiments 53 to 82, wherein the deletion or suppression is effected using Cas9-CRISPR.


Embodiment 84—The method as recited in any of embodiments 53 to 83, wherein the Cas9 is delivered into the cell as mRNA or protein.


Embodiment 85—The method as recited in any of embodiments 53 to 84, wherein the Cas9 is delivered into the cell as mRNA.


Embodiment 86—The method as recited in any of embodiments 53 to 85, wherein the Cas9 is delivered into the cell as protein.


Embodiment 87—The method as recited in any of embodiments 53 to 86, wherein a guide RNA (gRNA) targeting the gene to be deleted or suppressed is delivered contemporaneously with the Cas9.


Embodiment 88—The method as recited in any of embodiments 53 to 87, wherein the delivery is by electroporation.


Embodiment 89—The method as recited in any of embodiments 53 to 88, wherein the deletion or suppression is effected by suppression of the cytokine or chemokine or transcription factor gene transcript by transduction of one or more types of short hairpin RNAs (shRNA).


Embodiment 90—The method as recited in any of embodiments 531 to 89, wherein the deletion or suppression is effected by transducing a construct encoding a protein expression blocker (PEBL).


Embodiment 91—The method of any of embodiments 53 to 90, wherein the construct encodes an antibody-derived single-chain variable fragment specific for the cytokine, chemokine or TF gene.


Embodiment 92—The method of any of embodiments 53 to 91, wherein deletion of the cytokine, chemokine, or transcription factor gene does not reduce CAR-T-mediated killing.


Embodiment 93—The method of any of embodiments 53 to 92, wherein the CAR to be inserted comprises a donor template.


Embodiment 94—The method of any of embodiments 53 to 93, wherein donor template comprises an adeno-associated virus (AAV), a single-stranded DNA, or a double-stranded DNA.


Disclosed herein is a chimeric antigen receptor (CAR)-bearing immune effector cell that is deficient in a cytokine.


In certain embodiments, the cytokine deficiency is effected by ablation of a cytokine gene or a chemokine gene, or a transcription factor gene.


In certain embodiments, the ablation is effected by inserting the CAR into a locus of the cytokine/chemokine/transcription factor gene.


In certain embodiments, the CAR is part of a construct that also includes a selectable marker.


In certain embodiments, the cytokine deficiency is effected by deletion or suppression of the cytokine/chemokine/transcription factor gene, by Transcription Activator-like Effector Nucleases (TALENs), Zinc Finger Nucleases (ZFNs), or Clustered Regularly Interspaces Short Palindromic Repeats (CRISPR) editing.


In certain embodiments, the cytokine deficiency is effected by suppression of the cytokine/chemokine/transcription factor gene transcript by transfection of small interfering RNAs (siRNAs).


Also disclosed herein is a chimeric antigen receptor (CAR)-bearing immune effector cell expressing at least one CAR, wherein:

    • the at least one CAR is inserted into a locus of a cytokine/chemokine/transcription factor gene;
    • the cytokine/chemokine/transcription factor gene is deleted or suppressed by a method chosen from Transcription Activator-like Effector Nucleases (TALENs), Zinc Finger Nucleases (ZFNs), and Clustered Regularly Interspaces Short Palindromic Repeats (CRISPR) editing;
    • the cytokine is suppressed by expression of an scFv with an endoplasmic reticulum (ER) binding tether to bind the cytokine in the ER and prevent secretion; or
    • the cytokine/chemokine/transcription factor gene transcript is suppressed by small interfering RNAs (siRNAs) transfection.


In certain embodiments, the cell is chosen from a chimeric antigen receptor T cell (CAR-T), a CAR-bearing iNKT cell (iNKT-CAR), and a CAR-bearing natural killer (NK) cell (NK-CAR).


In certain embodiments, the cell is a CAR-T.


In certain embodiments, the cell is a dual or tandem CAR-T.


In certain embodiments, the cell is an iNKT-CAR.


In certain embodiments, the cell is a dual or tandem iNKT-CAR.


In certain embodiments, the cytokine contributes to the development of cytokine release syndrome.


In certain embodiments, the cytokine is chosen from MCP1 (CCL2), MCP-2, GM-CSF, G-CSF, M-CSF, IL-4, and IFNγ.


In certain embodiments, the cytokine is GM-CSF.


In certain embodiments, the cell is a GM-CSF deficient CAR-T cell.


In certain embodiments, the cell is a GM-CSF deficient iNKT-CAR cell.


In certain embodiments, the chimeric antigen receptor specifically binds at least one antigen expressed on a malignant T cell.


In certain embodiments, the antigen is selected from CD2, CD38, CD4, CD5, CD7, TCRA, and TCRβ.


In certain embodiments, the chimeric antigen receptor specifically binds at least one antigen expressed on a malignant B cell.


In certain embodiments, the antigen is selected from CD19 and CD20.


In certain embodiments, the chimeric antigen receptor specifically binds at least one antigen expressed on a malignant mesothelial cell.


In certain embodiments, the antigen is mesothelin.


In certain embodiments, the chimeric antigen receptor specifically binds at least one antigen expressed on a malignant plasma cell.


In certain embodiments, the antigen is selected from BCMA, CS1, CD38, and CD19.


In certain embodiments, the chimeric antigen receptor expresses the extracellular portion of the APRIL protein, the ligand for BCMA and TACI, effectively co-targeting both BCMA and TACI.


In certain embodiments, the CAR-T cell further comprises a suicide gene.


In certain embodiments, endogenous T cell receptor mediated signaling is negligible in the cell.


In certain embodiments, the cell does not induce alloreactivity or graft-versus-host disease.


In certain embodiments, the cell does not induce fratricide.


Also disclosed herein is a method of treatment of cancer in a patient, which has a reduced incidence of cytokine release syndrome and/or CAR-T associated neuropathy, comprising the administration of chimeric antigen receptor (CAR)-bearing immune effector cells as disclosed herein.


In certain embodiments, the cancer is a hematologic malignancy.


In certain embodiments, the hematologic malignancy is a T-cell malignancy.


In certain embodiments, the T cell malignancy is T-cell acute lymphoblastic leukemia (T-ALL).


In certain embodiments, the T cell malignancy is non-Hodgkin's lymphoma.


In certain embodiments, the hematologic malignancy is multiple myeloma.


In certain embodiments, the cancer is a solid tumor.


In certain embodiments, the cancer is cervical cancer, pancreatic cancer, ovarian cancer, mesothelioma, and lung cancer.


Also disclosed herein is a method of prevention or reduction of cytokine release syndrome, CAR-T associated neuropathy in a patient receiving chimeric antigen receptor T cell (CAR-T), CAR-bearing iNKT cell (iNKT-CAR), or CAR-bearing natural killer (NK) cell (NK-CAR) immunotherapy, comprising the administration of chimeric antigen receptor (CAR)-bearing immune effector cells as disclosed herein as the immunotherapy.


Also disclosed herein is a method of blocking the expression of a cytokine/chemokine/transcription factor gene in a chimeric antigen receptor T cell (CAR-T), CAR-bearing iNKT cell (iNKT-CAR), or CAR-bearing natural killer (NK) cell (NK-CAR), comprising the insertion of a CAR into a locus of the cytokine/chemokine/transcription factor gene.


CAR-Bearing Immune Effector Cells

A chimeric antigen receptor (CAR), is a recombinant fusion protein comprising: 1) an extracellular ligand-binding domain, i.e., an antigen-recognition domain, 2) a transmembrane domain, and 3) a signaling transducing domain.


Methods for CAR design, delivery and expression, and the manufacturing of clinical-grade CAR-T cell populations are known in the art. See, for example, Lee et al., Clin. Cancer Res., 2012, 18(10): 2780-90. An engineered chimeric antigen receptor polynucleotide that encodes for a CAR comprises: a signal peptide, an antigen recognition domain, at least one co-stimulatory domain, and a signaling domain.


The antigen-specific extracellular domain of a chimeric antigen receptor recognizes and specifically binds an antigen, typically a surface-expressed antigen of a malignancy. An “antigen-specific extracellular domain” (or, equivalently, “antigen-binding domain”) specifically binds an antigen when, for example, it binds the antigen with an affinity constant or affinity of interaction (KD) between about 0.1 pM to about 10 μM, preferably about 0.1 pM to about 1 μM, more preferably about 0.1 pM to about 100 nM. Methods for determining the affinity of interaction are known in the art. An antigen-specific extracellular domain suitable for use in a CAR of the present disclosure may be any antigen-binding polypeptide, a wide variety of which are known in the art. In some instances, the antigen-binding domain is a single chain Fv (scFv). Other antibody based recognition domains (cAb VHH (camelid antibody variable domains) and humanized versions thereof, lgNAR VH (shark antibody variable domains) and humanized versions thereof, sdAb VH (single domain antibody variable domains) and “camelized” antibody variable domains are suitable for use. In some instances, T-cell receptor (TCR) based recognition domains such as single chain TCR (scTv, single chain two-domain TCR containing VaVR) are also suitable for use.


A chimeric antigen receptor of the present disclosure also comprises an “intracellular domain” that provides an intracellular signal to the CAR-bearing immune effector cell upon antigen binding to the antigen-specific extracellular domain. The intracellular signaling domain of a chimeric antigen receptor of the present disclosure is responsible for activation of at least one of the effector functions of the T cell in which the chimeric receptor is expressed. The term “effector function” refers to a specialized function of a differentiated cell, such as an iNKT cell. An effector function of an iNKT cell, for example, may be NK transactivation, T cell activation and differentiation, B cell activation, dendritic cell activation and cross-presentation activity, and macrophage activation. Thus, the term “intracellular domain” refers to the portion of a CAR that transduces the effector function signal upon binding of an antigen to the extracellular domain and directs the iNKT cell to perform a specialized function. Non-limiting examples of suitable intracellular domains include the zeta chain of the T-cell receptor or any of its homologs (e.g., eta, delta, gamma, or epsilon), MB 1 chain, 829, Fe Rill, Fe R1, and combinations of signaling molecules, such as CD3ζ and CD28, CD27, 4-1 BB, DAP-10, OX40, and combinations thereof, as well as other similar molecules and fragments. Intracellular signaling portions of other members of the families of activating proteins may be used, such as FcγRIII and FcεRI. While usually the entire intracellular domain will be employed, in many cases it will not be necessary to use the entire intracellular polypeptide. To the extent that a truncated portion of the intracellular signaling domain may find use, such truncated portion may be used in place of the intact chain as long as it still transduces the effector function signal. The term intracellular domain is thus meant to include any truncated portion of the intracellular domain sufficient to transduce the effector function signal.


Typically, the antigen-specific extracellular domain is linked to the intracellular domain of the chimeric antigen receptor by a “transmembrane domain.” A transmembrane domain traverses the cell membrane, anchors the CAR to the T cell surface, and connects the extracellular domain to the intracellular signaling domain, thus impacting expression of the CAR on the T cell surface. Chimeric antigen receptors may also further comprise one or more costimulatory domain and/or one or more spacer. A “costimulatory domain” is derived from the intracellular signaling domains of costimulatory proteins that enhance cytokine production, proliferation, cytotoxicity, and/or persistence in vivo. A “peptide hinge” connects the antigen-specific extracellular domain to the transmembrane domain. The transmembrane domain is fused to the costimulatory domain, optionally a costimulatory domain is fused to a second costimulatory domain, and the costimulatory domain is fused to a signaling domain, not limited to CD3ζ. For example, inclusion of a spacer domain between the antigen-specific extracellular domain and the transmembrane domain, and between multiple scFvs in the case of tandem CAR, may affect flexibility of the antigen-binding domain(s) and thereby CAR function. Suitable transmembrane domains, costimulatory domains, and spacers are known in the art.


Engineered CARs may be introduced into CAR-bearing immune effector cells using retroviruses, which efficiently and stably integrate a nucleic acid sequence encoding the chimeric antigen receptor into the target cell genome. Other methods known in the art include, but are not limited to, lentiviral transduction, transposon-based systems, direct RNA transfection, and CRISPR/Cas systems (e.g., type I, type II, or type Ill systems using a suitable Cas protein such Cas3, Cas4, Cas5, Cas5e (or CasD), Cas6, Cas6e, Cas6f, Cas7, Cas8a1, Cas8a2, Cas8b, Cas8c, Cas9, Cas10, Cas1 Od, CasF, CasG, CasH, Csy1, Csy2, Csy3, Cse1 (or CasA), Cse2 (or CasB), Cse3 (or CasE), Cse4 (or CasC), Csc1, Csc2, Csa5, Csn2, Csm2, Csm3, Csm4, Csm5, Csm6, Cmr1, Cmr3, Cmr4, Cmr5, Cmr6, Csb1, Csb2, Csb3, Csx17, Csx14, Csx10, Csx16, CsaX, Csx3, Csz1, Csx15, Csf1, Csf2, Csf3, Csf4, and Cu1966, etc.). Zinc finger nucleases (ZFNs) and transcription activator-like effector nucleases (TALENs) may also be used. See, e.g., Shearer R F and Saunders D N, “Experimental design for stable genetic manipulation in mammalian cell lines: lentivirus and alternatives,” Genes Cells 2015 January; 20(1):1-10.


Manipulation of PI3K signaling can be used to prevent altered CAR-T cell differentiation due to constitutive CAR self-signaling and foster long-lived memory T cell development. pharmacologic blockade of PI3K during CAR-T manufacture and ex vivo expansion can abrogate preferential effector T cell development and restore CAR-T effector/memory ratio to that observed in empty vector transduced T cells, which can improve in vivo T cell persistence and therapeutic activity. Inhibition of p110δ PI3K can enhance efficacy and memory in tumor-specific therapeutic CD8 T cells, while inhibition of p110α PI3K can increase cytokine production and antitumor response.


This is proposed to be because the presence of a CAR on a T cell's surface can alter its activation and differentiation, even in the absence of ligand. Constitutive self-signaling through CAR, related to both the scFv framework and the signaling domains, can lead to aberrant T cell behavior, including altered differentiation and decreased survival. This is significant as the effectiveness of CAR-T cells in patients is directly associated with their in vivo longevity. The presence of the CD28 costimulatory domain increased CAR-T cell exhaustion induced by persistent CAR self-signaling; the 4-1BB costimulatory domain had a lesser effect. Furthermore, CD3-zeta significantly enhances the constitutive activation of the PI3K, AKT, mTOR, and glycolysis pathways, and fostered formation of short-lived effector cells over central/stem memory cells. See, e.g., Zhang W. et al., “Modulation of PI3K signaling to improve CAR T cell function,” Oncotarget, 2018 Nov. 9; 9(88): 35807-35808.


CAR Antigens. Suitable antigens to be genome-edited in the iNKT cells disclosed herein, and to be recognized by the CARs of iNKT-CARs disclosed herein, include antigens specific to hematologic malignancies. These can include T cell-specific antigens and/or antigens that are not specific to T cells. The antigen may be specifically bound by the chimeric antigen receptor of an iNKT-CARs cell, and the antigen for which the iNKT-CARs cell is deficient, is an antigen expressed on a malignant T cell, preferably an antigen that is overexpressed on malignant T cell (i.e., a T cell derived from a T-cell malignancy) in comparison to a nonmalignant T cell. Examples of such antigens include CD2, CD3ε, CD4, CD5, CD7, TRAC, and TCRβ.


T-cell malignancies comprise malignancies derived from T-cell precursors, mature T cells, or natural killer cells. Examples of T-cell malignancies include T-cell acute lymphoblastic leukemia/lymphoma (T-ALL), T-cell large granular lymphocyte (LGL) leukemia, human T-cell leukemia virus type 1-positive (HTLV-1+) adult T-cell leukemia/lymphoma (ATL), T-cell prolymphocytic leukemia (T-PLL), and various peripheral T-cell lymphomas (PTCLs), including but not limited to angioimmunoblastic T-cell lymphoma (AITL), ALK-positive anaplastic large cell lymphoma, and ALK-negative anaplastic large cell lymphoma.


Suitable CAR antigens can also include antigens found on the surface of a multiple myeloma cell, i.e., a malignant plasma cell, such as BCMA, CS1, CD38, and CD19. Alternatively, the CAR may be designed to express the extracellular portion of the APRIL protein, the ligand for BCMA and TACI, effectively co-targeting both BCMA and TACI for the treatment of multiple myeloma.


Additional examples of suitable antigens to be genome-edited in the iNKT cells disclosed herein, and to be recognized by the CARs of iNKT-CARs disclosed herein, are given below in Tables 1-10. These include CD2, CD3ε, CD4, CD5, CD7, TRAC, TCRβ, BCMA, CS1, and CD38.


Fratricide Resistance. The CAR-T, iNKT, NK and other CAR-bearing immune effector cells encompassed by the present disclosure are optionally deficient in one or more antigens to which the chimeric antigen receptor specifically binds and are therefore fratricide-resistant. In some embodiments, the one or more antigens of the cell is modified such the chimeric antigen receptor no longer specifically binds the one or more modified antigens. For example, the epitope of the one or more antigens recognized by the chimeric antigen receptor may be modified by one or more amino acid changes (e.g., substitutions or deletions) or the epitope may be deleted from the antigen. In other embodiments, expression of the one or more antigens is reduced in the cell by at least 50%, at least 60%, at least 70%, at least 80%, at least 90% or more. Methods for decreasing the expression of a protein are known in the art and include, but are not limited to, modifying or replacing the promoter operably linked to the nucleic acid sequence encoding the protein. In still other embodiments, the cell is modified such that the one or more antigens is not expressed, e.g., by deletion or disruption of the gene encoding the one or more antigens. In each of the above embodiments, the CAR-bearing immune effector cell may be deficient in one or preferably all the antigens to which the chimeric antigen receptor specifically binds. Methods for genetically modifying a cell to be deficient in one or more antigens are well known in art, and non-limiting examples are provided above. In an exemplary embodiment, CRISPR/cas9 gene editing can be used to modify a cell to be deficient in one or more antigens. Zinc finger nucleases (ZFNs) and transcription activator-like effector nucleases (TALENs) may also be used. See, e.g., Shearer RF and Saunders D N, “Experimental design for stable genetic manipulation in mammalian cell lines: lentivirus and alternatives,” Genes Cells 2015 January; 20(1):1-10.


Avoidance of Allogenicity. CAR-T, iNKT, NK and other CAR-bearing immune effector cells encompassed by the present disclosure may further be deficient in endogenous T cell receptor (TCR) signaling as a result of deleting a part of the T Cell Receptor (TCR)-CD3 complex. In various embodiments it may be desirable to eliminate or suppress endogenous TCR signaling in CAR-bearing immune effector cells disclosed herein. For example, decreasing or eliminating endogenous TCR signaling in CAR-T cells may prevent or reduce graft versus host disease (GvHD) when allogenic T cells are used to produce the CAR-T cells. Methods for eliminating or suppressing endogenous TCR signaling are known in the art and include, but are not limited to, deleting a part of the TCR-CD3 receptor complex, e.g., the TCR receptor alpha chain (TRAC), the TCR receptor beta chain (TRBC), CD3ε CD3γ CD3δ, and/or CD3ζ. Deleting a part of the TCR receptor complex may block TCR mediated signaling and may thus permit the safe use of allogeneic T cells as the source of CAR-T cells without inducing life-threatening GvHD.


Suicide Genes. Alternatively, or in addition, CAR-bearing immune effector cells encompassed by the present disclosure may further comprise one or more suicide genes. As used herein, “suicide gene” refers to a nucleic acid sequence introduced to a cell by standard methods known in the art that, when activated, results in the death of the cell. Suicide genes may facilitate effective tracking and elimination of the CAR-bearing immune effector cells in vivo if required. Facilitated killing by activating the suicide gene may occur by methods known in the art. Suitable suicide gene therapy systems known in the art include, but are not limited to, various the herpes simplex virus thymidine kinase (HSVtk)/ganciclovir (GCV) suicide gene therapy systems or inducible caspase 9 protein. In an exemplary embodiment, a suicide gene is a CD34/thymidine kinase chimeric suicide gene.


Components that may be included in a CAR as described herein are provided below in Tables 1 and 2.









TABLE 1







Amino acid sequences of different CAR components.










SEQ ID



Functional domains
NO: 
Amino acid sequence





CD8α signal peptide
SEQ ID
MALPVTALLLPLALLLHAARP



NO: 1






CD8α hinge
SEQ ID
TTTPAPRPPTPAPTIASQPLSLRPEACR



NO: 2
VPAAGGAHTRGLDFACD





CD28 Transmembrane
SEQ ID
FWVLVVVGGVLACYSLLVTVAFIIFWV


(Tm) domain
NO: 3






Surface
SEQ ID
MALPVTALLLPLALLLHAARPSQFRVSPLDRT


glycoprotein CD8
NO: 4
WNLGETVELKCQVLLSNPTSGCSWLFQPRGAA


alpha chain

ASPTFLLYLSQNKPKAAEGLDTQRFSGKRLGDT


isoform 1

FVLTLSDFRRENEGYYFCSALSNSIMYFSHFVP


precursor

VFLPAKPTTTPAPRPPTPAPTIASQPLSLRPEACR


(NP_001139345.1)

PAAGGAVHTRGLDFACDIYIWAPLAGTCGVLL




LSLVITLYCNHRNRRRVCKCPRPVVKSGDKPSL




SARYV





4-1BB costimulatory
SEQ ID
KRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFP


domain
NO: 5
EEEEGGCEL





CD28 costimulatory
SEQ ID
RSKRSRLLHSDYMNMTPRRPGPTRKHYQPYAP


domain
NO: 6
PRDFAAYRS





CD3 zeta (ζ)
SEQ ID
RVKFSRSADAPAYKQGQNQLYNELNLGRREEY



NO: 7
DVLDKRRGRDPEMGGKPRRKNPQEGLYNELQ




KDKMAEAYSEIGMKGERRRGKGHDGLYQGLS




TATKDTYDALHMQALPPR





P2A peptide
SEQ ID
GSGATNFSLLKQAGDVEENPGP



NO: 8






(GGGGS)4 linker
SEQ ID
GGGGSGGGGSGGGGSGGGGS



NO: 9






hCD34
SEQ ID
MPRGWTALCLLSLLPSGFMSLDNNGTATPELP



NO: 10
TQGTFSNVSTNVSYQETTTPSTLGSTSLHPVSQ




HGNEATTNITETTVKFTSTSVITSVYGNTNSSVQ




SQTSVISTVFTTPANVSTPETTLKPSLSPGNVSD




LSTTSTSLATSPTKPYTSSSPILSDIKAEIKCSG




IREVKLTQGICLEQNKTSSCAEFKKDRGEGLARV




LCGEEQADADAGAQVCSLLLAQSEVRPQCLLL




VLANRTEISSKLQLMKKHQSDLKKLGILDFTEQ




DVASHQSYSQKTLIALVTSGALLAVLGITGYFL




MNRRSWSPI





Human-Herpes Simplex
SEQ ID
MPRGWTALCLLSLLPSGFMSLDNNGTATPELP


Virus-1 (HSV)-
NO: 11
TQGTFSNVSTNVSYQETTTPSTLGSTSLHPVSQ


thymidine

HGNEATTNITETTVKFTSTSVITSVYGNTNSSVQ


kinase (TK)

SQTSVISTVFTTPANVSTPETTLKPSLSPGNVSD




LSTTSTSLATSPTKPYTSSSPILSDIKAEIKCSG




IREVKLTQGICLEQNKTSSCAEFKKDRGEGLARV




LCGEEQADADAGAQVCSLLLAQSEVRPQCLLL




VLANRTEISSKLQLMKKHQSDLKKLGILDFTEQ




DVASHQSYSQKTLIALVTSGALLAVLGITGYFL




MNRRSWSPTGEGGGGGDLGGVKLPHLFGKRL




VEARMASYPCHQHASAFDQAARSRGHSNRRT




ALRPRRQQEATEVRLEQKMPTLLRVYIDGPHG




MGKTTTTQLLVALGSRDDIVYVPEPMTYWQV




LGASETIANIYTTQHRLDQGEISAGDAAVVMTS




AQITMGMPYAVTDAVLAPHVGGEAGSSHAPPP




ALTLLLDRHPIAVMLCYPAARYLMGSMTPQAV




LAFVALIPPTLPGTNIVLGALPEDRHIDRLAKRQ




RPGERLDLAMLAAIRRVYGLLANTVRYLQGGG




SWWEDWGQLSGTAVPPQGAEPQSNAGPRPHIG




DTLFTLFRAPELLAPNGDLYNVFAWALDVLAK




RLRPMHVFILDYDQSPAGCRDALLQLTSGMVQ




THVTTPGSIPTICDLARTFAREMGEAN
















TABLE 2







Amino acid sequences of the variable heavy (VH)


and variable light (VL) chains of the scFvs.










SEQ ID



ScFv sequences
NO: 
Amino acid sequence





CD2 heavy chain variable
SEQ ID
EVKLEESGAELVKPGASVKLSCRTSGFNIKDTI


region (35.1 ATCC®HB-
NO: 12
HWVKQRPEQGLKWIGRIDPANGNTKYDPKFQ


222™)

DKATVTADTSSNTAYLQLSSLTSEDTAVYYCV




TYAYDGNWYFDVWGAGTAVTVSS





CD2 light chain variable
SEQ ID
DIKNITQSPSSMYVSLGERVTITCKASQDINSFL


region (35.1 ATCC®HB-
NO: 13
SWFQQKPGKSPKTLIYRANRLVDGVPSRFSGS


222™)

GSGQDYSLTISSLEYEDMEIYYCLQYDEFPYTF




GGGTKLEMKR





CD2 heavy chain variable
SEQ ID
EVQLEESGAELVRPGTSVKLSCKASGYTFTSY


region (OKT 11
NO: 14
WMHWIKQRPEQGLEWIGRIDPYDSETHYNEK


ATCC®CRL-8027™)

FKDKAILSVDKSSSTAYIQLSSLTSDDSAVYYC




SRRDAKYDGYALDYWGQGTSVTVSS





CD2 light chain variable
SEQ ID
DFVMTQAAPSVPVTPGESVSISCRSSKTLLHSN


region (OKT 11
NO: 15
GNTYLYWFLQRPGQSPQVLIYRMSNLASGVP


ATCC®CRL-8027™)

NRFSGSGSETTFTLRISRVEAEDVGIYYCMQHL




EYPYTFGGGTKLEIER





CD3 heavy chain variable
SEQ ID
GSQVQLQQSGAELARPGASVKMSCKASGYTF


region (OKT 3)
NO: 16
TRYTMHWVKQRPGQGLEWIGYINPSRGYTNY




NQKFKDKATLTTDKSSSTAYMQLSSLTSEDSA




VYYCARYYDDHYCLDYWGQGTTLTVSS





CD3 light chain variable
SEQ ID
QFVLTQSPAIMSASPGEKVTMTCSASSSVSYM


region (OKT 3)
NO: 17
NWYQQKSGTSPKRWIYDTSKLASGVPAHFRG




SGSGTSYSLTISGMEAEDAATYYCQQWSSNPF




TFGSGTKLEINR





CD3 heavy chain variable
SEQ ID
EVQLVESGGGLVQPGGSLRLSCAASGYSFTGY


region (UCHT1)
NO: 18
TMNWVRQAPGKCLEWVALINPYKGVSTYNQ




KFKDRFTISVDKSKNTAYLQMNSLRAEDTAV




YYCARSGYYGDSDWYFDVWGQGTLVTVSS





CD3 heavy chain variable
SEQ ID
DIQMTQSPSSLSASVGDRVTITCRASQDIRNYL


region (UCHT1)
NO: 19
NWYQQKPGKAPKLLIYYTSRLESGVPSRFSGS




GSGTDYTLTISSLQPEDFATYYCQQGNTLPWT




FGCGTKVEIK





CD7 heavy chain variable
SEQ ID
EVQLVESGGGLVKPGGSLKLSCAASGLTFSSY


region
NO: 20
AMSWVRQTPEKRLEWVASISSGGFTYYPDSV




KGRFTISRDNARNILYLQMSSLRSEDTAMYYC




ARDEVRGYLDVWGAGTTVTVS





CD7 light chain variable
SEQ ID
DIQMTQTTSSLSASLGDRVTISCSASQGISNYL


region
NO: 21
NWYQQKPDGTVKLLIYYTSSLHSGVPSRFSGS




GSGTDYSLTISNLEPEDIATYYCQQYSKLPYTF




GGGTKLEIKR





FTL3 heavy chain
SEQ ID
EVQLVQSGAEVKKPGASVKVSCKASGYTFTS


variable region (EB10)
NO: 22
YYMHWVRQAPGQGLEWMGIINPSGGSTSYAQ




KFQGRVTMTRDTSTSTVYMELSSLRSEDTAVY




YCARGVGAHDAFDIWGQGTTVTVSS





FTL3 light chain variable
SEQ ID
DVVMTQSPLSLPVTPGEPASISCRSSQSLLHSN


region (EB10)
NO: 23
GNNYLDWYLQKPGQSPQLLIYLGSNRASGVP




DRFSGSGSDTDFTLQISRVEAEDVGVYYCMQG




THPAISFGQGTRLEIK





FTL3 heavy chain
SEQ ID
EVQLVQSGAEVKKPGSSVKVSCKASGGTFSSY


variable region (NC7)
NO: 24
AISWVRQAPGQGLEWMGGIIPIFGTANYAQKF




QGRVTITADKSTSTAYMELSSLRSEDTAVYYC




ATFALFGFREQAFDIWGQGTTVTVSS





FTL3 light chain variable
SEQ ID
DIQMTQSPSSLSASVGDRVTITCRASQSISSYLN


region (NC7)
NO: 25
WYQQKPGKAPKLLIYAASSLQSGVPSRFSGSG




SGTDFTLTISSLQPEDLATYYCQQSYSTPFTFGP




GTKVDIK





FTL3 heavy chain
SEQ ID
EVQLVQSGAEVKKPGASVKVSCKASGYTFTS


variable region (D3-D4)
NO: 26
YYMHWARQAPGQGLEWMGIINPSGGSTSYAQ




KFQGRVTMTRDTSTSTVYMELSSLRSEDTAVY




YCARVVAAAVADYWGQGTLVTVS S





FTL3 light chain variable
SEQ ID
DVVMTQSPLSLPVTPGEPASISCRSSQSLLHSN


region (D3-D4)
NO: 27
GYNYLDWYLQKPGQSPQLLIYLGSNRASGVP




DRFSGSGSGTDFTLKISRVEAEDVGVYYCMQS




LQTPFTFGPGTKVDIK





CS1 heavy chain variable
SEQ ID
QVQLQQPGAELVRPGASVKLSCKASGYSFTTY


region
NO: 28
WMNWVKQRPGQGLEWIGMIHPSDSETRL




NQKFKDKATLTVDKSSSTAYMQLSSPTSEDSA




VYYCARSTMIATRAMDYWGQGTSVTVSS





CS1 light chain variable
SEQ ID
DIVMTQSQKSMSTSVGDRVSITCKASQDVITG


region
NO: 29
VAWYQQKPGQSPKLLIYSASYRYTGVPD




RFTGSGSGTDFTFTISNVQAEDLAVYYCQQHY




STPLTFGAGTKLELK





CD33 heavy chain
SEQ ID
QVQLQQPGAEVVKPGASVKMSCKASGYTFTS


variable region
NO: 30
YYIHWIKQTPGQGLEWVGVIYPGNDDISYNQK




FQGKATLTADKSSTTAYMQLSSLTSEDSAVYY




CAREVRLRYFDVWGQGTTVTVSSSG





CD33 light chain variable
SEQ ID
GSEIVLTQSPGSLAVSPGERVTMSCKSSQSVFF


region
NO: 31
SSSQKNYLAWYQQIPGQSPRLLIYWASTRESG




VPDRFTGSGSGTDFTLTISSVQPEDLAIYYCHQ




YLSSRTFGQGTKLEIKR









Mono CAR-T Cells

In certain embodiments, the disclosure provides an engineered T cell comprising a single CAR, that specifically binds an antigen or cell surface protein, wherein the T cell is optionally deficient in that antigen or cell surface protein (e.g., CD7CARTΔCD7 cell). In non-limiting examples, the deficiency in the antigen or cell surface protein resulted from (a) modification of antigen or cell surface protein expressed by the T cell such that the chimeric antigen receptors no longer specifically binds the modified antigen or cell surface protein (e.g., the epitope of the one or more antigens recognized by the chimeric antigen receptor may be modified by one or more amino acid changes (e.g., substitutions or deletions) or the epitope may be deleted from the antigen), (b) modification of the T cell such that expression of antigen or cell surface protein is reduced in the T cell by at least 50%, at least 60%, at least 70%, at least 80%, at least 90% or more, or (c) modification of the T cell such that antigen or cell surface protein is not expressed (e.g., by deletion or disruption of the gene encoding antigen or cell surface protein). In each of the above embodiments, the CAR-T cell may be deficient in one or preferably all the antigens or cell surface proteins to which the chimeric antigen receptor specifically binds. The methods to genetically modify a T cell to be deficient in one or more antigens or cell surface proteins are well known in art and non-limiting examples are provided herein. In embodiments described below, the CRISPR-Cas9 system is used to modify a T cell to be deficient in one or more antigens. Any of these may be accomplished by the methods disclosed herein. In further embodiments, the T cell comprises a suicide gene.


For example, the CAR for a CD7 specific CAR-T cell may be generated by cloning a commercially synthesized anti-CD7 single chain variable fragment (scFv) into a 3rd generation CAR backbone with CD28 and/or 4-1BB internal signaling domains. An extracellular hCD34 domain may be added after a P2A peptide to enable both detection of CAR following viral transduction and purification using anti-hCD34 magnetic beads. A similar method may be followed for making CARs specific for other malignant T cell antigens.


CAR-T cells encompassed by the present disclosure may further be deficient in endogenous T cell receptor (TCR) signaling as a result of deleting a part of the T Cell Receptor (TCR)-CD3 complex. In various embodiments it may be desirable to eliminate or suppress endogenous TCR signaling in CAR-T cells disclosed herein. For example, decreasing or eliminating endogenous TCR signaling in CAR-T cells may prevent or reduce graft versus host disease (GvHD) when allogenic T cells are used to produce the CAR-T cells. Methods for eliminating or suppressing endogenous TCR signaling are known in the art and include, but are not limited to, deleting a part of the TCR-CD3 receptor complex, e.g., the TCR receptor alpha chain (TRAC), the TCR receptor beta chain (TCRβ) or subtypes thereof, TCRδ, TCRγ, CD3ε, CD3γ, and/or CD3δ. Deleting a part of the TCR receptor complex may block TCR mediated signaling and may thus permit the safe use of allogeneic T cells as the source of CAR-T cells without inducing life-threatening GvHD.


In addition, the CAR-T cells encompassed by the present disclosure may further comprise one or more suicide genes as described herein.


In a similar manner, other mono-CAR-T cells may be constructed and are given below in Table 3.









TABLE 3







Mono CARs and CAR-Ts












Antigen Target
Antigen Deletion/



Example
of CAR-T cells
Suppression







M1
APRIL




M2
APRIL
APRIL



M3
APRIL
APRIL + TRAC



M4
APRIL
APRIL + CD3ε



M5
APRIL
CD3ε



M6
BCMA




M7
CD117




M8
CD117
CD117



M9
CD123




M10
CD123
CD123



M11
CD135




M12
CD135
CD135



M13
CD138




M14
CD19




M15
CD1a




M16
CD1a
CD3ε



M17
CD1a
TRAC



M18
CD1a
CD1a + TRAC



M19
CD1a
CD1a + CD3ε



M20
CD2




M21
CD2
CD2



M22
CD2
CD2 + TRAC



M23
CD2
CD2 + CD3ε



M24
CD20




M25
CD21




M26
CD22




M27
CD23




M28
CD3




M29
CD3
CD3ε



M30
CD3
CD3ε + TRAC



M31
CD33




M32
CD33
CD33



M33
CD371




M34
CD371
CD371



M35
CD38




M36
CD38
CD38



M37
CD4




M38
CD4
CD4



M39
CD4
CD4 + TRAC



M40
CD4
CD4 + CD3ε



M41
CD5




M42
CD5
CD5



M43
CD5
CD5 + TRAC



M44
CD5
CD5 + CD3ε



M45
CD56




M46
CD56
CD56



M47
CD56
CD56 + TRAC



M48
CD56
CD56 + CD3ε



M49
CD56
CD3ε



M50
CD56
TRAC



M51
CD7




M52
CD7
CD7



M53
CD7
CD7 + TRAC



M54
CD7
CD7 + CD3ε



M55
CD79A




M56
CD79B




M57
CS1




M58
CS1
CS1



M59
Tim-3




M60
Tim-3
Tim-3



M61
Tim-3
Tim-3 + TRAC



M62
Tim-3
TRAC



M63
Tim-3
CD3ε



M64
Tim-3
Tim-3 + CD3ε










Disclosed are embodiments of CAR amino acid sequences that can be expressed on the surface of a genome-edited CAR-T cell derived from a cytotoxic T cell, a memory T cell, or a gamma delta (γδ) T cell.









TABLE 4







Amino Acid Sequences of Mono Chimeric Antigen Receptors (CARs).









Mono CAR
SEQ ID



Constructs
NO: 
Amino acid sequence





CD7-CAR-4-
SEQ ID
MALPVTALLLPLALLLHAARPDIQMTQTTSSLSASLGDRVT


1BB_CD34
NO: 32
ISCSASQGISNYLNWYQQKPDGTVKLLIYYTSSLHSGVPSRF




SGSGSGTDYSLTISNLEPEDIATYYCQQYSKLPYTFGGGTKL




EIKRGGGGSGGGGSGGGGSGGGGSEVQLVESGGGLVKPG




GSLKLSCAASGLTFSSYAMSWVRQTPEKRLEWVASISSGGF




TYYPDSVKGRFTISRDNARNILYLQMSSLRSEDTAMYYCA




RDEVRGYLDVWGAGTTVTVSPRASTTTPAPRPPTPAPTIAS




QPLSLRPEACRPAAGGAVHTRGLDFACDFWVLVVVGGVL




ACYSLLVTVAFIIFWVKRGRKKLLYIFKQPFMRPVQTTQEE




DGCSCRFPEEEEGGCELRVKFSRSADAPAYKQGQNQLYNE




LNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNEL




QKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTY




DALHMQALPPRRTDGSGATNFSLLKQAGDVEENPGPVSEA




MPRGWTALCLLSLLPSGFMSLDNNGTATPELPTQGTFSNVS




TNVSYQETTTPSTLGSTSLHPVSQHGNEATTNITETTVKFTS




TSVITSVYGNTNSSVQSQTSVISTVFTTPANVSTPETTLKPSL




SPGNVSDLSTTSTSLATSPTKPYTSSSPILSDIKAEIKCSGI




REVKLTQGICLEQNKTSSCAEFKKDRGEGLARVLCGEEQADA




DAGAQVCSLLLAQSEVRPQCLLLVLANRTEISSKLQLMKK




HQSDLKKLGILDFTEQDVASHQSYSQKTLIALVTSGALLAV




LGITGYFLMNRRSWSPI





CD7-CAR-4-
SEQ ID
MALPVTALLLPLALLLHAARPDIQMTQTTSSLSASLGDRVT


1BB_CD34_TK
NO: 33
ISCSASQGISNYLNWYQQKPDGTVKLLIYYTSSLHSGVPSRF




SGSGSGTDYSLTISNLEPEDIATYYCQQYSKLPYTFGGGTKL




EIKRGGGGSGGGGSGGGGSGGGGSEVQLVESGGGLVKPG




GSLKLSCAASGLTFSSYAMSWVRQTPEKRLEWVASISSGGF




TYYPDSVKGRFTISRDNARNILYLQMSSLRSEDTAMYYCA




RDEVRGYLDVWGAGTTVTVSPRASTTTPAPRPPTPAPTIAS




QPLSLRPEACRPAAGGAVHTRGLDFACDFWVLVVVGGVL




ACYSLLVTVAFIIFWVKRGRKKLLYIFKQPFMRPVQTTQEE




DGCSCRFPEEEEGGCELRVKFSRSADAPAYKQGQNQLYNE




LNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNEL




QKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTY




DALHMQALPPRRTDGSGATNFSLLKQAGDVEENPGPVSEA




MPRGWTALCLLSLLPSGFMSLDNNGTATPELPTQGTFSNVS




TNVSYQETTTPSTLGSTSLHPVSQHGNEATTNITETTVKFTS




TSVITSVYGNTNSSVQSQTSVISTVFTTPANVSTPETTLKPSL




SPGNVSDLSTTSTSLATSPTKPYTSSSPILSDIKAEIKCSGIRE




VKLTQGICLEQNKTSSCAEFKKDRGEGLARVLCGEEQADA




DAGAQVCSLLLAQSEVRPQCLLLVLANRTEISSKLQLMKK




HQSDLKKLGILDFTEQDVASHQSYSQKTLIALVTSGALLAV




LGITGYFLMNRRSWSPTGEGGGGGDLGGVKLPHLFGKRLV




EARMASYPCHQHASAFDQAARSRGHSNRRTALRPRRQQE




ATEVRLEQKMPTLLRVYIDGPHGMGKTTTTQLLVALGSRD




DIVYVPEPMTYWQVLGASETIANIYTTQHRLDQGEISAGDA




AVVMTSAQITMGMPYAVTDAVLAPHVGGEAGSSHAPPPA




LTLLLDRHPIAVMLCYPAARYLMGSMTPQAVLAFVALIPPT




LPGTNIVLGALPEDRHIDRLAKRQRPGERLDLAMLAAIRRV




YGLLANTVRYLQGGGSWWEDWGQLSGTAVPPQGAEPQS




NAGPRPHIGDTLFTLFRAPELLAPNGDLYNVFAWALDVLA




KRLRPMHVFILDYDQSPAGCRDALLQLTSGMVQTHVTTPG




SIPTICDLARTFAREMGEAN





CD7-CAR-
SEQ ID
MALPVTALLLPLALLLHAARPDIQMTQTTSSLSASLGDRVT


CD28_CD34
NO: 34
ISCSASQGISNYLNWYQQKPDGTVKLLIYYTSSLHSGVPSRF




SGSGSGTDYSLTISNLEPEDIATYYCQQYSKLPYTFGGGTKL




EIKRGGGGSGGGGSGGGGSGGGGSEVQLVESGGGLVKPG




GSLKLSCAASGLTFSSYAMSWVRQTPEKRLEWVASISSGGF




TYYPDSVKGRFTISRDNARNILYLQMSSLRSEDTAMYYCA




RDEVRGYLDVWGAGTTVTVSPRASTTTPAPRPPTPAPTIAS




QPLSLRPEACRPAAGGAVHTRGLDFACDFWVLVVVGGVL




ACYSLLVTVAFIIFWVRSKRSRLLHSDYMNMTPRRPGPTRK




HYQPYAPPRDFAAYRSRVKFSRSADAPAYKQGQNQLYNE




LNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNEL




QKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTY




DALHMQALPPRRTDGSGATNFSLLKQAGDVEENPGPVSEA




MPRGWTALCLLSLLPSGFMSLDNNGTATPELPTQGTFSNVS




TNVSYQETTTPSTLGSTSLHPVSQHGNEATTNITETTVKFTS




TSVITSVYGNTNSSVQSQTSVISTVFTTPANVSTPETTLKPSL




SPGNVSDLSTTSTSLATSPTKPYTSSSPILSDIKAEIKCSGIRE




VKLTQGICLEQNKTSSCAEFKKDRGEGLARVLCGEEQADA




DAGAQVCSLLLAQSEVRPQCLLLVLANRTEISSKLQLMKK




HQSDLKKLGILDFTEQDVASHQSYSQKTLIALVTSGALLAV




LGITGYFLMNRRSWSPI





CD7-CAR-
SEQ ID
MALPVTALLLPLALLLHAARPDIQMTQTTSSLSASLGDRVT


CD28_CD34_TK
NO: 35
ISCSASQGISNYLNWYQQKPDGTVKLLIYYTSSLHSGVPSRF




SGSGSGTDYSLTISNLEPEDIATYYCQQYSKLPYTFGGGTKL




EIKRGGGGSGGGGSGGGGSGGGGSEVQLVESGGGLVKPG




GSLKLSCAASGLTFSSYAMSWVRQTPEKRLEWVASISSGGF




TYYPDSVKGRFTISRDNARNILYLQMSSLRSEDTAMYYCA




RDEVRGYLDVWGAGTTVTVSPRASTTTPAPRPPTPAPTIAS




QPLSLRPEACRPAAGGAVHTRGLDFACDFWVLVVVGGVL




ACYSLLVTVAFIIFWVRSKRSRLLHSDYMNMTPRRPGPTRK




HYQPYAPPRDFAAYRSRVKFSRSADAPAYKQGQNQLYNE




LNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNEL




QKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTY




DALHMQALPPRRTDGSGATNFSLLKQAGDVEENPGPVSEA




MPRGWTALCLLSLLPSGFMSLDNNGTATPELPTQGTFSNVS




TNVSYQETTTPSTLGSTSLHPVSQHGNEATTNITETTVKFTS




TSVITSVYGNTNSSVQSQTSVISTVFTTPANVSTPETTLKPSL




SPGNVSDLSTTSTSLATSPTKPYTSSSPILSDIKAEIKCSGIRE




VKLTQGICLEQNKTSSCAEFKKDRGEGLARVLCGEEQADA




DAGAQVCSLLLAQSEVRPQCLLLVLANRTEISSKLQLMKK




HQSDLKKLGILDFTEQDVASHQSYSQKTLIALVTSGALLAV




LGITGYFLMNRRSWSPTGEGGGGGDLGGVKLPHLFGKRLV




EARMASYPCHQHASAFDQAARSRGHSNRRTALRPRRQQE




ATEVRLEQKMPTLLRVYIDGPHGMGKTTTTQLLVALGSRD




DIVYVPEPMTYWQVLGASETIANIYTTQHRLDQGEISAGDA




AVVMTSAQITMGMPYAVTDAVLAPHVGGEAGSSHAPPPA




LTLLLDRHPIAVMLCYPAARYLMGSMTPQAVLAFVALIPPT




LPGTNIVLGALPEDRHIDRLAKRQRPGERLDLAMLAAIRRV




YGLLANTVRYLQGGGSWWEDWGQLSGTAVPPQGAEPQS




NAGPRPHIGDTLFTLFRAPELLAPNGDLYNVFAWALDVLA




KRLRPMHVFILDYDQSPAGCRDALLQLTSGMVQTHVTTPG




SIPTICDLARTFAREMGEAN





CD79B-CAR-
SEQ ID
MALPVTALLLPLALLLHAARPGSDIQLTQSPSSLSASVGDR


CD28_CD34
NO: 36
VTITCKASQSVDYEGDSFLNWYQQKPGKAPKLLIYAASNL




ESGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSNEDPL




TFGQGTKVEIKRGGGGSGGGGSGGGGSGGGGSGGGGSEV




QLVESGGGLVQPGGSLRLSCAASGYTFSSYWIEWVRQAPG




KGLEWIGEILPGGGDTNYNEIFKGRATFSADTSKNTAYLQM




NSLRAEDTAVYYCTRRVPIRLDYWGQGTLVTVSSPRASTT




TPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFAC




DFWVLVVVGGVLACYSLLVTVAFIIFWVRSKRSRLLHSDY




MNMTPRRPGPTRKHYQPYAPPRDFAAYRSRVKFSRSADAP




AYKQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPR




RKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGL




YQGLSTATKDTYDALHMQALPPRRTDGSGATNFSLLKQAG




DVEENPGPVSEAMPRGWTALCLLSLLPSGFMSLDNNGTAT




PELPTQGTFSNVSTNVSYQETTTPSTLGSTSLHPVSQHGNEA




TTNITETTVKFTSTSVITSVYGNTNSSVQSQTSVISTVFTTPA




NVSTPETTLKPSLSPGNVSDLSTTSTSLATSPTKPYTSSSPILS




DIKAEIKCSGIREVKLTQGICLEQNKTSSCAEFKKDRGEGLA




RVLCGEEQADADAGAQVCSLLLAQSEVRPQCLLLVLANRT




EISSKLQLMKKHQSDLKKLGILDFTEQDVASHQSYSQKTLI




ALVTSGALLAVLGITGYFLMNRRSWSPTGEGGGGGFKRDL




GGVKLPHLFGKRLVEARMASYPCHQHASAFDQAARSRGH




SNRRTALRPRRQQEATEVRLEQKMPTLLRVYIDGPHGMGK




TTTTQLLVALGSRDDIVYVPEPMTYWQVLGASETIANIYTT




QHRLDQGEISAGDAAVVMTSAQITMGMPYAVTDAVLAPH




VGGEAGSSHAPPPALTLLLDRHPIAVMLCYPAARYLMGSM




TPQAVLAFVALIPPTLPGTNIVLGALPEDRHIDRLAKRQRPG




ERLDLAMLAAIRRVYGLLANTVRYLQGGGSWWEDWGQL




SGTAVPPQGAEPQSNAGPRPHIGDTLFTLFRAPELLAPNGD




LYNVFAWALDVLAKRLRPMHVFILDYDQSPAGCRDALLQ




LTSGMVQTHVTTPGSIPTICDLARTFAREMGEAN





CD2-CAR-
SEQ ID
MALPVTALLLPLALLLHAARPDIVMTQAAPSVPVTPGESVS


CD28_CD34
NO: 37
ISCRSSKTLLHSNGNTYLYWFLQRPGQSPQVLIYRMSNLAS




GVPNRFSGSGSETTFTLRISRVEAEDVGIYYCMQHLEYPYT




FGGGTKLEIERGGGGSGGGGSGGGGSGGGGSEVQLEESGA




ELVRPGTSVKLSCKASGYTFTSYWMHWIKQRPEQGLEWIG




RIDPYDSETHYNEKFKDKAILSVDKSSSTAYIQLSSLTSDDS




AVYYCSRRDAKYDGYALDYWGQGTSVTVSSPRASTTTPA




PRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDF




WVLVVVGGVLACYSLLVTVAFIIFWVRSKRSRLLHSDYMN




MTPRRPGPTRKHYQPYAPPRDFAAYRSRVKFSRSADAPAY




KQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRK




NPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQ




GLSTATKDTYDALHMQALPPRRTDGSGATNFSLLKQAGDV




EENPGPVSEAMPRGWTALCLLSLLPSGFMSLDNNGTATPEL




PTQGTFSNVSTNVSYQETTTPSTLGSTSLHPVSQHGNEATT




NITETTVKFTSTSVITSVYGNTNSSVQSQTSVISTVFTTPANV




STPETTLKPSLSPGNVSDLSTTSTSLATSPTKPYTSSSPILSDI




KAEIKCSGIREVKLTQGICLEQNKTSSCAEFKKDRGEGLAR




VLCGEEQADADAGAQVCSLLLAQSEVRPQCLLLVLANRTE




ISSKLQLMKKHQSDLKKLGILDFTEQDVASHQSYSQKTLIA




LVTSGALLAVLGITGYFLMNRRSWSPI





CD2-CAR-4-
SEQ ID
MALPVTALLLPLALLLHAARPDIVMTQAAPSVPVTPGESVS


1BB_CD34
NO: 38
ISCRSSKTLLHSNGNTYLYWFLQRPGQSPQVLIYRMSNLAS




GVPNRFSGSGSETTFTLRISRVEAEDVGIYYCMQHLEYPYT




FGGGTKLEIERGGGGSGGGGSGGGGSGGGGSEVQLEESGA




ELVRPGTSVKLSCKASGYTFTSYWMHWIKQRPEQGLEWIG




RIDPYDSETHYNEKFKDKAILSVDKSSSTAYIQLSSLTSDDS




AVYYCSRRDAKYDGYALDYWGQGTSVTVSSPRASTTTPA




PRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDF




WVLVVVGGVLACYSLLVTVAFIIFWVKRGRKKLLYIFKQP




FMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAY




KQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRK




NPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQ




GLSTATKDTYDALHMQALPPRRTDGSGATNFSLLKQAGDV




EENPGPVSEAMPRGWTALCLLSLLPSGFMSLDNNGTATPEL




PTQGTFSNVSTNVSYQETTTPSTLGSTSLHPVSQHGNEATT




NITETTVKFTSTSVITSVYGNTNSSVQSQTSVISTVFTTPANV




STPETTLKPSLSPGNVSDLSTTSTSLATSPTKPYTSSSPILSDI




KAEIKCSGIREVKLTQGICLEQNKTSSCAEFKKDRGEGLAR




VLCGEEQADADAGAQVCSLLLAQSEVRPQCLLLVLANRTE




ISSKLQLMKKHQSDLKKLGILDFTEQDVASHQSYSQKTLIA




LVTSGALLAVLGITGYFLMNRRSWSPI





CD3-CD28-CD34
SEQ ID
MALPVTALLLPLALLLHAARPGSQVQLQQSGAELARPGAS



NO: 39
VKMSCKASGYTFTRYTMHWVKQRPGQGLEWIGYINPSRG




YTNYNQKFKDKATLTTDKSSSTAYMQLSSLTSEDSAVYYC




ARYYDDHYCLDYWGQGTTLTVSSGGGGSGGGGSGGGGS




GGGGSQIVLTQSPAIMSASPGEKVTMTCSASSSVSYMNWY




QQKSGTSPKRWIYDTSKLASGVPAHFRGSGSGTSYSLTISG




MEAEDAATYYCQQWSSNPFTFGSGTKLEINRPRASTTTPAP




RPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDFW




VLVVVGGVLACYSLLVTVAFIIFWVRSKRSRLLHSDYMNM




TPRRPGPTRKHYQPYAPPRDFAAYRSRVKFSRSADAPAYK




QGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKN




PQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQG




LSTATKDTYDALHMQALPPRRTDGSGATNFSLLKQAGDVE




ENPGPVSEAMPRGWTALCLLSLLPSGFMSLDNNGTATPELP




TQGTFSNVSTNVSYQETTTPSTLGSTSLHPVSQHGNEATTNI




TETTVKFTSTSVITSVYGNTNSSVQSQTSVISTVFTTPANVST




PETTLKPSLSPGNVSDLSTTSTSLATSPTKPYTSSSPILSDI




KAEIKCSGIREVKLTQGICLEQNKTSSCAEFKKDRGEGLARVL




CGEEQADADAGAQVCSLLLAQSEVRPQCLLLVLANRTEISS




KLQLMKKHQSDLKKLGILDFTEQDVASHQSYSQKTLIALV




TSGALLAVLGITGYFLMNRRSWSPI









Tandem CAR-T Cells

A tandem CAR-T cell (tCAR-T), is a T cell with a single chimeric antigen polypeptide comprising two distinct extracellular ligand-binding (antigen/protein recognition) domains capable of interacting with two different cell surface molecules (e.g., antigen/protein), wherein the extracellular ligand-binding domains are linked together by one or more flexible linkers and share one or more costimulatory domains, wherein the binding of the first or second extracellular ligand-binding domain will signal through one or more the costimulatory domains(s) and a signaling transducing domain.


In certain embodiments, the T cell is deficient in one or more antigens or cell surface proteins (e.g., CD7 and CD2 for a CD7*CD2-tCARΔCD7ΔCD2 cell, or CD2 for a CD3*CD2-tCARΔCD3ΔCD2 cell). In non-limiting examples, the deficiency in the antigen(s) or cell surface protein(s) resulted from (a) modification of antigen or cell surface protein expressed by the T cell such that the chimeric antigen receptor no longer specifically binds the modified antigen(s) or cell surface protein(s) (e.g., the epitope of the one or more antigens recognized by the chimeric antigen receptor may be modified by one or more amino acid changes (e.g., substitutions or deletions) or the epitope may be deleted from the antigen), (b) modification of the T cell such that expression of antigen(s) or cell surface protein(s) is/are reduced in the T cell by at least 50%, at least 60%, at least 70%, at least 80%, at least 90% or more, or (c) modification of the T cell such that antigen(s) or cell surface protein(s) is/are not expressed (e.g., by deletion or disruption of the gene encoding antigen or cell surface protein). In each of the above embodiments, the CAR-T cell may be deficient in one or preferably all the antigens or cell surface proteins to which the chimeric antigen receptor specifically binds. The methods to genetically modify a T cell to be deficient in one or more antigens or cell surface proteins are well known in art and non-limiting examples are provided herein. In embodiments described below, the CRISPR-Cas9 system is used to modify a T cell to be deficient in one or more antigen(s) or cell surface protein(s). Any of these may be accomplished by the methods disclosed herein. In further embodiments, the T cell comprises a suicide gene.


A tCAR for a genome-edited, tandem CAR-T cell, i.e., CD2*CD3-tCARTΔCD2ΔCD3ε, may be generated by cloning a commercially synthesized anti-CD2 single chain variable fragment (scFv) and an anti-CD3 single chain variable fragment (scFv), separated by a peptide linker, into a lentiviral vector containing, e.g., a 2nd or 3rd generation CAR backbone with CD28 and/or 4-1BB internal signaling domains. An extracellular hCD34 domain may be added after a P2A peptide to enable both detection of CAR following viral transduction and purification using anti-hCD34 magnetic beads. A similar method may be followed for making tCARs specific for other malignant T cell antigens.


Tandem CARs may have different linker structures, i.e., be linear or hairpin, and the hairpin linker may optionally comprise a (Cys=Cys) double-stranded bond (DSB).


A linear tandem CAR-T cell comprises a chimeric antigen receptor (CAR) polypeptide comprising a first signal peptide, a first extracellular ligand-binding domain, a second extracellular ligand-binding domain, a hinge region, a transmembrane domain, one or more co-stimulatory domains, and a signaling transducing domain, wherein the first extracellular ligand-binding antigen recognition domain and the second extracellular ligand-binding antigen recognition domain have affinities for different cell surface molecules, i.e., antigens on a cancer cell, for example, a malignant T cell, B cell, or plasma cell; and wherein the linear tandem CAR-T cell possesses one or more genetic modifications, deletions, or disruptions resulting in reduced expression of the cell surface molecules in the linear tandem CAR-T cell.


In another embodiment, the signal peptide is the signal peptide from human CD8α.


In a third embodiment, the first extracellular ligand-binding domain comprises a single chain antibody fragment (scFv), comprising the light (VL) and the heavy (VH) variable fragment, designated VH1 and VL1 and joined by a linker (e.g., GGGGS (SEQ ID NO: 3065)). In some embodiments, this linker peptide is repeated 2, 3, 4, 5 or 6 times. In some embodiments. the first antigen recognition domain can be selected from: 1) VH1—(GGGGS)3-4 (SEQ ID NO: 3066)—VL1 or 2) VL1—(GGGGS)4 (SEQ ID NO: 3066)—VH1.


In some embodiments, the second extracellular ligand-binding domain comprises a single chain antibody fragment (scFv), comprising the light (VL) and the heavy (VH) variable fragment, designated VH2 and VL2 and joined by a linker (e.g., GGGGS (SEQ ID NO: 3065)). In some embodiments, this linker peptide is repeated 2, 3, 4, 5 or 6 times. In some embodiments, the first antigen recognition domain can be selected from: 1) VH2-(GGGGS)3-4 (SEQ ID NO: 3066)—VL2 or 2) VL2—(GGGGS)3-4 (SEQ ID NO: 3066)—VH2.


In further embodiments, the first antigen recognition domain and second antigen recognition domain are connected by a short linker peptide of 5 amino acids (GGGGS (SEQ ID NO: 3065)). In some embodiments, this linker peptide is repeated 2, 3, 4, 5, or 6 times.


Tandem CAR Constructs

In one embodiment, the first extracellular ligand-binding domain antigen recognition comprises a single chain antibody fragment (scFv), comprising the heavy (VH) and the light (VL) variable fragment, designated VH1 and VL1, and joined by a linker (e.g., GGGGS (SEQ ID NO: 3065)), targets a cell surface molecule, i.e., an antigen expressed on a malignant cell.


In certain embodiments, the heavy (VH) and the light (VL) variable fragment, designated VH1 and VL1, targeting an antigen expressed on a malignant T cell is selected from BCMA, CS1, CD38, CD138, CD19, CD33, CD123, CD371, CD117, CD135, Tim-3, CD5, CD7, CD2, CD4, CD3, CD79A, CD79B, APRIL, CD56, and CD1a.


In certain embodiments, the second extracellular ligand-binding domain antigen recognition comprises a single chain antibody fragment (scFv), comprising the heavy (VH) and the light (VL) variable fragment, designated VH2 and VL2, and joined by a linker (e.g., GGGGS (SEQ ID NO: 3065)), and targets a cell surface molecule, i.e., an antigen, expressed on a malignant cell.


In certain embodiments, the heavy (VH) and the light (VL) variable fragments, designated VH2 and VL2, targeting an antigen expressed on a malignant T cell is selected from BCMA, CS1, CD38, CD138, CD19, CD33, CD123, CD371, CD117, CD135, Tim-3, CD5, CD7, CD2, CD4, CD3, CD79A, CD79B, APRIL, CD56, and CD1a and differs from the variable heavy (VH1) and light sequences (VL1) of the first extracellular ligand-binding domain of the CAR molecule.


Additional examples of tandem CARs are given below in Table 5.









TABLE 5







Tandem CARs and CAR-Ts









Example
Antigen Target CAR-T cell
Antigen Deletion/Suppression





T1
APRILxBCMA



T2
APRILxCD19



T3
APRILxCD38



T4
APRILxCD38
CD38


T5
APRILxCS1



T6
APRILxCS1
CS1


T7
BCMAxCD19



T8
BCMAxCD38



T9
BCMAxCD38
CD38


T10
BCMAxCS1



T11
BCMAxCS1
CS1


T12
CD138xAPRIL



T13
CD138xBCMA



T14
CD138xCD19



T15
CD138xCD38



T16
CD138xCD38
CD38


T17
CD138xCD79A



T18
CD138xCD79B



T19
CD138xCS1



T20
CD138xCS1
CS1


T21
CD19xCD38



T22
CD19xCD38
CD38


T23
CD2xCD3ε



T24
CD2xCD3ε
CD2


T25
CD2xCD3ε
CDε


T26
CD2xCD3ε
CD2 and CD3ε


T27
CD2xCD4



T28
CD2xCD4
CD2


T29
CD2xCD4
CD4


T30
CD2xCD4
CD2 and CD4


T31
CD2xCD4
CD2 and TRAC


T32
CD2xCD4
CD4 and TRAC


T33
CD2xCD4
CD2 and CD4 and TRAC


T34
CD2xCD5



T35
CD2xCD5
CD2


T36
CD2xCD5
CD5


T37
CD2xCD5
CD2 and CD5


T38
CD2xCD5
CD2 and TRAC


T39
CD2xCD5
CD5 and TRAC


T40
CD2xCD5
CD2 and CD5 and TRAC


T41
CD2xCD7



T42
CD2xCD7
CD2


T43
CD2xCD7
CD7


T44
CD2xCD7
CD2 and CD7


T45
CD2xCD7
CD2 and TRAC


T46
CD2xCD7
CD7 and TRAC


T47
CD2xCD7
CD2 and CD7 and TRAC


T48
CD3εxCD4



T49
CD3εxCD4
CD3ε


T50
CD3εxCD4
CD4


T51
CD3εxCD4
CD3ε and CD4


T52
CD3εxCD5



T53
CD3εxCD5
CD3ε


T54
CD3εxCD5
CD5


T55
CD3εxCD5
CD3ε and CD5


T56
CD3εxCD7



T57
CD3εxCD7
CD3ε


T58
CD3εxCD7
CD7


T59
CD3εxCD7
CD3ε and CD7


T60
CD4xCD5



T61
CD4xCD5
CD4


T62
CD4xCD5
CD5


T63
CD4xCD5
CD4 and CD5


T64
CD4xCD5
CD4 and TRAC


T65
CD4xCD5
CD5 and TRAC


T66
CD4xCD5
CD4 and CD5 and TRAC


T67
CD4xCD7



T68
CD4xCD7
CD4


T69
CD4xCD7
CD7


T70
CD4xCD7
CD4 and CD7


T71
CD4xCD7
CD4 and TRAC


T72
CD4xCD7
CD4 and TRAC


T73
CD4xCD7
CD4 and CD7 and TRAC


T74
CD5xCD7



T75
CD5xCD7
CD5


T76
CD5xCD7
CD7


T77
CD5xCD7
CD5 and CD7


T78
CD5xCD7
CD5 and TRAC


T79
CD5xCD7
CD7 and TRAC


T80
CD5xCD7
CD5 and CD7 and TRAC


T81
CD79AxAPRIL



T82
CD79AxBCMA



T83
CD79AxCD19



T84
CD79AxCD38



T85
CD79AxCD38
CD38


T86
CD79AxCD79B



T87
CD79AxCS1



T88
CD79AxCS1
CS1


T89
CD79BxAPRIL



T90
CD79BxBCMA



T91
CD79BxCD19



T92
CD79BxCD38



T93
CD79BxCD38
CD38


T94
CD79BxCD79A



T95
CD79BxCS1



T96
CD79BxCS1
CS1


T97
CS1xCD19



T98
CS1xCD19
CS1


T99
CS1xCD38



T100
CS1xCD38
CS1


T101
CS1xCD38
CD38


T102
CS1xCD38
CS1 and CD38


T103
TCRβxCD2



T104
TCRβxCD2
TCRβ


T105
TCRβxCD2
CD2


T106
TCRβxCD2
TCRβ and CD2


T107
TCRβxCD3ε



T108
TCRβxCD3ε
TCRβ


T109
TCRβxCD3ε
CDε


T110
TCRβxCD3ε
TCRβ and CD3ε


T111
TCRβxCD4



T112
TCRβxCD4
TCRβ


T113
TCRβxCD4
CD4


T114
TCRβxCD4
TCRβ and CD4


T115
TCRβxCD5



T116
TCRβxCD5
TCRβ


T117
TCRβxCD5
CD5


T118
TCRβxCD5
TCRβ and CD5


T119
TCRβxCD7



T120
TCRβxCD7
TCRβ


T121
TCRβxCD7
CD7


T122
TCRβxCD7
TCRβ and CD7


T123
TRACxCD2



T124
TRACxCD2
TRAC


T125
TRACxCD2
CD2


T126
TRACxCD2
TRAC and CD2


T127
TRACxCD3ε



T128
TRACxCD3ε
TRAC


T129
TRACxCD3ε
CDε


T130
TRACxCD3ε
TRAC and CD3ε


T131
TRACxCD4



T132
TRACxCD4
TRAC


T133
TRACxCD4
CD4


T134
TRACxCD4
TRAC and CD4


T135
TRACxCD5



T136
TRACxCD5
TRAC


T137
TRACxCD5
CD5


T138
TRACxCD5
TRAC and CD5


T139
TRACxCD7



T140
TRACxCD7
TRAC


T141
TRACxCD7
CD7


T142
TRACxCD7
TRAC and CD7









In some embodiments, hairpin tandem CAR constructs may be provided herein, such as including, but not limited to, a construct incorporating the VH and VL domains of CD2 and CD3 scFvs (Table 6).









TABLE 6





Hairpin Tandem CAR Constructs Targeting CD2 and CD3.






















Clone 5
Clone 6
Clone 7
Clone 8
Clone 13
Clone 14
Clone 15
Clone 16





CD8a
CD8a
CD8a
CD8a
CD8a
CD8a
CD8a
CD8a





CD3-VL
CD3-VL
CD3-VL
CD3-VL
CD2-VL
CD2-VL
CD3-VL
CD3-VL





GGGGS4
GGGGS4
GGGGS4
GGGGS4
GGGGS4
GGGGS4
GGGGS4
GGGGS4


(SEQ ID
(SEQ ID
(SEQ ID
(SEQ ID
(SEQ ID
(SEQ ID
(SEQ ID
(SEQ ID


NO: 9)
NO: 9)
NO: 9)
NO: 9)
NO: 9)
NO: 9)
NO: 9)
NO: 9)





CD2-VL
CD2-VL
CD2-VL
CD2-VL
CD3-VL
CD3-VL
CD2-VL
CD2-VL





(GGGGS)10
(GGGGS)4
(GGGG
(GGG
(GGG
(GGGGS
(GGGG
(GGGG


(SEQ
GGGG
S)10
GS)4G
GS)10
)4GGGG
S)10
S)4GGGG


ID NO:
P(GGGG
(SEQ
GGGP(G
(SEQ
P(GGGG
(SEQ
P(GGGG


3067)
S)4
ID NO: 
GGGS)4
ID NO: 
S)4
ID NO: 
S)4



(SEQ
3067)
(SEQ
3067)
(SEQ
3067)
(SEQ



ID NO: 

ID NO: 

ID NO: 

ID NO: 



3068)

3068)

3068)

3068)





CD2-VH
CD2-VH
CD2-VH
CD2-VH
CD3-VH
CD3-VH
CD2-VH
CD2-VH





GGGGS4
GGGGS4
GGGGS4
GGGGS4
GGGGS4
GGGGS4
GGGGS4
GGGGS4


(SEQ ID
(SEQ ID
(SEQ ID
(SEQ ID
(SEQ ID
(SEQ ID
(SEQ ID
(SEQ ID


NO: 9)
NO: 9)
NO: 9)
NO: 9)
NO: 9)
NO: 9)
NO: 9)
NO: 9)





CD3-VH
CD3-VH
CD3-VH
CD3-VH
CD2-VH
CD2-VH
CD3-VH
CD3-VH





CD28
CD28
CD28
CD28
CD28
CD28
CD28
CD28


Tm
Tm
Tm
Tm
Tm
Tm
Tm
Tm


CD28
CD28
CD28
CD28
CD28
CD28
CD28
CD28


CD3z(1-2)
CD3z(1-2)
CD3z(1-2)
CD3z(1-2)
CD3z(1-2)
CD3z(1-2)
CD3z(1-2)
CD3z(1-2)


P2A
P2A
P2A
P2A
P2A
P2A
P2A
P2A


CD34
CD34
CD34
CD34
CD34
CD34
CD34
CD34









Dual CAR-T Cells

In certain embodiments, the disclosure provides an engineered T cell with two distinct chimeric antigen receptor polypeptides with affinity to different antigen(s) or cell surface protein(s) expressed within the same effector cell, wherein each CAR functions independently. The CAR may be expressed from single or multiple polynucleotide sequences that specifically bind different antigen(s) or cell surface protein(s), wherein the T cell is deficient in the antigen(s) or cell surface protein(s) to which the CARs bind (e.g., CD7*CD2-dCARΔCD7ΔCD2 cell). In non-limiting examples, the deficiency in the antigen(s) or cell surface protein(s) resulted from (a) modification of antigen or cell surface protein expressed by the T cell such that the chimeric antigen receptor no longer specifically binds the modified antigen(s) or cell surface protein(s) (e.g., the epitope of the one or more antigens recognized by the chimeric antigen receptor may be modified by one or more amino acid changes (e.g., substitutions or deletions) or the epitope may be deleted from the antigen), (b) modification of the T cell such that expression of antigen(s) or cell surface protein(s) is/are reduced in the T cell by at least 50%, at least 60%, at least 70%, at least 80%, at least 90% or more, or (c) modification of the T cell such that antigen(s) or cell surface protein(s) is/are not expressed (e.g., by deletion or disruption of the gene encoding antigen or cell surface protein). In each of the above embodiments, the CAR-T cell may be deficient in one or preferably all the antigens or cell surface proteins to which the chimeric antigen receptor specifically binds. The methods to genetically modify a T cell to be deficient in one or more antigens or cell surface proteins are well known in art and non-limiting examples are provided herein. In embodiments described below, the CRISPR-Cas9 system is used to modify a T cell to be deficient in one or more antigen(s) or cell surface protein(s). Any of these may be accomplished by the methods disclosed herein. In further embodiments, the T cell comprises a suicide gene.


A dCAR for a genome-edited, dual CAR-T cell, i.e., CD2*CD38-dCARTΔCD2ΔCD3ε, may be generated by cloning a commercially synthesized anti-CD2 single chain variable fragment into a lentiviral vector containing, e.g., a 2nd or 3rd generation CAR backbone with CD28 and/or 4-1BB internal signaling domains and cloning a commercially synthesized anti-CD38 single chain variable into the same lentiviral vector containing an additional 2nd or 3rd generation CAR backbone with CD28 and/or 4-1BB internal signaling domains resulting in a plasmid from which the two CAR constructs are expressed from the same vector. An extracellular hCD34 domain may be added after a P2A peptide to enable both detection of CAR following viral transduction and purification using anti-hCD34 magnetic beads. A similar method may be followed for making tCARs specific for other malignant T cell antigens.


In a similar manner, other dual CARs may be constructed and are given below in Tables 5-7.


In one embodiment, a dual CAR-T cell comprises (i) a first chimeric antigen receptor (CAR) polypeptide comprising a first signal peptide, a first antigen recognition domain, a first hinge region, a first transmembrane domain, a first co-stimulatory domain, and a first signaling domain; and (ii) a second chimeric antigen receptor polypeptide comprising a second signaling peptide, a second antigen recognition domain, a second hinge region, a second transmembrane domain, a second co-stimulatory domain, and a second signaling domain; wherein the first antigen recognition domain and the second antigen recognition domain have affinities for different target antigens; and wherein the dual CAR-T cell possesses one or more genetic disruptions resulting in reduced expression of the target antigen in the dual CAR-T cell.


In a second embodiment, the first signal peptide is a CD8a signal sequence.


In a third embodiment, the first antigen recognition domain is fusion protein of the variable regions of immunoglobulin heavy and light chains, designated VH1 and VL1, for the first antigen recognition domain, connected by a short linker peptide of 5 amino acids (GGGGS (SEQ ID NO: 3065)). In some embodiments, this linker peptide is repeated 3 or 4 times. In some embodiments, the first antigen recognition domain can be selected from VH1—(GGGGS)3-4 (SEQ ID NO: 3066)—VL1 or VL1—(GGGGS)3-4 (SEQ ID NO: 3066)—VH1.


In some embodiments, the first hinge region comprises CD8a.


In some embodiments, the first transmembrane domain is CD8 or CD28.


In some embodiments, the first co-stimulatory domain comprises 4-1BB, CD28, or a combination of both, in either order, i.e., 4-1BB-CD28 or CD28-4-1BB.


In some embodiments, the first signaling domain is CD3ζ or a CD3ζ bi-peptide, i.e., CD3ζ-CD3ζ.


In some embodiments, the second signal peptide is a CD8a signal sequence of SEQ NO: 1.


In some embodiments, the second antigen recognition domain is fusion protein of the variable regions of immunoglobulin heavy and light chains, designated VH2 and VL2, for the second antigen recognition domain, connected by a short linker peptide of 5 amino acids (GGGGS (SEQ ID NO: 3065)). In some embodiments, this linker peptide is repeated 3 or 4 times. In some embodiments, the second antigen recognition domain can be selected from VH2—(GGGGS)3-4 (SEQ ID NO: 3066)—VL2 or VL2—(GGGGS)3-4 (SEQ ID NO: 3066)—VH1


In some embodiments, the second hinge region comprises CD8a.


In some embodiments, the second transmembrane domain is CD8 or CD28.


In some embodiments, the second co-stimulatory domain comprises 4-1BB, CD28, or a combination of both, in either order, i.e., 4-1BB-CD28 or CD28-4-1BB.


In some embodiments, the second signaling domain is CD3ζ or a CD3ζ bi-peptide, i.e., CD3ζ-CD3ζ.


In some embodiments, the CAR polypeptide comprises a first antigen recognition domain fusion protein of VH1—(GGGGS)3-4 (SEQ ID NO: 3066)—VL1 and a second antigen recognition domain fusion protein of VH2—(GGGGS)3-4 (SEQ ID NO: 3066)—VH2,


In some embodiments, the CAR polypeptide comprises a first antigen recognition domain fusion protein of VL1—(GGGGS)3-4 (SEQ ID NO: 3066)—VH1 and a second antigen recognition domain fusion protein of VL2—(GGGGS)3-4 (SEQ ID NO: 3066)—VH2.


In some embodiments, the CAR polypeptide comprises a first antigen recognition domain fusion protein of VL2—(GGGGS)3-4 (SEQ ID NO: 3066)—VL2 and a second antigen recognition domain fusion protein of VH1—(GGGGS)3-4 (SEQ ID NO: 3066)—VL1.


In some embodiments, the CAR polypeptide comprises a first antigen recognition domain fusion protein of VL2—(GGGGS)3-4 (SEQ ID NO: 3066)—VH2 and a second antigen recognition domain fusion protein of VL1—(GGGGS)3-4 (SEQ ID NO: 3066)—VH1.


In some embodiments, the CAR polypeptide comprises a first antigen recognition domain fusion protein of VH1—(GGGGS)3-4 (SEQ ID NO: 3066)—VL1 and a second antigen recognition domain fusion protein of VL2—(GGGGS)3-4 (SEQ ID NO: 3066)—VH2.


In some embodiments, the CAR polypeptide comprises a first antigen recognition domain fusion protein of VL1—(GGGGS)3-4 (SEQ ID NO: 3066)—VH1 and a second antigen recognition domain fusion protein of VH2—(GGGGS)3-4 (SEQ ID NO: 3066)—VL2.


In some embodiments, the CAR polypeptide comprises a first antigen recognition domain fusion protein of VH2—(GGGGS)3-4 (SEQ ID NO: 3066)—VL2 and a second antigen recognition domain fusion protein of VL1—(GGGGS)3-4 (SEQ ID NO: 3066)—VH1.


In some embodiments, the CAR polypeptide comprises a first antigen recognition domain fusion protein of VL2—(GGGGS)3-4 (SEQ ID NO: 3066)—VH2 and a second antigen recognition domain fusion protein of VH1—(GGGGS)3-4 (SEQ ID NO: 3066)—VL1.


In some embodiments, the CAR polypeptide comprises at least one high efficiency cleavage site, wherein the high efficiency cleavage site is selected from P2A, T2A, E2A, and F2A.


In some embodiments, the CAR polypeptide comprises a suicide gene.


In some embodiments, the CAR polypeptide comprises a mutant cytokine receptor.


In some embodiments, the dual CAR-T cell targets two antigens selected from CD5, CD7, CD2, CD4, CD3, CD33, CD123 (IL3RA), CD371 (CLL-1; CLEC12A), CD117 (c-kit), CD135 (FLT3), BCMA, CS1, CD38, CD79A, CD79B, CD138, and CD19, APRIL, and TACI.


Additional examples of dual CARs are given below in Table 7.









TABLE 7







Dual CARs and dCAR-Ts










Antigen Targets of



Example
CARs in dCAR-T cell
Antigen Deletion/Suppression





D1
APRILxBCMA



D2
APRILxCD19



D3
APRILxCD38



D4
APRILxCD38
CD38


D5
APRILxCS1



D6
APRILxCS1
CS1


D7
BCMAxCD19



D8
BCMAxCD38



D9
BCMAxCD38
CD38


D10
BCMAxCS1



D11
BCMAxCS1
CS1


D12
CD138xAPRIL



D13
CD138xBCMA



D14
CD138xCD19



D15
CD138xCD38



D16
CD138xCD38
CD38


D17
CD138xCD79A



D18
CD138xCD79B



D19
CD138xCS1



D20
CD138xCS1
CS1


D21
CD19xCD38



D22
CD19xCD38
CD38


D23
CD2xCD3ε



D24
CD2xCD3ε
CD2


D25
CD2xCD3ε
CD3ε


D26
CD2xCD3ε
CD2 and CD3ε


D27
CD2xCD4



D28
CD2xCD4
CD2


D29
CD2xCD4
CD4


D30
CD2xCD4
CD2 and CD4


D31
CD2xCD4
CD2 and TRAC


D32
CD2xCD4
CD4 and TRAC


D33
CD2xCD4
CD2 and CD4 and TRAC


D34
CD2xCD5



D35
CD2xCD5
CD2


D36
CD2xCD5
CD5


D37
CD2xCD5
CD2 and CD5


D38
CD2xCD5
CD2 and TRAC


D39
CD2xCD5
CD5 and TRAC


D40
CD2xCD5
CD2 and CD5 and TRAC


D41
CD2xCD7



D42
CD2xCD7
CD2


D43
CD2xCD7
CD7


D44
CD2xCD7
CD2 and CD7


D45
CD2xCD7
CD2 and TRAC


D46
CD2xCD7
CD7 and TRAC


D47
CD2xCD7
CD2 and CD7 and TRAC


D48
CD3εxCD4



D49
CD3εxCD4
CD3ε


D50
CD3εxCD4
CD4


D51
CD3εxCD4
CD3ε and CD4


D52
CD3εxCD5



D53
CD3εxCD5
CD3ε


D54
CD3εxCD5
CD5


D55
CD3εxCD5
CD3ε and CD5


D56
CD3εxCD7



D57
CD3εxCD7
CD3ε


D58
CD3εxCD7
CD7


D59
CD3εxCD7
CD3ε and CD7


D60
CD4xCD5



D61
CD4xCD5
CD4


D62
CD4xCD5
CD5


D63
CD4xCD5
CD4 and CD5


D64
CD4xCD5
CD4 and TRAC


D65
CD4xCD5
CD5 and TRAC


D66
CD4xCD5
CD4 and CD5 and TRAC


D67
CD4xCD7



D68
CD4xCD7
CD4


D69
CD4xCD7
CD7


D70
CD4xCD7
CD4 and CD7


D71
CD4xCD7
CD4 and TRAC


D72
CD4xCD7
CD7 and TRAC


D73
CD4xCD7
CD4 and CD7 and TRAC


D74
CD5xCD7



D75
CD5xCD7
CD5


D76
CD5xCD7
CD7


D77
CD5xCD7
CD5 and CD7


D78
CD5xCD7
CD5 and TRAC


D79
CD5xCD7
CD7 and TRAC


D80
CD5xCD7
CD5 and CD7 and TRAC


D81
CD79AxAPRIL



D82
CD79AxBCMA



D83
CD79AxCD19



D84
CD79AxCD38



D85
CD79AxCD38
CD38


D86
CD79AxCD79B



D87
CD79AxCS1



D88
CD79AxCS1
CS1


D89
CD79BxAPRIL



D90
CD79BxBCMA



D91
CD79BxCD19



D92
CD79BxCD38



D93
CD79BxCD38
CD38


D94
CD79BxCD79A



D95
CD79BxCS1



D96
CD79BxCS1
CS1


D97
CS1xCD19



D98
CS1xCD19
CS1


D99
CS1xCD38



D100
CS1xCD38
CS1


D101
CS1xCD38
CD38


D102
CS1xCD38
CS1 and CD38


D103
TCRβxCD2



D104
TCRβxCD2
TCRβ


D105
TCRβxCD2
CD2


D106
TCRβxCD2
TCRβ and CD2


D107
TCRβxCD3ε



D108
TCRβxCD3ε
TCRβ


D109
TCRβxCD3ε
CD3ε


D110
TCRβxCD3ε
TCRβ and CD3ε


D111
TCRβxCD4



D112
TCRβxCD4
TCRβ


D113
TCRβxCD4
CD4


D114
TCRβxCD4
TCRβ and CD4


D115
TCRβxCD5



D116
TCRβxCD5
TCRβ


D117
TCRβxCD5
CD5


D118
TCRβxCD5
TCRβ and CD5


D119
TCRβxCD7



D120
TCRβxCD7
TCRβ


D121
TCRβxCD7
CD7


D122
TCRβxCD7
TCRβ and CD7


D123
TRACxCD2



D124
TRACxCD2
TRAC


D125
TRACxCD2
CD2


D126
TRACxCD2
TRAC and CD2


D127
TRACxCD3ε



D128
TRACxCD3ε
TRAC


D129
TRACxCD3ε
CDε


D130
TRACxCD3ε
TRAC and CD3ε


D131
TRACxCD4



D132
TRACxCD4
TRAC


D133
TRACxCD4
CD4


D134
TRACxCD4
TRAC and CD4


D135
TRACxCD5



D136
TRACxCD5
TRAC


D137
TRACxCD5
CD5


D138
TRACxCD5
TRAC and CD5


D139
TRACxCD7



D140
TRACxCD7
TRAC


D141
TRACxCD7
CD7


D142
TRACxCD7
TRAC and CD7









Cytokine/Chemokine/Transcription Factor Gene Deletion or Suppression

Cytokine release syndrome (CRS) is caused by a large, rapid release of cytokines from immune cells in response to immunotherapy (or other immunological stimulus). Accordingly, reducing the level of cytokines released would prevent or reduce the development and/or maintenance of CRS. As disclosed herein, this can be accomplished by modifying, disrupting, or deleting one or more cytokine/chemokine/transcription factor genes. One method to accomplish this is genetic ablation (gene silencing) in which gene expression is abolished through the alteration or deletion of genetic sequence information. This can be accomplished using known genetic engineering tools in the art such as Transcription Activator-like Effector Nucleases (TALENs), Zinc Finger Nucleases (ZFNs), CRISPR, and also by transfection of small interfering RNAs (siRNAs).


Another technique is expression of an scFv with an endoplasmic reticulum (ER) binding tether to bind the cytokine in the ER and prevent secretion. Specific constructs, named protein expression blockers (PEBLs), prevent transport of targeted proteins to the cell membrane. PEBL constructs can be readily combined with other gene modification systems for ex vivo cell processing of immune cells. A short hairpin RNA or small hairpin RNA (shRNA/Hairpin Vector) is an artificial RNA molecule with a tight hairpin turn that can be used to silence target gene expression, i.e., of antigens, via RNA interference (RNAi). Expression of shRNA in cells is typically accomplished by delivery of plasmids or through viral or bacterial vectors.


Cytokines or chemokines that can be deleted from immune effector cells as disclosed herein, e.g., by targeted transduction of a CAR into the gene sequence of the cytokine, include without limitation the following: XCL1, XCL2, CCL1, CCL2, CCL3, CCL4, CCL5, CCL7, CCL8, CCL11, CCL13, CCL14, CCL15, CCL16, CCL17, CCL18, CCL19, CCL20, CCL21, CCL22, CCL23, CCL24, CCL25, CCL26, CCL27, CXCL1, CXCL2, CXCL3, CXCL4, CXCL5, CXCL6, CXCL7, CXCL8, CXCL9, CXCL10, CXCL11, CXCL12, CXCL13, CXCL14, CX3CL1, IL-1α, IL-1β, IL-1RA, IL-18, IL-2, IL-4, IL-7, IL-9, IL-13, IL-15, IL-3, IL-5, GM-CSF, IL-6, IL-11, G-CSF, IL-12, LIF, OSM, IL-10, IL-20, IL-14, IL-16, IL-17, IFN-α, IFN-β, IFN-γ, CD154, LT-β, TNF-α, TNF-β, 4-1BBL, APRIL, CD70, CD153, CD178, GITRL, LIGHT, OX40L, TALL-1, TRAIL, TWEAK, TRANCE, TGF-β1, TGF-β2, TGF-β3, Epo, Tpo, Flt-3L, SCF, M-CSF, MSP, A2M, ACKR1, ACKR2, ACKR3, ACVR1, ACVR2B, ACVRL1, ADIPOQ, AGER, AGRN, AIMP1, AREG, BMP1, BMP10, BMP15, BMP2, BMP3, BMP4, BMP5, BMP6, BMP7, BMP8A, BMP8B, BMPR2, C10 orf99, C1 QTNF4, C5, CCL28, CCR1, CCR2, CCR3, CCR5, CCR6, CCR7, CD109, CD36, CD4, CD40LG, CD74, CER1, CHRD, CKLF, CLCF1, CMTM1, CMTM2, CMTM3, CMTM4, CMTM5, CMTM6, CMTM7, CMTM8, CNTF, CNTFR, COPS5, CRLF1, CSF1, CSF1R, CSF2, CSF3, CSF3R, CTF1, CX3CR1, CXCL16, CXCL17, CXCR1, CXCR2, CXCR3, CXCR4, CXCR6, EBI3, EDN1, ELANE, ENG, FAM3B, FAM3C, FAM3D, FAS, FASLG, FGF2, FLT3LG, FZD4, GBP1, GDF1, GDF10, GDF11, GDF15, GDF2, GDF3, GDF5, GDF6, GDF7, GDF9, GPI, GREM1, GREM2, GRN, HAX1, HFE2, HMGB1, HYAL2, IFNA10, IFNA14, IFNA16, IFNA2, IFNA5, IFNA6, IFNA8, IFNAR1, IFNAR2, IFNB1, IFNE, IFNG, IFNGR1, IFNK, IFNL1, IFNL3, IFNW1, IL10RA, IL11RA, IL12A, IL12B, IL12RB1, IL17A, IL17B, IL17C, IL17D, IL17F, IL18BP, IL-19, IL1F10, IL1R1, IL1R2, IL1 RAPL1, IL1RL1, IL1RN, IL20RA, IL20RB, IL21, IL22, IL22RA1, IL22RA2, IL23A, IL23R, IL24, IL25, IL26, IL27, IL2RA, IL2RB, IL2RG, IL31, IL31RA, IL32, IL33, IL34, IL36A, IL36B, IL36G, IL36RN, IL37, IL6R, IL6ST, INHA, INHBA, INHBB, INHBC, INHBE, ITGA4, ITGAV, ITGB1, ITGB3, KIT, KITLG, KLHL20, LEFTY1, LEFTY2, LIFR, LTA, LTB, LTBP1, LTBP3, LTBP4, MIF, MINOS1-, MSTN, NAMPT, NBL1, NDP, NLRP7, NODAL, NOG, NRG1, NRP1, NRP2, OSMR, PARK7, PDPN, PF4, PF4V1, PGLYRP1, PLP2, PPBP, PXDN, SCG2, SCGB3A1, SECTM1, SLURP1, SOSTDC1, SP100, SPP1, TCAP, TGFBR1, TGFBR2, TGFBR3, THBS1, THNSL2, THPO, TIMP1, TNF, TNFRSF11, TNFRSF1A, TNFRSF9, TNFRSF10, TNFSF11, TNFSF12, TNFSF12-, TNFSF13, TNFSF13B, TNFSF14, TNFSF15, TNFSF18, TNFSF4, TNFSF8, TNFSF9, TRIM16, TSLP, TWSG1, TXLNA, VASN, VEGFA, VSTM1, WFIKKN1, WFIKKN2, WNT1, WNT2, WNT5A, WNT7A, and ZFP36.


In some embodiments, the cytokine is chosen from cytokine is chosen from MCP1 (CCL2), MCP-2, GM-CSF, G-CSF, M-CSF, Il-4, and IFNγ.


In certain embodiments, transcription factors that can be deleted from immune effector cells as disclosed herein include AHR, BCL6, FOXP3, GATA3, MAF, RORC, SPI1, and TBX21.


The sequences of these genes are known and available in the art and can include, for example, those provided in Table 10.


Selection of Priority Target Genes

Cytokine/chemokine gene targets of specific interest for deletion or suppression (e.g., ablation, knock-out, KO) in CAR-T cells for the mitigation of CRS have been categorized into groups based on biological function. CRS development is dependent upon CAR-T cell activation and subsequent cytokine release, which initiates a dysregulated immune system in the recipient of the CAR-T cell therapy. Several studies have indicated that recipient myeloid activation is necessary for the development of CRS.


The first group of potential genes to KO in CAR-T are surface receptors that, when engaged with myeloid cells in normal immunological responses activate the myeloid cells (e.g., CD40L). The second group are cytokines that are released from CAR-T cells that activate myeloid cells (e.g., GM-CSF). In both these categories, the goal is to prevent CAR-T cell signaling, which will activate recipient myeloid cells and initiate CRS.


The third category of targets are endogenous T cell receptors that increase T cell activation (potentially in the absence of tumor target) that are integrated into the CAR-T receptor (e.g., endogenous CD28). The aim is to curtail activation of the CAR-T from non-tumor interactions, such as activated myeloid cells that could engage with CD28 on activated T cells, thus amplifying T cell cytokine production and subsequent myeloid activation.


The fourth and fifth categories of gene KO targets are transcription factors and cytokines that drive CAR-T cell differentiation and subsequent functional characteristics. CAR-T cells that are phenotypically similar to normal cytotoxic T cells (CTL, typically identified by CD8 expression) are capable of direct tumor killing through T cell mediated effector functions. CTL are supported and maintained by T helper cells (expressing CD4). Importantly, subsets of T helper cells can support CTL (i.e., Th1 cells) or inhibit (i.e., Th2 cells). Other T cells, such as Tregs can also inhibit CTL development and function. The aim is to target cytokines or transcription factors in the CAR-T population that would lead to CAR-T differentiation into non-cytotoxic T cell populations. Additionally, Th2 cells produce cytokines (such as GM-CSF and IL-4), which are indicative markers of CRS. It is likely that CAR-T phenotypes, which cannot optimally kill tumor cells, will be activated via CAR-T receptors, produce signals that drive CRS in the host, increase the time required for tumor killing, and require higher CAR-T cell doses than optimized “killing” products. Thus, knocking out or ablating (or suppressing) transcription factors (such as GATA3) or cytokines (such as IL-4) will prevent (or reduce) Th2 bias and reduce CRS.


Of additional interest regarding both myeloid activating cytokines and optimized CAR-T cell differentiation is the potential to mitigate CAR-T neurotoxicity. Neurotoxicity occurs in a small population of CAR-T recipients with CRS. Research has shown that CRS patients that progress to develop neurotoxicity have increased IL-5 and ferritin levels (Santomasso et al., 2017 and Philip et al., 2019). These two biomarkers indicate that mast cell activation may be involved with CNS complications. IL-5 is a key cytokine produced by Th2 cells, eosinophils and mast cells. Excessive levels of ferritin are observed in mastocytosis and several diseases involving mast cell dysregulation. Additionally, many mast cell diseases include neurological dysregulation. Given raised levels of IL-5, ferritin, and neurological toxicity in some patients, reducing mast cell activation by cytokines such as IL-5, or preventing Th2 cell development which drives IL-5-dependent mast cell activation, may also mitigate neurological complications seen in CRS recipients. With respect to CNS complications, CX3CR1 and OX40 were also highlighted, due to previously demonstrated high T cell burden in the CNS. CX3CR1 is a T cell chemokine receptor that principally directs T cells and potentially CAR-T cells into the CNS. OX40 is the T cell receptor which facilitates activation by cell-cell interaction with OX40L on eosinophils and mast cells.


Thus, in certain embodiments, the cytokine is chosen from CCL2 (MCP1), MCP-2, GM-CSF, G-CSF, M-CSF, Il-4, and IFNγ.


Indications and Standards of Care in CAR-T Therapy

In some embodiment, the genome-edited immune effector cells disclosed herein, and/or generated using the methods disclosed herein, express one or more chimeric antigen receptors (CARs) and can be used as a medicament, i.e., for the treatment of disease. In many embodiments, the cells are CAR-T cells.


Cells disclosed herein, and/or generated using the methods disclosed herein, may be used in immunotherapy and adoptive cell transfer, for the treatment, or the manufacture of a medicament for treatment, of cancers, autoimmune diseases, infectious diseases, and other conditions.


The cancer may be a hematologic malignancy or solid tumor. Hematologic malignancies include leukemias, lymphomas, multiple myeloma, and subtypes thereof. Lymphomas can be classified various ways, often based on the underlying type of malignant cell, including Hodgkin's lymphoma (often cancers of Reed-Sternberg cells, but also sometimes originating in B cells; all other lymphomas are non-Hodgkin's lymphomas), B-cell lymphomas, T-cell lymphomas, mantle cell lymphomas, Burkitt's lymphoma, follicular lymphoma, and others as defined herein and known in the art.


B-cell lymphomas include, but are not limited to, diffuse large B-cell lymphoma (DLBCL), chronic lymphocytic leukemia (CLL)/small lymphocytic lymphoma (SLL), and others as defined herein and known in the art.


T-cell lymphomas include T-cell acute lymphoblastic leukemia/lymphoma (T-ALL), peripheral T-cell lymphoma (PTCL), T-cell chronic lymphocytic leukemia (T-CLL) Sezary syndrome, and others as defined herein and known in the art.


Leukemias include Acute myeloid (or myelogenous) leukemia (AML), chronic myeloid (or myelogenous) leukemia (CML), acute lymphocytic (or lymphoblastic) leukemia (ALL), chronic lymphocytic leukemia (CLL) hairy cell leukemia (sometimes classified as a lymphoma), and others as defined herein and known in the art.


Plasma cell malignancies include lymphoplasmacytic lymphoma, plasmacytoma, and multiple myeloma.


In some embodiments, the medicament can be used for treating cancer in a patient, particularly for the treatment of solid tumors such as melanomas, neuroblastomas, gliomas or carcinomas such as tumors of the brain, head and neck, breast, lung (e.g., non small cell lung cancer, NSCLC), reproductive tract (e.g., ovary), upper digestive tract, pancreas, liver, renal system (e.g., kidneys), bladder, prostate and colorectum.


In another embodiment, the medicament can be used for treating cancer in a patient, particularly for the treatment of hematologic malignancies selected from multiple myeloma and acute myeloid leukemia (AML) and for T-cell malignancies selected from T-cell acute lymphoblastic leukemia (T-ALL), non-Hodgkin's lymphoma, and T-cell chronic lymphocytic leukemia (T-CLL).


In some embodiments, the cells may be used in the treatment of autoimmune diseases such as lupus, autoimmune (rheumatoid) arthritis, multiple sclerosis, transplant rejection, Crohn's disease, ulcerative colitis, dermatitis, and the like. In some embodiments, the cells are chimeric autoantibody receptor T-cells, or CAAR-Ts displaying antigens or fragments thereof, instead of antibody fragments; in this version of adoptive cell transfer, the B cells that cause autoimmune diseases will attempt to attack the engineered T cells, which will respond by killing them.


In some embodiments, the cells may be used in the treatment of infectious diseases such as HIV and tuberculosis.


In another embodiment, the CAR-T cells of the present disclosure can undergo robust in vivo T cell expansion and can persist for an extended amount of time.


In some embodiments, the treatment of a patient with CAR-T cells of the present disclosure can be ameliorating, curative or prophylactic. It may be either part of an autologous immunotherapy or part of an allogenic immunotherapy treatment. By autologous, it is meant that cells, cell line or population of cells used for treating patients are originating from said patient or from a Human Leucocyte Antigen (HLA) compatible donor. By allogeneic, is meant that the cells or population of cells used for treating patients are not originating from the patient but from a donor.


The treatment of cancer with CAR-T cells of the present disclosure may be in combination with one or more therapies selected from antibody therapy, chemotherapy, cytokine therapy, dendritic cell therapy, gene therapy, hormone therapy, radiotherapy, laser light therapy, and radiation therapy.


The administration of CAR-T cells or a population of CAR-T cells of the present disclosure of the present disclosure be carried out by aerosol inhalation, injection, ingestion, transfusion, implantation or transplantation. The CAR-T cells compositions described herein, i.e., mono CAR, dual CAR, tandem CARs, may be administered to a patient subcutaneously, intradermally, intratumorally, intranodally, intramedullary, intramuscularly, by intravenous or intralymphatic injection, or intraperitoneally. In one embodiment, the cell compositions of the present disclosure are preferably administered by intravenous injection.


The administration of CAR-T cells or a population of CAR-T cells can consist of the administration of 104-109 cells per kg body weight, preferably 105 to 106 cells/kg body weight including all integer values of cell numbers within those ranges. The CAR-T cells or a population of CAR-T cells can be administrated in one or more doses. In another embodiment, the effective amount of CAR-T cells or a population of CAR-T cells are administrated as a single dose. In another embodiment, the effective amount of cells are administered as more than one dose over a period time. Timing of administration is within the judgment of a health care provider and depends on the clinical condition of the patient. The CAR-T cells or a population of CAR-T cells may be obtained from any source, such as a blood bank or a donor. While the needs of a patient vary, determination of optimal ranges of effective amounts of a given CAR-T cell population(s) for a particular disease or conditions are within the skill of the art. An effective amount means an amount which provides a therapeutic or prophylactic benefit. The dosage administered will be dependent upon the age, health and weight of the patient recipient, type of concurrent treatment, if any, frequency of treatment, and the nature of the effect desired.


In another embodiment, the effective amount of CAR-T cells or a population of CAR-T cells or composition comprising those CAR-T cells are administered parenterally. The administration can be an intravenous administration. The administration of CAR-T cells or a population of CAR-T cells or composition comprising those CAR-T cells can be directly done by injection within a tumor.


In one embodiment of the present disclosure, the CAR-T cells or a population of the CAR-T cells are administered to a patient in conjunction with, e.g., before, simultaneously or following, any number of relevant treatment modalities, including but not limited to, treatment with cytokines, or expression of cytokines from within the CAR-T, that enhance T-cell proliferation and persistence and, include but not limited to, IL-2, IL-7, and IL-15.


In a second embodiment, the CAR-T cells or a population of CAR-T cells of the present disclosure may be used in combination with agents that inhibit immunosuppressive pathways, including but not limited to, inhibitors of TGF-β, interleukin 10 (IL-10), adenosine, VEGF, indoleamine 2,3 dioxygenase 1 (IDO1), indoleamine 2,3-dioxygenase 2 (IDO2), tryptophan 2-3-dioxygenase (TDO), lactate, hypoxia, arginase, and prostaglandin E2.


In another embodiment, the CAR-T cells or a population of CAR-T cells of the present disclosure may be used in combination with T-cell checkpoint inhibitors, including but not limited to, anti-CTLA4 (Ipilimumab) anti-PD1 (Pembrolizumab, Nivolumab, Cemiplimab), anti-PDL1 (Atezolizumab, Avelumab, Durvalumab), anti-PDL2, anti-BTLA, anti-LAG3, anti-TIM3, anti-VISTA, anti-TIGIT, and anti-KIR.


In another embodiment, the CAR-T cells or a population of CAR-T cells of the present disclosure may be used in combination with T cell agonists, including but not limited to, antibodies that stimulate CD28, ICOS, OX-40, CD27, 4-1BB, CD137, GITR, and HVEM


In another embodiment, the CAR-T cells or a population of CAR-T cells of the present disclosure may be used in combination with therapeutic oncolytic viruses, including but not limited to, retroviruses, picornaviruses, rhabdoviruses, paramyxoviruses, reoviruses, parvoviruses, adenoviruses, herpesviruses, and poxviruses.


In another embodiment, the CAR-T cells or a population of CAR-T cells of the present disclosure may be used in combination with immunostimulatory therapies, such as toll-like receptors agonists, including but not limited to, TLR3, TLR4, TLR7 and TLR9 agonists.


In another embodiment, the CAR-T cells or a population of CAR-T cells of the present disclosure may be used in combination with stimulator of interferon gene (STING) agonists, such as cyclic GMP-AMP synthase (cGAS).


Immune effector cell aplasia, particularly T cell aplasia is also a concern after adoptive cell transfer therapy. When the malignancy treated is a T-cell malignancy, and CAR-T cells target a T cell antigen, normal T cells and their precursors expressing the antigen will become depleted, and the immune system will be compromised. Accordingly, methods for managing these side effects are attendant to therapy. Such methods include selecting and retaining non-malignant T cells or precursors, either autologous or allogeneic (optionally engineered not to cause rejection or be rejected), for later expansion and re-infusion into the patient, after CAR-T cells are exhausted or deactivated. Alternatively, CAR-T cells which recognize and kill subsets of TCR-bearing cells, such as normal and malignant TRBC1+, but not TRBC2+ cells, or alternatively, TRBC2+, but not TRBC1+ cells, may be used to eradicate a T cell malignancy while preserving sufficient normal T cells to maintain normal immune system function.


Definitions

As used herein, the terms below have the meanings indicated. Other definitions may occur throughout the specification.


When ranges of values are disclosed, and the notation “from n1 . . . to n2” or “between n1 . . . and n2” is used, where n1 and n2 are the numbers, then unless otherwise specified, this notation is intended to include the numbers themselves and the range between them. This range may be integral or continuous between and including the end values. By way of example, the range “from 2 to 6 carbons” is intended to include two, three, four, five, and six carbons, since carbons come in integer units. Compare, by way of example, the range “from 1 to 3 μM (micromolar),” which is intended to include 1 μM, 3 μM, and everything in between to any number of significant figures (e.g., 1.255 μM, 2.1 μM, 2.9999 μM, etc.).


The term “about,” as used herein, is intended to qualify the numerical values which it modifies, denoting such a value as variable within a margin of error. When no particular margin of error, such as a standard deviation to a mean value given in a chart or table of data, is recited, the term “about” should be understood to mean that range which would encompass the recited value and the range which would be included by rounding up or down to that figure as well, taking into account significant figures.


The term “activation” (and other conjugations thereof) in reference to cells is generally understood to be synonymous with “stimulating” and as used herein refers to treatment of cells that results in expansion of cell populations. In T cells, activation is often accomplished by exposure to CD2 and CD28 (and sometimes CD2 as well) agonists, typically antibodies, optionally coated onto magnetic beads or conjugated to a colloidal polymeric matrix.


The term “antigen” as used herein is a cell surface protein recognized by (i.e., that is the target of) T cell receptor or chimeric antigen receptor. In the classical sense antigens are substances, typically proteins, that are recognized by antibodies, but the definitions overlap insofar as the CAR comprises antibody-derived domains such as light (VL) and heavy (VH) chains recognizing one or more antigen(s).


The term “cancer” refers to a malignancy or abnormal growth of cells in the body. Many different cancers can be characterized or identified by particular cell surface proteins or molecules. Thus, in general terms, cancer in accordance with the present disclosure may refer to any malignancy that may be treated with an immune effector cell, such as a CAR-T cell as described herein, in which the immune effector cell recognizes and binds to the cell surface protein on the cancer cell. As used herein, cancer may refer to a hematologic malignancy, such as multiple myeloma, a T-cell malignancy, or a B cell malignancy. T cell malignancies may include, but are not limited to, T-cell acute lymphoblastic leukemia (T-ALL) or non-Hodgkin's lymphoma. A cancer may also refer to a solid tumor, such as including, but not limited to, cervical cancer, pancreatic cancer, ovarian cancer, mesothelioma, and lung cancer.


A “cell surface protein” as used herein is a protein (or protein complex) expressed by a cell at least in part on the surface of the cell. Examples of cell surface proteins include the TCR (and subunits thereof) and CD7.


The term “combination therapy” means the administration of two or more therapeutic agents to treat a therapeutic condition or disorder described in the present disclosure. Such administration encompasses co-administration of these therapeutic agents in a substantially simultaneous manner, such as in a single capsule having a fixed ratio of active ingredients or in multiple, separate capsules for each active ingredient. In addition, such administration also encompasses use of each type of therapeutic agent in a sequential manner. In either case, the treatment regimen will provide beneficial effects of the drug combination in treating the conditions or disorders described herein.


The term “composition” as used herein refers to an immunotherapeutic cell population combination with one or more therapeutically acceptable carriers.


The term “disease” as used herein is intended to be generally synonymous, and is used interchangeably with, the terms “disorder,” “syndrome,” and “condition” (as in medical condition), in that all reflect an abnormal condition of the human or animal body or of one of its parts that impairs normal functioning, is typically manifested by distinguishing signs and symptoms, and causes the human or animal to have a reduced duration or quality of life.


The term “donor template” refers to the reference genomic material that the cell uses as a template to repair the a double-stranded break through the homology-directed repair (HDR) DNA repair pathway. The donor template contains the piece of DNA to be inserted into the genome (containing the gene to be expressed, CAR, or marker) with two homology arms flanking the site of the double-stranded break. In some embodiments, a donor template may be an adeno-associated virus, a single-stranded DNA, or a double-stranded DNA.


The term “fratricide” as used herein means a process which occurs when a CAR-T cell (or other CAR-bearing immune effector cell) becomes the target of, and is killed by, another CAR-T cell comprising the same chimeric antigen receptor as the target of CAR-T cell, because the targeted cell expresses the antigen specifically recognized by the chimeric antigen receptor on both cells. CAR-T comprising a chimeric antigen receptor which are deficient in an antigen to which the chimeric antigen receptor specifically binds will be “fratricide-resistant.”


The term “genome-edited” or “gene-edited” as used herein means having a gene or portion of the genome added, deleted, or modified (e.g., disrupted) to be non-functional. Thus, in certain embodiments, a “genome-edited T cell” is a T cell that has had a gene such as a CAR recognizing at least one antigen added; and/or has had a gene such as the gene(s) to the antigen(s) that are recognized by the CAR deleted, and/or has had the gene to the TCR or a subunit thereof disrupted.


A “healthy donor,” as used herein, is one who does not have a malignancy (particularly a hematologic malignancy, e.g., a T-cell malignancy).


As used herein, an “immature dendritic cell” or “iDC” refers to an immature dendritic cell.


The term “therapeutically acceptable” refers to substances which are suitable for use in contact with the tissues of patients without undue toxicity, irritation, and allergic response, are commensurate with a reasonable benefit/risk ratio, and/or are effective for their intended use.


The term “therapeutically effective” is intended to qualify the amount of active ingredients used in the treatment of a disease or disorder or on the effecting of a clinical endpoint.


The term “patient” is generally synonymous with the term “subject” and includes all mammals including humans.


A “malignant B cell” is a B cell derived from a B-cell malignancy. B cell malignancies include, without limitation, (DLBCL), chronic lymphocytic leukemia (CLL)/small lymphocytic lymphoma (SLL), and B cell-precursor acute lymphoblastic leukemia (ALL).


The term “exposing to,” as used herein, in the context of bringing compositions of matter (such as antibodies) into intimate contact with other compositions of matter (such as cells), is intended to be synonymous with “incubated with,” and no lengthier period of time in contact is intended by the use of one term instead of the other.


A “malignant T cell” is a T cell derived from a T-cell malignancy. The term “T-cell malignancy” refers to a broad, highly heterogeneous grouping of malignancies derived from T-cell precursors, mature T cells, or natural killer cells. Non-limiting examples of T-cell malignancies include T-cell acute lymphoblastic leukemia/lymphoma (T-ALL), human T-cell leukemia virus type 1-positive (HTLV-1+) adult T-cell leukemia/lymphoma (ATL), T-cell prolymphocytic leukemia (T-PLL), Adult T-cell lymphoma/leukemia (HTLV-1 associated), Aggressive NK-cell leukemia, Anaplastic large-cell lymphoma (ALCL), ALK positive, Anaplastic large-cell lymphoma (ALCL), ALK negative, Angioimmunoblastic T-cell lymphoma (AITL), Breast implant-associated anaplastic large-cell lymphoma, Chronic lymphoproliferative disorder of NK cells, Extra nodal NK/T-cell lymphoma, nasal type, Enteropathy-type T-cell lymphoma, Follicular T-cell lymphoma, Hepatosplenic T-cell lymphoma, Indolent T-cell lymphoproliferative disorder of the GI tract, Monomorphic epitheliotrophic intestinal T-cell lymphoma, Mycosis fungoides, Nodal peripheral T-cell lymphoma with TFH phenotype, Peripheral T-cell lymphoma (PTCL), NOS, Primary cutaneous a/p T-cell lymphoma, Primary cutaneous CD8+ aggressive epidermotropic cytotoxic T-cell lymphoma, Primary cutaneous acral CD8+ T-cell lymphoma, Primary cutaneous CD4+ small/medium T-cell lymphoproliferative disorders [Primary cutaneous anaplastic large-cell lymphoma (C-ALCL), lymphoid papulosis], Sezary syndrome, Subcutaneous, panniculitis-like T-cell lymphoma, Systemic EBV+ T-cell lymphoma of childhood, and T-cell large granular lymphocytic leukemia (LGL).


A “malignant plasma cell” is a plasma cell derived from a plasma cell malignancy. The term “plasma-cell malignancy” refers to a malignancy in which abnormal plasma cells are overproduced. Non-limiting examples of plasma cell malignancies include lymphoplasmacytic lymphoma, plasmacytoma, and multiple myeloma.


As used herein, “suicide gene” refers to a nucleic acid sequence introduced to a CAR-T cell by standard methods known in the art, that when activated result in the death of the CAR-T cell. If required suicide genes may facilitate the tracking and elimination, i.e., killing, of CAR-T cells in vivo. Facilitated killing of CAR-T cells by activating a suicide gene can be accomplished by standard methods known in the art. Suicide gene systems known in the art include, but are not limited to, several herpes simplex virus thymidine kinase (HSVtk)/ganciclovir (GCV) suicide gene therapy systems and inducible caspase 9 proteins. In one embodiment, the suicide gene is a chimeric CD34/thymidine kinase.


As used herein, an “immune effector cell” is a leukocyte that can modulate an immune response. Immune effector cells include T cells, B cells, natural killer (NK) cells, iNKT cells (invariant T-cell receptor alpha natural killer T cells), and macrophages. T cell receptor (TCR)-bearing immune effector cells include, of course, T cells, but also cells which have been engineered to express a T cell receptor. Immune effector cells may be obtained or derived/generated from any appropriate source, such as including, but not limited to, healthy donors, peripheral blood mononuclear cells, cord blood, and induced pluripotent stem cells (iPSC).


As used herein, a “CAR-bearing immune effector cell” is an immune effector cell which has been transduced with at least one CAR. A “CAR-T cell” is a T cell which has been transduced with at least one CAR; CAR-T cells can be mono, dual, or tandem CAR-T cells. CAR-T cells can be autologous, meaning that they are engineered from a subject's own cells, or allogeneic, meaning that the cells are sourced from a healthy donor, and in many cases, engineered so as not to provoke a host-vs-graft or graft-vs-host reaction.


A “chimeric antigen receptor” or “CAR” as used herein and generally used in the art, refers to a recombinant fusion protein that has an extracellular ligand-binding domain, a transmembrane domain, and a signaling transducing domain that directs the cell to perform a specialized function upon binding of the extracellular ligand-binding domain to a component present on the target cell. For example, a CAR can have an antibody-based specificity for a desired antigen (e.g., tumor antigen) with a T cell receptor-activating intracellular domain to generate a chimeric protein that exhibits specific anti-target cellular immune activity. First-generation CARs include an extracellular ligand-binding domain and signaling transducing domain, commonly CD3ζ or FcεRIγ. Second generation CARs are built upon first generation CAR constructs by including an intracellular costimulatory domain, commonly 4-1BB or CD28. These costimulatory domains help enhance CAR-T cell cytotoxicity and proliferation compared to first generation CARs. The third generation CARs include multiple costimulatory domains, primarily to increase CAR-T cell proliferation and persistence. Chimeric antigen receptors are distinguished from other antigen binding agents by their ability both to bind MHC-independent antigens and transduce activation signals via their intracellular domain.


The term “CAR-iNKT cell” (equivalently, iNKT-CAR) means an iNKT cell that expresses a chimeric antigen receptor. A dual iNKT-CAR cell (equivalently, iNKT-dCAR) is an iNKT-CAR cell that expresses two distinct chimeric antigen receptor polypeptides with affinity to different target antigens expressed within the same effector cell, wherein each CAR functions independently. The CAR may be expressed from a single or multiple polynucleotide sequences. A tandem iNKT-CAR cell (equivalently, iNKT-tCAR) is an iNKT-CAR cell with a single chimeric antigen polypeptide containing two distinct antigen recognition domains with affinity to different targets, wherein the antigen recognition domains are linked through a peptide linker and share common costimulatory domain(s), and wherein binding of either antigen recognition domain will signal though a common costimulatory domains(s) and signaling domain.


The term “chimeric antigen receptor T cell” (equivalently, CAR-T) means an T cell that expresses a chimeric antigen receptor.


The term dual CAR-T (dCAR-T), means a CAR-T cell that expresses cells two distinct chimeric antigen receptor polypeptides with affinity to different target antigen expressed within the same effector cell, wherein each CAR functions independently. The CAR may be expressed from single or multiple polynucleotide sequences.


The term tandem CAR-T (tCAR-T) means a single chimeric antigen polypeptide containing two distinct antigen recognition domains with affinity to different targets wherein the antigen recognition domain is linked through a peptide linker and share common costimulatory domain(s), wherein the binding of either antigen recognition domain will signal through a common co-stimulatory domains(s) and signaling domain.


As used herein, a chimeric antigen receptor natural killer (NK) cell (equivalently, NK-CAR) would have a meaning analogous to the definitions of CAR-T and iNKT-CAR.


As used herein, a chimeric antigen receptor macrophage (equivalently, CAR-macrophage) would have a meaning analogous to the definitions of CAR-T, iNKT-CAR, and NK-CAR.


As used herein, the term “cytokine release syndrome” refers to a condition that may occur after treatment with some types of immunotherapy, such as monoclonal antibodies and CAR-T or other CAR-bearing immune effector cells. Cytokine release syndrome is caused by a large, rapid release of cytokines into the blood from immune cells affected by the immunotherapy. Symptoms of CRS include fever, fatigue, loss of appetite, muscle and joint pain, nausea, vomiting, diarrhea, rashes, fast breathing, rapid heartbeat, low blood pressure, seizures, headache, confusion, delirium, hallucinations, tremor, and loss of coordination. CRS can manifest along a spectrum of mild to fatal, and can be ranked by severity as follows:

    • Grade 1: Mild reaction, infusion interruption not indicated; intervention not indicated
    • Grade 2: Therapy or infusion interruption indicated but responds promptly to symptomatic treatment (e.g., antihistamines, NSAIDS, narcotics, IV fluids); prophylactic medications indicated for <=24 hrs
    • Grade 3: Prolonged (e.g., not rapidly responsive to symptomatic medication and/or brief interruption of infusion); recurrence of symptoms following initial improvement; hospitalization indicated for clinical sequelae (e.g., renal impairment, pulmonary infiltrates)
    • Grade 4: Life-threatening consequences; pressor or ventilatory support indicated
    • Grade 5: Death


      See, e.g., “Common Terminology Criteria for Adverse Events (CTCAE) Version v4.03,” National Institutes of Health and National Cancer Institute, Jun. 14, 2010; Lee D W et al., “Current concepts in the diagnosis and management of cytokine release syndrome,” Blood 2014 124(2):188-95.


As used herein, the term “CAR-T associated neuropathy” means neuropathy that arises subsequent to administration of CAR-T therapy to a patient, often after intervening cytokine release syndrome has occurred and subsided. The term is relatively new, mainly because CAR-T therapy is relatively new; see, e.g., Vasthie P and Breitbart W S, “Chimeric antigen receptor T-cell neuropsychiatric toxicity in acute lymphoblastic leukemia,” Palliat Support Care. 2017 August; 15(4): 499-503. Accordingly, CAR-T associated neuropathy should be understood at this time to be equivalent to the term “CAR-bearing immune effector cell associated neuropathy,” since similar neuropathy could arise from therapy with, e.g., iNKT-CARs or NK-CARs.


As used herein, a “cytokine” is one of a class of small (˜5-20 kDa), soluble signaling proteins that are that are synthesized and secreted by certain cells of the immune system at variable, and occasionally locally high, concentrations and by binding to receptors on other cells, send signals to and have an effect on those cells. A “chemokine” is a chemotactic cytokine, i.e., a subspecies of cytokine that is able to induce chemotaxis in nearby responsive cells.


As used herein, to be “deficient” in a cytokine or protein means to lack sufficient quantity of the cytokine or protein for the cytokine or protein to elicit its normal effect. A cell that is “deficient” in GM-CSF, for example, (a “GM-CSF deficient” cell) could be entirely lacking in GM-CSF, but it also could express such a negligible quantity of GM-CSF that the GM-CSF present could not contribute in any meaningful way to the development or maintenance of cytokine release syndrome.


The term “deletion” as used herein in reference to the effect of editing on a gene or its protein product, means alteration or loss of part the sequence of DNA encoding the protein so as to reduce or prevent expression of the protein product. The term “suppression” in the same context means to reduce expression of the protein product; and the term “ablation” in the same context means to knock out (KO) or prevent expression of the protein product. Deletion encompasses suppression and ablation.


As used herein, a “secretable protein” is s protein secreted by a cell which has an effect on other cells. By way of example, secretable proteins include cytokines, chemokines, and transcription factors.


As used herein, a “selectable marker” refers to a marker that allows distinguishing between different cell types, such as a cell into which a CAR has been successfully inserted (i.e., a gene-edited or modified cell). Selectable markers are well known in the art and materials and methods for their use are readily available. In some embodiments, a selectable marker appropriate in accordance with the present disclosure may be a fluorescent protein gene, such as including, but not limited to, a green fluorescence (GFP) gene or a yellow fluorescent protein (YFP) gene. In some embodiments, a selectable marker may be a splice variant of a CD34 gene, such as a truncated CD34 (tCD34) gene or a truncated EGFR (tEGFR) gene. In some embodiments, a selectable marker described herein, such as GFP, or others known and available in the art, may be inserted alone into a gene as described herein (i.e., without a CAR), or may be inserted as a component of a construct comprising the selectable marker and a CAR.


As used herein, a “short hairpin RNA” or “small hairpin RNA” (shRNA) is an artificial RNA molecule, often about 80 base pairs in length and with a tight hairpin turn, that can be used to silence target gene expression via processing within the cell into siRNA which in turn knocks down gene expression. ShRNAs can be incorporated into genomic DNA, and provide stable and long-lasting expression.


As used herein, “transduction” is the process by which foreign DNA is introduced into a cell by a virus or viral vector such as a plasmid, for example by short hairpin RNAs (shRNAs); it often provides long-lasting or permanent silencing of a gene. It may be accomplished by methods known in the art, including electroporation.


Transfection is the process of deliberately introducing purified nucleic acids into eukaryotic cells, for example small interfering RNAs (siRNAs); it produces transient silencing of a gene by RNA interference with mRNA transcripts. Transduction is the process by which foreign DNA is introduced into a cell by a virus or viral vector such as a plasmid, for example by short hairpin RNAs (shRNAs); it often provides long-lasting or permanent silencing of a gene. Both may be accomplished by methods known in the art, including electroporation.


EXAMPLES

The invention is further illustrated by the following examples.


Example 1—Method of Blocking Gene Translation by CAR Insertion


FIG. 1 shows the deletion of a cytokine gene by using Cas9/CRISPR to target a locus in a cytokine gene, then use homology-directed repair to insert the CAS construct. Optionally, the CAS construct would contain a marker, such as a selectable marker as described herein. One challenge with this concept is that is hard to select and sort out the cells have been edited from those that still express the cytokine/chemokine. One way to overcome this is to insert the CAR construct containing a marker, into gene that has been deleted. It has previously been demonstrated that inserting CAR19 into the TRAC gene allows the selection of TRAC-negative, CAR-positive cells. This would allow sorting of cells that have both expression of the car and have the cytokine deleted. Another alternative would be to express a shRNA from the CAR construct that would degrade the RNA encoding for the cytokine without altering the genome. A marker on the CAR construct (such as the truncated CD34 marker) could be used to select cells that downregulate the cytokine and express the CAR, i.e., CAR+=Cytokine negative.


As shown in FIG. 2, CAR-bearing immune effector cells (e.g., CAR-T) would be activated for two days prior to gene editing to delete the target cytokine gene and transduction of CAR.


Example 2—General Method of Making Genome-Edited CAR-Bearing Immune Effector Cells

Further details regarding the manner of making CAR-bearing immune effector cells are known in the art, e.g., as disclosed in WO2018027036A1.


The following general steps may be taken to provide CAR-bearing immune effector cells. As those of skill in the art will recognize, certain of the steps may be conducted sequentially or out of the order listed below, though perhaps leading to different efficiency.


Step 1. Cells are harvested, isolated, and purified, for example using magnetic selection with a labelled antibody-coated magnetic beads that bind to a cell-specific protein (available from, e.g., Miltenyi Biotec). For T cells, anti-CD3/CD28 beads could be used. Other purification techniques are known in the art and could be used.


Step 3. Cells are thereafter activated. There are several ways to activate immune effector cells. For example, T cells may be activated using antiCD3/CD28 beads for two days prior to bead removal. Alternatively, an antibody could be used.


Step 4. The antigen that is the target of the CAR may be deleted from the cell surface or its expression suppressed to prevent subsequent fratricide. Target deletion may be accomplished by electroporation with Cas9 mRNA and gRNA against the target(s). Other techniques, however, could be used to suppress expression of the target. These include other genome editing techniques such as TALENs, ZFNs, RNA interference, and eliciting of internal binding of the antigen to prevent cell surface expression. Deletion of the target may not be required in every circumstance. Examples of gRNAs that may be used include those shown in Tables 8-10, as well as others known in the art.









TABLE 8







Guide RNA sequences for use in removing surface antigens on immune effector cells








Target



gene
gRNA sequence





CD7
5′_2′OMe(A(ps)U(pS)C(ps))ACGGAGGUCAAUGUCUAGUUUUAGAGCUAGA



AAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUG



GCACCGAGUCGGUGC2′OMe(U(ps)U(ps)U(ps)U_3′ (SEQ ID NO: 40)





CD7g10
5′_2′OMe(G(ps)U(ps)A(ps))GACAUUGACCUCCGUGAGUUUUAGAGCUAGA



AAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUG



GCACCGAGUCGGUGC2′OMe(U(ps)U(ps)U(ps)U_3′ (SEQ ID NO: 41)





CD7g4
5′_2′OMe(A(ps)U(ps)C(ps))ACGGAGGUCAAUGUCUAGUUUUAGAGCUAGA



AAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUG



GCACCGAGUCGGUGC2′OMe(U(ps)U(ps)U(ps)U _3′ (SEQ ID NO: 42)





TRACg
5′_2′OMe(G(ps)A(ps)G(ps))AAUCAAAAUCGGUGAAUGUUUUAGAGCUAGA



AAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUG



GCACCGAGUCGGUGC2′OMe(U(ps)U(ps)U(ps)U_3′ (SEQ ID NO: 43)





CS1
5′_2′OMe(G(ps)A(ps)C(ps))CAAUCUGACAUGCUGCAGUUUUAGAGCUAGA



AAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUG



GCACCGAGUCGGUGC2′OMe(U(ps)U(ps)U(ps)U_3′ (SEQ ID NO: 44)





CD2
5′_2′OMe(A(ps)C(ps)A(ps))GCUGACAGGCUCGACACGUUUUAGAGCUAGA



AAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUG



GCACCGAGUCGGUGC2′OMe(U(ps)U(ps)U(ps) U_3′ (SEQ ID NO: 45)





CD2g
5′_2′OMe(G(ps)A(ps)G(ps))AAUCAAAAUCGGUGAAUGUUUUAGAGCUAGA



AAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUG



GCACCGAGUCGGUGC2′OMe(U(ps)U(ps)U(ps) U 3′ (SEQ ID NO: 46)





CD3ϵg
5′_2′OMe(A(ps)G(ps)G(ps))GCAUGUCAAUAUUACUGGUUUUAGAGCUAGA



AAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUG



GCACCGAGUCGGUGC2′OMe(U(ps)U(ps)U(ps) U 3′ (SEQ ID NO: 47)





CD5
5′_2′OMe(C(ps)G(ps)U(ps))UCCAACUCGAAGUGCCAGUUUUAGAGCUAGA



AAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUG



GCACCGAGUCGGUGC2′OMe(U(ps)U(ps)U(ps))U3′ (SEQ ID NO: 48)





CD5g
5′_2′OMe(C(ps)G(ps)U(ps))uCCAACUCGAAGUGCCAGUUUUAGAGCUAGA



AAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUG



GCACCGAGUCGGUGC2′OMe(U(ps)U(ps)U(ps)U_3′ (SEQ ID NO: 49)





RNA; (ps) indicate phosphorothioate.


Underlined bases denote target sequence.






Step 5. Cells may then be transduced with a CAR targeted to (i.e., that recognizes) one or more antigen or protein targets, for example with a lentivirus containing a CAR construct. Any other suitable method of transduction/transfection may be used, for example transfection using DNA-integrating viral or non-viral vectors containing transposable elements, or transient expressing of non-DNA integrating polynucleotides, such as mRNA, or insertion of CAR polynucleotide into site of nuclease activity using homologous or non-homologous recombination.


Step 6. CAR-bearing immune effector cells are then cultured to expand their population.


Example 3—Method of Making Genome-Edited Tandem CAR-Bearing Immune Effector Cells

In a variation of the protocol in the example above, a tCAR cell recognizing two antigens can be made. In Step 4, the two antigens can be deleted from the cell surface, or suppressed as described above, but electroporation with gRNA for each of the two targets and Cas9 mRNA. In Step 5, cells are then transduced with a CAR that recognizes two targets.


Example 4—Method of Making Genome-Edited Dual CAR-Bearing Immune Effector Cells

In a variation of the protocol in the example above, a dCAR cell targeting two antigens can be made. This variation would contain two separate CARs, each recognizing a different antigen.


Example 5—Method of Making and Testing a Genome-Edited CAR-T Cell with Suppressed Expression of Cytokine or Chemokine

The following steps may be taken to provide a genome-edited CAR-T cell with suppressed expression and/or secretion of a specific cytokine or chemokine. This example describes the making of a CD19 targeting CAR-T which is deficient in expression of GM-CSF. As those of skill in the art will recognize, certain of the steps may be conducted sequentially or out of the order listed below, though perhaps leading to different efficiency.


Tumor is injected into SCID-Beige mice (3e6 Raji containing Luciferase) if performing in vivo CRS experiment. This should be completed 3 weeks prior to infusion of CAR-T into mice.


T Cell Activation (Day 0).


T cells are purified via leukapheresis chamber using a Miltenyi human PanT isolation kit, then resuspended in media. Cells are counted and the number of human T cell activation CD3/CD28 beads required to obtain 3:1 bead:cell ratio determined. Beads are washed 2× with T cell media, then cells diluted at 1.256 cells/mL in hXcyte media. Human T cell activation CD3/CD28 beads are added. Into each well of a 6-well plate are aliquoted 4 mL/well of 1.256 cell/mL solution. Cells are incubated at 37° C.


CRISPR (Day 2).


Target deletion may be accomplished by electroporating with Cas9 mRNA and gRNA against the target(s). Other techniques, however, could be used to suppress expression of the target. These include other genome editing techniques such as TALENs, RNA interference, and eliciting of internal binding of the antigen to prevent cell surface expression. Examples of gRNAs that may be used include those shown in Tables 8-10, as well as others known in the art.


















Nucleofection


Sample ID
gRNA#1
Cas9
Buffer P3








20 μg gGM-CSF
15 μg Cas9 mRNA
100 μl
















TABLE 9







Guide RNA sequences for use in reducing CRS








Target



gene
gRNA sequence





GM-CSF
5′_2′OMe(U(ps)A(ps)C(ps))UCAGGUU



CAGGAGACGCGUUUUAGAGCUAGAAAUAGCAA



GUUAAAAUAAGGCUAGUCCGUUAUCAACUUGA



AAAAGUGGCACCGAGUCGGUGC 2′OMe



(U(ps)U(ps)U(ps)U _3′ (SEQ ID NO: 50)





RNA; (ps) indicate phosphorothioate.


Underlined bases denote target sequence.













TABLE 10







Additional guide RNA sequences for use in


reducing the incidence of CRS











Target






Gene

SEQ ID




Symbol
Guide RNA Sequence
NO: 
Location
Gene ID No.














A2M
GACAAAUCAAUCUACAAACC
56
chr12
ENST00000318602.11





A2M
GGUCCGCUUCUUAAAUUCUU
57
chr12
ENST00000318602.11





A2M
UCCUCAUUGGAUGAAGACUU
58
chr12
ENST00000318602.1





ACKR1
AACUGAGAACUCAAGUCAGC
59
chr1
ENST00000368122.2





ACKR1
GAAGGAAUCAUUCACACCAU
60
chr1
ENST00000368122.2





ACKR1
ACACUGGUGAGGAUGAAGAA
61
chr1
ENST00000368122.2





ACKR2
UCGGCAUCCUCAGUGGCGAG
62
chr3
ENST00000422265.5





ACKR2
GCAUGAAGGCCACUUCAUCC
63
chr3
ENST00000422265.5





ACKR2
AAAUCAGGCUAUAGAAGACU
64
chr3
ENST00000422265.5





ACKR3
UAGAGCAGGACGCUUUUGUU
65
chr2
ENST00000272928.3





ACKR3
CGGCAUGAUUGCCAACUCCG
66
chr2
ENST00000272928.3





ACKR3
CCCACAGGUCGGCAAUGGCC
67
chr2
ENST00000272928.3





ACVR1
UCUUUCCCUGCUCAUAAACC
68
chr2
ENST00000263640.7





ACVR1
UCCUCACUGAGCAUCAACGA
69
chr2
ENST00000263640.7





ACVR1
CUCUACAUGUGUGUGUGUGA
70
chr2
ENST00000263640.7





ACVR2B
CUACAACGCCAACUGGGAGC
71
chr3
ENST00000352511.4





ACVR2B
GCCAGAGCUGUUGCGCCAGG
72
chr3
ENST00000352511.4





ACVR2B
AUGACUUCAACUGCUACGAU
73
chr3
ENST00000352511.4





ACVRL1
AGACCCUGUGAAGCCGUCUC
74
chr12
ENST00000388922.8





ACVRL1
GGCCUACCUGCCGGGGGGCC
75
chr12
ENST00000388922.8





ACVRL1
GGGGCUGCGGGAACUUGCAC
76
chr12
ENST00000388922.8





ADIPOQ
GGGCUCAGGAUGCUGUUGCU
77
chr3
ENST00000320741.6





ADIPOQ
CGUGGUUUCCUGGUCAUGAC
78
chr3
ENST00000320741.6





ADIPOQ
GGGGGCCUGCACAGGUUGGA
79
chr3
ENST00000320741.6





AGER
ACGGACUCGGUAGUUGGACU
80
chr6
ENST00000375076.8





AGER
CUGUCGGGAUCCAGGAUGAG
81
chr6
ENST00000375076.8





AGER
UCCCCAGGGAGGAGGCCCCU
82
chr6
ENST00000375076.8





AGRN
CGGGGAAUGCUGUGCGGCUU
83
chr1
ENST00000379370.6





AGRN
GAAGAGCCCGUGCCCCAGCG
84
chr1
ENST00000379370.6





AGRN
GCAUUCGUUGCUGUAGGUGG
85
chr1
ENST00000379370.6





AHR
UUGAUUCCUUCAGCUGGGAU
86
chr7
ENST00000242057.8





AHR
GUAAAGCCAAUCCCAGCUGA
87
chr7
ENST00000242057.8





AHR
UUGACUUGAUUCCUUCAGCU
88
chr7
ENST00000242057.8





AHR
UUUGACUUGAUUCCUUCAGC
89
chr7
ENST00000242057.8





AIMP1
AACCAAUUCGAAGAUCCAGA
90
chr4
ENST00000358008.7





AIMP1
UAUGUGGAAGAAGUAGAUGU
91
chr4
ENST00000358008.7





AIMP1
CAAGAGGAACAUGAUUCACC
92
chr4
ENST00000358008.7





AREG
GGUCCAAUCCAGCAGCAUAA
93
chr4
ENST00000395748.7





AREG
CCACAGUGCUGAUGGAUUUG
94
chr4
ENST00000395748.7





AREG
AGAAGGCAUUUCACUCACAG
95
chr4
ENST00000395748.7





BCL6
CUGUAAAGAUGCUAUAGAAC
96
chr3
ENST00000406870.6





BCL6
CACUAAGGUUGCAUUUCAAC
97
chr3
ENST00000406870.6





BCL6
GAUCUAGAUUGAUCACACUA
98
chr3
ENST00000406870.6





BCL6
AAUCCCUCAGGGUUGAUCUC
99
chr3
ENST00000406870.6





BMP1
CAGCGGCAGGCGGGCCACGC
100
chr8
ENST00000306385.9





BMP1
GGCCGACUACACCUAUGACC
101
chr8
ENST00000306385.9





BMP1
GGCGCUCACCCGCCUUGCAG
102
chr8
ENST00000306385.9





BMP10
GAAACUCAUCCUUCAUGCUC
103
chr2
ENST00000295379.1





BMP10
AGGGACAUAUCUUCUUCCAG
104
chr2
ENST00000295379.1





BMP10
CAGAAACCAAGUAAGCUGCC
105
chr2
ENST00000295379.1





BMP15
CGUGCUUUUCAUGGAACACA
106
chrX
ENST00000252677.3





BMP15
CCCUACUUUGCCCCUGAUUG
107
chrX
ENST00000252677.3





BMP15
AGUGAAUGCCCUAGGAGCCG
108
chrX
ENST00000252677.3





BMP2
CGCAGGUCGACCAUGGUGGC
109
chr20
ENST00000378827.4





BMP2
GGGCCGCAGGAAGUUCGCGG
110
chr20
ENST00000378827.4





BMP2
GCCGCAACUCGAACUCGCUC
111
chr20
ENST00000378827.4





BMP3
AGGCUGCUCUUUCUGUGGCU
112
chr4
ENST00000282701.2





BMP3
GGCACAGCUUUGCGGAGCUC
113
chr4
ENST00000282701.2





BMP3
CGAGCUGCAGCCGCAAGACA
114
chr4
ENST00000282701.2





BMP4
UUCGUGGUGGAAGCUCCUCA
115
chr14
ENST00000245451.8





BMP4
GAAGAGCAGAUCCACAGCAC
116
chr14
ENST00000245451.8





BMP4
CACUCUUGCUAGGCUGCGGG
117
chr14
ENST00000245451.8





BMP5
ACUGAGUACUCCGACUCUUC
118
chr6
ENST00000370830.3





BMP5
AGACCCAGACCAUUUUCACC
119
chr6
ENST00000370830.3





BMP5
CGAAAGACGGGAAAUACAAA
120
chr6
ENST00000370830.3





BMP6
GAUGCAGAAGGAGAUCUUGU
121
chr6
ENST00000283147.6





BMP6
GGCUGUUGGAGGCCGUGCAG
122
chr6
ENST00000283147.6





BMP6
CCUCGCGGAGAGCCCCCUCC
123
chr6
ENST00000283147.6





BMP7
UCUCAUUGUCGAAGCGUUCC
124
chr20
ENST00000395863.7





BMP7
GCCGUGACAGCUUCCCCUUC
125
chr20
ENST00000395863.7





BMP7
UCUUCCAGUGGAACAUGACA
126
chr20
ENST00000395863.7





BMP8A
CCAGAAGCCAGAGCGGUCCG
127
chr1
ENST00000331593.5





BMP8A
UGGCGGUGCUCGGGCUACCC
128
chr1
ENST00000331593.5





BMP8A
UGGCGGUGCUCGGGCUACCC
129
chr1
ENST00000331593.5





BMP8B
CGGUGCUCGGGCUGCCUGGG
130
chr1
ENST00000372827.7





BMP8B
CCCAGGAGCCAGAGCGGGCC
131
chr1
ENST00000372827.7





BMP8B
CCCAGGAGCCAGAGCGGGCC
132
chr1
ENST00000372827.7





BMPR2
CACUUGCAGCUGAUUGGCCG
133
chr2
ENST00000374580.8





BMPR2
UUUCGUUGAUAAAAUUCUGA
134
chr2
ENST00000374580.8





BMPR2
CUCAUCUCCAACUAUAAAGC
135
chr2
ENST00000374580.8





C10orf99
GGAAAGGACUAGAAGCCUCA
136
chr10
ENST00000372126.3





C10orf99
CCUACCUUCUGUGGAGAAGA
137
chr10
ENST00000372126.3





C10orf99
CUCUGAUCAGGAUUUGUUGG
138
chr10
ENST00000372126.3





C1QTNF4
CCAUCUCCGACGUGCCCUCC
139
chr11
ENST00000302514.3





C1QTNF4
CAGCUCAGAGGAUCCCGGGC
140
chr11
ENST00000302514.3





C1QTNF4
CCCAGCAGGCCCAGCAGAAG
141
chr11
ENST00000302514.3





C5*
UCCAGCACCGUCACUCCAGA
142
chr9
ENST00000223642.2





C5*
ACACGUGUUACACUUUUGCU
143
chr9
ENST00000223642.2





C5*
GAUUCGCUUGACCAGUUGGU
144
chr9
ENST00000223642.2





CCL1*
AGAAACGUGGUCAGCCACUC
145
chr17
ENST00000225842.3





CCL1*
CUCUUGCUGUCCACAUCUUC
146
chr17
ENST00000225842.3





CCL1*
GCAGAUCAUCACCACAGCCC
147
chr17
ENST00000225842.3





CCL11
AGGUCUCCGCAGCACUUCUG
148
chr17
ENST00000305869.3





CCL11
CUGGCCCAGCGAGCCCCUGG
149
chr17
ENST00000305869.3





CCL11
UAACCACCUCCAGAGCUACU
150
chr17
ENST00000305869.3





CCL13*
AUGUGAAGCAGCAAGUAGAU
151
chr17
ENST00000225844.6





CCL13*
CUUCUGGGGACACCUGCUGG
152
chr17
ENST00000225844.6





CCL13*
AACUUUGGAUUGGGGAAUGU
153
chr17
ENST00000225844.6





CCL14
CAGGAGUGAGGUUGGGGCUC
154
chr17
ENST00000618404.4





CCL14
GGCAUUGCACUCACGUGAGG
155
chr17
ENST00000618404.4





CCL14
CCACAGCAUGAAGAUCUCCG
156
chr17
ENST00000618404.4





CCL15*
UCCCAGAGCUUGAGUUACCA
157
chr17
ENST00000617897.1





CCL15*
CACCAUUUGUGAACUGGGCC
158
chr17
ENST00000617897.1





CCL15*
CACCAUUUGUGAACUGGGCC
159
chr17
ENST00000617897.1





CCL16
GGACGUGCUUACAUGAUUGC
160
chr17
ENST00000611905.1





CCL16
GUUGCCAAGGAGACUAGUGG
161
chr17
ENST00000611905.1





CCL16
UCCUUCAGAAGUUCCUGAGU
162
chr17
ENST00000611905.1





CCL17
GCUCCUCUCCCUGCAGCUCG
163
chr16
ENST00000219244.8





CCL17
UGGCUCCCUUGAAGUACUCC
164
chr16
ENST00000219244.8





CCL17
UGGAGCAGUCCUCAGAUGUC
165
chr16
ENST00000219244.8





CCL18
GAGGCAGCAGAGCUCUUUGU
166
chr17
ENST00000616054.1





CCL18
UGGCUUGGGGCACUGGGGGC
167
chr17
ENST00000616054.1





CCL18
CCAUCCUCCCUCCCGAGGGG
168
chr17
ENST00000616054.1





CCL19
CCCACAACUCACACUACAGC
169
chr9
ENST00000311925.6





CCL19
AAGUUCCUCACGAUGUACCC
170
chr9
ENST00000311925.6





CCL19
CAUUGGUGCCACUCAGAGUU
171
chr9
ENST00000311925.6





CCL2
GUUAUAACAGCAGGUGACUG
172
chr17
ENST00000225831.4





CCL2
UUCUUUGGGACACUUGCUGC
173
chr17
ENST00000225831.4





CCL2
GGCUUGUCCCUUGCUCCACA
174
chr17
ENST00000225831.4





CCL20
CAAACUCUUGGUACAGCACA
175
chr2
ENST00000358813.4





CCL20
CACCUCUGCGGCGAAUCAGA
176
chr2
ENST00000358813.4





CCL20
CAGUUGAGCUCAAGGCUAAA
177
chr2
ENST00000358813.4





CCL21
CCCUGCAGGCAGUGAUGGAG
178
chr9
ENST00000259607.6





CCL21
CCUCCCUGCCUUGGUACCUU
179
chr9
ENST00000259607.6





CCL21
GGCUCUGAGCCUCCUUAUCC
180
chr9
ENST00000259607.6





CCL22
ACCCCUCCCCUAGGCCCCUA
181
chr16
ENST00000219235.4





CCL22
GACGGUAACGGACGUAAUCA
182
chr16
ENST00000219235.4





CCL22
AGGCCUCGGGCAGGAGUCUG
183
chr16
ENST00000219235.4





CCL23*
UCGUUUCAAAGUAACUCUCC
184
chr17
ENST00000615050.1





CCL23*
AGCAGUCAGCACUAGUAGCA
185
chr17
ENST00000615050.1





CCL23*
GUGCUCUGGAGGAGAAAGAU
186
chr17
ENST00000615050.1





CCL24
CCUCAAGGCAGGAGUGAUGU
187
chr7
ENST00000222902.6





CCL24
GAGAAUUCCUGAGAACCGAG
188
chr7
ENST00000222902.6





CCL24
CUCUCCUCUUCUAGGCUCUG
189
chr7
ENST00000222902.6





CCL25
UGUGGUUGCAGGUGUCUUUG
190
chr19
ENST00000390669.7





CCL25
GGGCUGUGCUCCGGCGCGCC
191
chr19
ENST00000390669.7





CCL25
GGCCCCACUCACAUCGCAGC
192
chr19
ENST00000390669.7





CCL26
CCAUCCUGUUGCAUGACUGA
193
chr7
ENST00000005180.8





CCL26
CCAGUAACAGCUGCUCCCAG
194
chr7
ENST00000005180.8





CCL26
GUAUUGGAAGCAGCAGGUCU
195
chr7
ENST00000005180.8





CCL27
GAGCUCACACGAAAGCCUGG
196
chr9
ENST00000259631.4





CCL27
ACUGAGGAAGGUCAUCCAGG
197
chr9
ENST00000259631.4





CCL27
AGGCAGUGCUGGGUGGCAGU
198
chr9
ENST00000259631.4





CCL28
AAUCCUUCCAGCACAGAUUU
199
chr5
ENST00000361115.4





CCL28
GUGCCCCCACUCACCUUCUG
200
chr5
ENST00000361115.4





CCL28
CAGGCAGGAAUGCAGCAGAG
201
chr5
ENST00000361115.4





CCL3
GCCACAAGAAAAGAUUGAUG
202
chr17
ENST00000613922.1





CCL3
ACUCACGUGAUGCAGAGAAC
203
chr17
ENST00000613922.1





CCL3
UCACCUGCUCAGAAUCAUGC
204
chr17
ENST00000613922.1





CCL4*
UGAGAGCGCUGGAGAGCAGA
205
chr17
ENST00000615863.1





CCL4*
UGAGAGCGCUGGAGAGCAGA
206
chr17
ENST00000615863.1





CCL4*
UGAGAGCGCUGGAGAGCAGA
207
chr17
ENST00000615863.1





CCL5
GACUCACACGACUGCUGGGU
208
chr17
ENST00000603197.5





CCL5
UGAUGUGGGCACGGGGCAGU
209
chr17
ENST00000603197.5





CCL5
CUUUCCUCUUCCAGAUUCCU
210
chr17
ENST00000603197.5





CCL7
AUUGAUAAAUCUGUAGCAGC
211
chr17
ENST00000378569.2





CCL7
CCACAUACAUUACAGCUUCC
212
chr17
ENST00000378569.2





CCL7
CUAGUCCUAUUCCCUGCCCA
213
chr17
ENST00000378569.2





CCL8*
AAAGCAGCAGGUGAUUGGAA
214
chr17
ENST00000394620.1





CCL8*
CACCAACAUCCAAUGUCCCA
215
chr17
ENST00000394620.1





CCL8*
CCCCCAUUCAAAAGUUCUGA
216
chr17
ENST00000394620.1





CCR1
CCUGGUUGGAAACAUCCUGG
217
chr3
ENST00000296140.3





CCR1
AGGCCCUCUCGUUCACCUUC
218
chr3
ENST00000296140.3





CCR1
UCAUAGUCCUCUGUGGUGUU
219
chr3
ENST00000296140.3





CCR2
GAUAAACCGAGAACGAGAUG
220
chr3
ENST00000445132.2





CCR2
ACCUUUUUUGAUUAUGAUUA
221
chr3
ENST00000445132.2





CCR2
GUAGAGCGGAGGCAGGAGUU
222
chr3
ENST00000445132.2





CCR3
CUAGAUACAGUUGAGACCUU
223
chr3
ENST00000357422.2





CCR3
UAUCAGCUUUUUCACAGAGC
224
chr3
ENST00000357422.2





CCR3
GGUGUUCACUGUGGGCCUCU
225
chr3
ENST00000357422.2





CCR5
UUUUGCAGUUUAUCAGGAUG
226
chr3
ENST00000292303.4





CCR5
AAAACAGGUCAGAGAUGGCC
227
chr3
ENST00000292303.4





CCR5
UGUAUUUCCAAAGUCCCACU
228
chr3
ENST00000292303.4





CCR6
UUGUUCUUACUCUCCCAUUC
229
chr6
ENST00000341935.9





CCR6
GCCUUUUAGCAACUUGCACG
230
chr6
ENST00000341935.9





CCR6
CCGGUACAUCGCCAUUGUAC
231
chr6
ENST00000341935.9





CCR7
GGCCGCGCUGUAGGCCCAGA
232
chr17
ENST00000246657.2





CCR7
GAGCAGGUAGGUAUCGGUCA
233
chr17
ENST00000246657.2





CCR7
UAGGCCCACGAAACAAAUGA
234
chr17
ENST00000246657.2





CD109
UCCGGGCCUGAUGAUCCCUG
235
chr6
ENST00000287097.5





CD109
GCCUUCACAGUCACCUGUGA
236
chr6
ENST00000287097.5





CD109
CAAAGACUCCUUCUGCUUCC
237
chr6
ENST00000287097.5





CD27**
CCUUCACGAGGAAUGUUCCU
238
chr12
ENST00000266557.3





CD27**
UCCUUCACGAGGAAUGUUCC
239
chr12
ENST00000266557.3





CD27**
GCUGGUCACAGUCCUUCACG
240
chr12
ENST00000266557.3





CD27**
ACUGAGCAGCCUUUCUAUGC
241
chr12
ENST00000266557.3





CD28***
CACCAAAAUCUUGUUUCCUG
242
ch2
ENST00000458610.6





CD28***
UCACCAAAAUCUUGUUUCCU
243
ch2
ENST00000458610.6





CD28***
UUGUCGUACGCUACAAGCAU
244
ch2
ENST00000458610.6





CD28***
AUUGUCGUACGCUACAAGCA
245
ch2
ENST00000458610.6





CD36
CAAGAAAAAUGGGCUGUGAC
246
chr7
ENST00000309881.11





CD36
GGUGCUGUCCUGGCUGUGUU
247
chr7
ENST00000309881.11





CD36
UAUCCAGAAGACAAUUAAAA
248
chr7
ENST00000309881.11





CD4**
AGUGCAAUGUAGGAGUCCAA
249
chr12
ENST00000011653.8





CD4**
CUGGAGCUCCAGCUGAGACA
250
chr12
ENST00000011653.8





CD4**
UUUUGAACUCCACCUUCUUC
251
chr12
ENST00000011653.8





CD40LG**
CAAAAUAGAUAGAAGAUGAA
252
chrX
ENST00000370629.6





CD40LG**
ACGAUACAGAGAUGCAACAC
253
chrX
ENST00000370629.6





CD40LG**
CCAGUUUGAAGGCUUUGUGA
254
chrX
ENST00000370629.6





CD70
AGCGCUGGAUGCACACCACG
255
chr19
ENST00000245903.3





CD70
AGCCCGCAGGACGCACCCAU
256
chr19
ENST00000245903.3





CD70
CGCCGCGGCGAUGCCGGAGG
257
chr19
ENST00000245903.3





CD74
UGCUCACACAUACCUCUCCG
258
chr5
ENST00000009530.11





CD74
UGUUGGAGAUAAGGUCGCGC
259
chr5
ENST00000009530.11





CD74
AGAUGCACAGGAGGAGAAGC
260
chr5
ENST00000009530.11





CD8A**
CCGGAACUGGCUCGGCCUGG
261
ch2
ENST00000409511.6





CD8A**
CACCCGGAACUGGCUCGGCC
262
ch2
ENST00000409511.6





CD8A**
GGCGACACCCGGAACUGGCU
263
ch2
ENST00000409511.6





CD8A**
CGCCAGGCCGAGCCAGUUCC
264
ch2
ENST00000409511.6





CER1
GGCACUGCGACAAACAGAUC
265
chr9
ENST00000380911.3





CER1
AAAGAGAACUCUGAUUCUGG
266
chr9
ENST00000380911.3





CER1
CUGCUGGUACUCCUGCCUCU
267
chr9
ENST00000380911.3





CHRD
GCCGGCGGGGCCGGGAGGCU
268
chr3
ENST00000204604.5





CHRD
UCCCGGCCGGCCCGCGGCGC
269
chr3
ENST00000204604.5





CHRD
CUGCCCGUUCGGGGAGCGGC
270
chr3
ENST00000204604.5





CKLF
GUGAUAACAAUAUAUGGUUC
271
chr16
ENST00000264001.8





CKLF
GACUUGAUCGAUUAAUGAAG
272
chr16
ENST00000264001.8





CKLF
GUAGGAAAAAGUAAAAUUUA
273
chr16
ENST00000264001.8





CLCF1
CAUAGGUCCCAGCCAAGCUG
274
chr11
ENST00000312438.7





CLCF1
CAUAGGUUUUCUGGAUGGAG
275
chr11
ENST00000312438.7





CLCF1
GGUGCCAGAGCACCGUGCAC
276
chr11
ENST00000312438.7





CMTM1
GACUCAGGUUUGGCGUGUUC
277
chr16
ENST00000379500.6





CMTM1
UACGCUGCCGCUACUUGCCA
278
chr16
ENST00000379500.6





CMTM1
UGCGGGGUGCUUCCGGGGUG
279
chr16
ENST00000379500.6





CMTM2
CGAGUCAGUCAUGGCACCUA
280
chr16
ENST00000268595.2





CMTM2
UCCACCCGGGGCCAAACCCG
281
chr16
ENST00000268595.2





CMTM2
AAAGGCGGUGCAGGACCAUA
282
chr16
ENST00000268595.2





CMTM3
CCGGACCCCGAGCCUGCCGG
283
chr16
ENST00000361909.8





CMTM3
GGAAAGCCCGCGCCGGCAGC
284
chr16
ENST00000361909.8





CMTM3
UCCCGCCGCACUCACCGACU
285
chr16
ENST00000361909.8





CMTM4
CCGCCCGGCACCUACCACUU
286
chr16
ENST00000394106.6





CMTM4
GGUAGUCGGGGUCGCAGCGC
287
chr16
ENST00000394106.6





CMTM4
GCUGCUGGCGCCCGAGAUCA
288
chr16
ENST00000394106.6





CMTM5
GCUCAGUGCUCGAGAUCGCC
289
chr14
ENST00000359320.7





CMTM5
CCAGGGCUUCGCGGUGGACA
290
chr14
ENST00000359320.7





CMTM5
UUACCAGCUCGGUUUCCAGC
291
chr14
ENST00000359320.7





CMTM6
UUGAGAACGCGCCGGAGCAA
292
chr3
ENST00000205636.3





CMTM6
CGCUCCGGGGGCCUCUGGCG
293
chr3
ENST00000205636.3





CMTM6
CGGCCCGAGGCGAUGGAGAA
294
chr3
ENST00000205636.3





CMTM7
GGAGCUCCGCACACAGAUGA
295
chr3
ENST00000334983.9





CMTM7
AAUGAUCCUCGCCUUUUACC
296
chr3
ENST00000334983.9





CMTM7
CGACAGGGGCCAGCUGAUAC
297
chr3
ENST00000334983.9





CMTM8
GUGUGCGAGCGGGCGCGCUG
298
chr3
ENST00000307526.3





CMTM8
CAGCAGCUUCGCCUACGACC
299
chr3
ENST00000307526.3





CMTM8
CCCGUCGGCACUCACGAUCU
300
chr3
ENST00000307526.3





CNTF
AUCAACCUGGACUCUGCGGA
301
chr11
ENST00000361987.5





CNTF
GGAAGGUACGAUAAGCUUGA
302
chr11
ENST00000361987.5





CNTF
GUGCAUUUUACCCCAACCGA
303
chr11
ENST00000361987.5





CNTFR
CCAGCUGAGAGCCGUUGAGC
304
chr9
ENST00000351266.8





CNTFR
GGGAUGCUGCGGUGACGUGG
305
chr9
ENST00000351266.8





CNTFR
CAGGCGCUCGUACUGCACAU
306
chr9
ENST00000351266.8





COPS5
UAGUCCAGGGCUUCGCCGCC
307
chr8
ENST00000357849.8





COPS5
CUGAGCUUCCUGCAUGUUGU
308
chr8
ENST00000357849.8





COPS5
CCUCGGCGAUGGCGGCGUCC
309
chr8
ENST00000357849.8





CRLF1
CCAGCGUGAGUACAUCGGAG
310
chr19
ENST00000392386.7





CRLF1
CAUAGGGCGUAAAGAGAGCC
311
chr19
ENST00000392386.7





CRLF1
ACUCCUCACAUGUGUUGUCC
312
chr19
ENST00000392386.7





CSF1*
GCCUUCUUAAGGUAGCACAC
313
chr1
ENST00000329608.10





CSF1*
GGUGUUAUCUCUGAAGCGCA
314
chr1
ENST00000329608.10





CSF1*
GUCAUGCUCUUCAUAAUCCU
315
chr1
ENST00000329608.10





CSF1R
CACCUUUCUGCACUUUCAGC
316
chr5
ENST00000286301.7





CSF1R
CAAUGCAGUGCCCUGAUGGG
317
chr5
ENST00000286301.7





CSF1R
CCAGGGCGAGAAGGAGUAGU
318
chr5
ENST00000286301.7





CSF2*
CCACAGUGCCCAAGAGCAGC
319
chr5
ENST00000296871.3





CSF2*
CAGGGCUGCGUGCUGGGGCU
320
chr5
ENST00000296871.3





CSF2*
UAGAGACACUGCUGCUGAGA
321
chr5
ENST00000296871.3





CSF3
UGUGCCACAGCAGCAGCUGC
322
chr17
ENST00000225474.6





CSF3
GAAGCUCUGGGGCAGGGAGC
323
chr17
ENST00000225474.6





CSF3
GGGCGAUGGCGCAGCGCUCC
324
chr17
ENST00000225474.6





CSF3R
GCAAUAGCAACAAGACCUGG
325
chr1
ENST00000361632.8





CSF3R
AUAGCCCUGAGGCCAUGGAU
326
chr1
ENST00000361632.8





CSF3R
GCAUGGAGUCUGGUCAGAGC
327
chr1
ENST00000361632.8





CTF1
UCUCCGCAGGUGCAGCUCCA
328
chr16
ENST00000279804.2





CTF1
CCGGCCACCGGCAGCCGCGG
329
chr16
ENST00000279804.2





CTF1
AGCCGCAGCCGCUCGUGCAC
330
chr16
ENST00000279804.2





CX3CL1
GUGCCGACCCGAAGGAGCAA
331
chr16
ENST00000006053.6





CX3CL1
CUUCGAGAAGCAGAUCGGCG
332
chr16
ENST00000006053.6





CX3CL1
CGCCUGUGGCUUCGGGCUCC
333
chr16
ENST00000006053.6





CX3CR1**
CUACCAACAAAUUUCCCACC
334
chr3
ENST00000399220.2





CX3CR1**
UUGGGGACAUCGUGGUCUUU
335
chr3
ENST00000399220.2





CX3CR1**
AAGUUUUCUGUCACUGAUUC
336
chr3
ENST00000399220.2





CXCL1
GGAGAGAGCAGCGCGGGCCA
337
chr4
ENST00000395761.3





CXCL1
CUGGCCGGCGCGCAGCAGGU
338
chr4
ENST00000395761.3





CXCL1
GUGCCUACCCCAGCCGCGUC
339
chr4
ENST00000395761.3





CXCL10
UACAGUAUAUAAUUACAACC
340
chr4
ENST00000306602.2





CXCL10
CGUGGACAAAAUUGGCUUGC
341
chr4
ENST00000306602.2





CXCL10
UUGAUUACUAAUGCUGAUGC
342
chr4
ENST00000306602.2





CXCL11
GCUGAAGAUGACAAUGGUGC
343
chr4
ENST00000306621.7





CXCL11
GUUGUUACUUGGGUACAUUA
344
chr4
ENST00000306621.7





CXCL11
AGCGUCCUCUUUUGAACAUG
345
chr4
ENST00000306621.7





CXCL12
CUACACCUUUAAUAAGACUC
346
chr10
ENST00000343575.10





CXCL12
UCAAGACUUACACAAUCUGA
347
chr10
ENST00000343575.10





CXCL12
UGACGUUGGCUCUGGCAACA
348
chr10
ENST00000343575.10





CXCL13
AGGUCUAUUACACAAGCUUG
349
chr4
ENST00000286758.4





CXCL13
UUCGAUCAAUGAAGCGUCUA
350
chr4
ENST00000286758.4





CXCL13
ACUUACAUGAUUUCUUUUCU
351
chr4
ENST00000286758.4





CXCL14
AAUUGGCGCUUGGGUUCCCC
352
chr5
ENST00000337225.5





CXCL14
CUGUACACCGCGCGUGUGGA
353
chr5
ENST00000337225.5





CXCL14
GAGGGGCGCGGCGUGGGAGC
354
chr5
ENST00000337225.5





CXCL16
GCGCUGAGUGGACUGCAAGG
355
chr17
ENST00000293778.10





CXCL16
UACCAGCCCCCCAAUUUCUC
356
chr17
ENST00000293778.10





CXCL16
AAUGUGGACAUGCUUACUCG
357
chr17
ENST00000293778.10





CXCL17
AUUGGUCUCAGAGGGGCCCA
358
chr19
ENST00000601181.5





CXCL17
GACCUUUGCACUCACAUUCU
359
chr19
ENST00000601181.5





CXCL17
CUUCCGACAGGGGUCGCCAG
360
chr19
ENST00000601181.5





CXCL2
GCAGUGGCAGCGGCAGCGAU
361
chr4
ENST00000508487.2





CXCL2
UUCUUCCCUAGGAGCGCCCC
362
chr4
ENST00000508487.2





CXCL2
UUCUUCCCUAGGAGCGCCCC
363
chr4
ENST00000508487.2





CXCL3*
UGGCAGCGGAAGCGCGGGGC
364
chr4
ENST00000296026.4





CXCL3*
GGGACCUUACAUUCACACUU
365
chr4
ENST00000296026.4





CXCL3*
GGGACCUUACAUUCACACUU
366
chr4
ENST00000296026.4





CXCL5
CCCUUUAUAGGGCAGGUUGC
367
chr4
ENST00000296027.4





CXCL5
GCGCCAUGCGCUCUCACCGC
368
chr4
ENST00000296027.4





CXCL5
GAGCUCCUUGUGCGCGCUGU
369
chr4
ENST00000296027.4





CXCL6*
GCCCGAAGGACCCGGGACAC
370
chr4
ENST00000226317.9





CXCL6*
GGGGCCCCGGCGGCGUCAGC
371
chr4
ENST00000226317.9





CXCL6*
UUUUUAUAGGGCAGGUUGCU
372
chr4
ENST00000226317.9





CXCL8
CAACAGGUGCAGUUUUGCCA
373
chr4
ENST00000307407.7





CXCL8
AAAUUUGGGGUGGAAAGGUU
374
chr4
ENST00000307407.7





CXCL8
UACUUACAUAAUUUCUGUGU
375
chr4
ENST00000307407.7





CXCL9*
UUUCAAUUUUCUCGCAGGAA
376
chr4
ENST00000264888.5





CXCL9*
AUUGUAGGUGGAUAGUCCCU
377
chr4
ENST00000264888.5





CXCL9*
GGAACCCCAGUAGUGAGAAA
378
chr4
ENST00000264888.5





CXCR1
GAUGGUAAGCCUGGCGGAAA
379
chr2
ENST00000295683.2





CXCR1
UUGGUCAAGUUUGUUUGUCU
380
chr2
ENST00000295683.2





CXCR1
CUACAGUGGCAUCCUGCUGU
381
chr2
ENST00000295683.2





CXCR2
GUGACAGCUUUGAAGAUUUC
382
chr2
ENST00000318507.6





CXCR2
AUCUAGUAGAAAAGGGGGCA
383
chr2
ENST00000318507.6





CXCR2
UGUGGUCAUUAUCUAUGCCC
384
chr2
ENST00000318507.6





CXCR3{circumflex over ( )}
GCAGAAAGAGGAGGCUGUAG
385
chrX
ENST00000373693.3





CXCR3{circumflex over ( )}
UACCUCCCCGCCCUGCCCAC
386
chrX
ENST00000373693.3





CXCR3{circumflex over ( )}
AAGAGCUGAAGUUCUCCAGG
387
chrX
ENST00000373693.3





CXCR4{circumflex over ( )}
CCCAAAGUACCAGUUUGCCA
388
chr2
ENST00000409817.1





CXCR4{circumflex over ( )}
AGAGGAGGUCGGCCACUGAC
389
chr2
ENST00000409817.1





CXCR4{circumflex over ( )}
UGGAUUGGUCAUCCUGGUCA
390
chr2
ENST00000409817.1





CXCR6**
GGAAGUCUUGAUGCUCCUCC
391
chr3
ENST00000304552.4





CXCR6**
GGUGUUUGUCUGUGGUCUGG
392
chr3
ENST00000304552.4





CXCR6**
CCGUCAGGCUCUGCAACUUA
393
chr3
ENST00000304552.4





EBB
CACCCUGUGCAGGCUCGGCA
394
chr19
ENST00000221847.5





EBB
GGAUGUCCAGCUGUUCUCCA
395
chr19
ENST00000221847.5





EBB
AGCUGCUGCUGGAGCCCCAG
396
chr19
ENST00000221847.5





EDN1
UUUCUCUCCCCAGCAGUCUU
397
chr6
ENST00000379375.5





EDN1
CCACUCCCAGUCCACCCUGG
398
chr6
ENST00000379375.5





EDN1
UCUGCCACCUGGACAUCAUU
399
chr6
ENST00000379375.5





ELANE
CACAAUCUCCGAGGCCAGCG
400
chr19
ENST00000263621.1





ELANE
GUGUCCCUGCAGCUGCGCGG
401
chr19
ENST00000263621.1





ELANE
GCGGCCGACAUGACGAAGUU
402
chr19
ENST00000263621.1





ENG
CCGGGCCACUCGGCCGGGUA
403
chr9
ENST00000344849.4





ENG
CGCCUUCCAAGUGGCAGCCC
404
chr9
ENST00000344849.4





ENG
CGUGCGGCCCAUGUCCUGGC
405
chr9
ENST00000344849.4





EPO
CUGUUCUAGAAUGUCCUGCC
406
chr7
ENST00000252723.2





EPO
CCCUCUGGGCCUCCCAGUCC
407
chr7
ENST00000252723.2





EPO
CCUGGAGAGGUACCUCUUGG
408
chr7
ENST00000252723.2





FAM3B
UUACAGCUCCAGUCCCCAAA
409
chr21
ENST00000357985.6





FAM3B
UGUAGGCAUAGGUGUCAGAU
410
chr21
ENST00000357985.6





FAM3B
GUUAUCCUCAAAGCAGAUUU
411
chr21
ENST00000357985.6





FAM3C
CUGAUAGUGCAUUUUACUUU
412
chr7
ENST00000359943.7





FAM3C
AUUGUUCUUACCAUUUGCCA
413
chr7
ENST00000359943.7





FAM3C
UCCCCCUUCAGUUUAAUGAG
414
chr7
ENST00000359943.7





FAM3D
GUGUCUUAGGUACUUACAUG
415
chr3
ENST00000358781.6





FAM3D
UUUGCGUUUAAAAUCUGCAG
416
chr3
ENST00000358781.6





FAM3D
GAGGUUAAAAAGUACAAGUG
417
chr3
ENST00000358781.6





FAS
CACUUGGGCAUUAACACUUU
418
chr10
ENST00000355740.6





FAS
UACAGUUGAGACUCAGAACU
419
chr10
ENST00000355740.6





FAS
GUGUAACAUACCUGGAGGAC
420
chr10
ENST00000355740.6





FASLG
UGGGGAUAUGGGUAAUUGAA
421
chr1
ENST00000367721.2





FASLG
AACUGUGCCUGGAGGGGCCC
422
chr1
ENST00000367721.2





FASLG
GGUGGUGGCCUCCUUUGACC
423
chr1
ENST00000367721.2





FGF2
CCGCAGGGACCAUGGCAGCC
424
chr4
ENST00000608478.1





FGF2
GGGUCCUUGAAGUGGCCGGG
425
chr4
ENST00000608478.1





FGF2
ACGGCCGAGUUGACGGGGUC
426
chr4
ENST00000608478.1





FLT3LG
CUCUAGGAGGAGCUCUGCGG
427
chr19
ENST00000594009.5





FLT3LG
GAGCGGCUCAAGACUGUCGC
428
chr19
ENST00000594009.5





FLT3LG
GCUGACCUGAAAGGCACAUU
429
chr19
ENST00000594009.5





FOXP3
AGGACCCGAUGCCCAACCCC
430
chrX
ENST00000376207.8





FOXP3
CCGAGGGCUUGCCAGGCCUG
431
chrX
ENST00000376207.8





FOXP3
CCGAUGCCCAACCCCAGGCC
432
chrX
ENST00000376207.8





FOXP3
CCCCAGGCCUGGCAAGCCCU
433
chrX
ENST00000376207.8





FZD4
UCGUCCCCGAAGCCCCGCGC
434
chr11
ENST00000531380.1





FZD4
CAGACUGAGACCGACGCCCC
435
chr11
ENST00000531380.1





FZD4
CGAUGCUGGCCAUGGCCUGG
436
chr11
ENST00000531380.1






GATA3


GAGCACAGCCGAGGCCAUGG


437


chr10


ENST00000346208.4







GATA3


CUGGUCCGCCGUCACCUCCA


438


chr10


ENST00000346208.4







GATA3


AGCCGAGGCCAUGGAGGUGA


439


chr10


ENST00000346208.4







GATA3


CGAGGCCAUGGAGGUGACGG


440


chr10


ENST00000346208.4






GBP1
UCCUCACAUCUUCAUAAUGG
441
chr1
ENST00000370473.4





GBP1
CAAGCUGGCUGGAAAGAAAA
442
chr1
ENST00000370473.4





GBP1
UUCUGCCAUUACACAGCCUA
443
chr1
ENST00000370473.4





GDF1
CGUCCGCCGCGAGCCAGACC
444
chr19
ENST00000247005.7





GDF1
GGCCGGUUCCCCCGGUCAUG
445
chr19
ENST00000247005.7





GDF1
GGGGCACGGGGGCGCGGGUC
446
chr19
ENST00000247005.7





GDF10
CCAUGGCUCAUGUCCCCGCU
447
chr10
ENST00000580279.1





GDF10
CGUUGUUUCUGCUGUUGCUC
448
chr10
ENST00000580279.1





GDF10
CGCGGGCAGUGCGGACCAGG
449
chr10
ENST00000580279.1





GDF11
ACGGGGCAGCCGUCCGGCUC
450
chr12
ENST00000257868.9





GDF11
UCUGCGACUUGAUGCUCUCU
451
chr12
ENST00000257868.9





GDF11
CACCACCUCGCGGCUGAUGU
452
chr12
ENST00000257868.9





GDF15
CACCGUCCUGAGUUCUUGCC
453
chr19
ENST00000252809.3





GDF15
GCUCGCCUCGGCCAGAGACA
454
chr19
ENST00000252809.3





GDF15
CUCUCGGAAUCUGGAGUCUU
455
chr19
ENST00000252809.3





GDF2
GCGGGCCUAAGAUGUGUCCU
456
chr10
ENST00000581492.2





GDF2
CUGCAGUGGCUUCCCCUGUA
457
chr10
ENST00000581492.2





GDF2
AGGCACCCCCAGUGGGCUGU
458
chr10
ENST00000581492.2





GDF3
AAAAUUUUCUUCAAGAUAUA
459
chr12
ENST00000329913.3





GDF3
CCUUAUCUAAGCCCAGAAAU
460
chr12
ENST00000329913.3





GDF3
GUUAAUUCUGGCUUUGGGCC
461
chr12
ENST00000329913.3





GDF5
CCCUGGCCUGAAGACGUUCC
462
chr20
ENST00000374369.7





GDF5
UGACUUGGGCCAGAGACCCC
463
chr20
ENST00000374369.7





GDF5
GGUACCAAAGCAAGAAAGUG
464
chr20
ENST00000374369.7





GDF6
GCGGCUUCGCAUGCCCUUGG
465
chr8
ENST00000287020.6





GDF6
GGAUUUGCCCGGUUUCCAGC
466
chr8
ENST00000287020.6





GDF6
GCCCGCCAUGGAUACUCCCA
467
chr8
ENST00000287020.6





GDF7
AGCGCCUGCCGCCCCCGCGA
468
chr2
ENST00000272224.4





GDF7
GCUGGGCCGGUCCGGAGCCC
469
chr2
ENST00000272224.4





GDF7
UGCGGGCGCCGCGGCUGUCC
470
chr2
ENST00000272224.4





GDF9
AUACCUGUUACCUGGUCUCC
471
chr5
ENST00000378673.2





GDF9
AGAGCCGAACAGUGUUGUAG
472
chr5
ENST00000378673.2





GDF9
CUAUAAGACAUAUGCUACCA
473
chr5
ENST00000378673.2





GPI
GUCCCGGGUGAGAGCGGCCA
474
chr19
ENST00000356487.9





GPI
UCAGCUCGGAGCGGUGCUCG
475
chr19
ENST00000356487.9





GPI
CGGCCCGCCCGCACCUGAAG
476
chr19
ENST00000356487.9





GREM1
UAUGAGCCGCACAGCCUACA
477
chr15
ENST00000622074.1





GREM1
CCCUUCAGCAGCCGGCAGCA
478
chr15
ENST00000622074.1





GREM1
UUGUGCUGGGCCUUGUCUGG
479
chr15
ENST00000622074.1





GREM2
CGGUGACCACCAGGGCCUCC
480
chr1
ENST00000318160.4





GREM2
GCAGCAACAACUCGGAGAGA
481
chr1
ENST00000318160.4





GREM2
GCUUUCCCUGUCCUUGUUCC
482
chr1
ENST00000318160.4





GRN
CAGGCAGACCAUGUGGACCC
483
chr17
ENST00000053867.7





GRN
GCUGGAACGCGGUGCCCAGA
484
chr17
ENST00000053867.7





GRN
AGCUGGCUCCUCCGGGGUCC
485
chr17
ENST00000053867.7





HAX1
CACCUUUCUGCAGAACUUCC
486
chr1
ENST00000328703.11





HAX1
UACGGGAGGGACAGACACUU
487
chr1
ENST00000328703.11





HAX1
CCAGUCUGGUGCUGGUUGGG
488
chr1
ENST00000328703.11





HFE2
AGGAGCGGAGGGCUCGACAG
489
chr1
ENST00000336751.10





HFE2
UCAUCAGGAGCACUUCGAGG
490
chr1
ENST00000336751.10





HFE2
CGUACUCAGCAUUGCAGCGG
491
chr1
ENST00000336751.10





HMGB1*
GAUACUCACGGAGGCCUCUU
492
chr13
ENST00000339872.8





HMGB1*
GAUACUCACGGAGGCCUCUU
493
chr13
ENST00000339872.8





HMGB1*
GAUACUCACGGAGGCCUCUU
494
chr13
ENST00000339872.8





HYAL2
GCGUGGGCCACAGUCCUGUG
495
chr3
ENST00000357750.8





HYAL2
UGCUGUGGGCUUGAGCUCCA
496
chr3
ENST00000357750.8





HYAL2
GCAGCCCCCAGCAUGCGGGC
497
chr3
ENST00000357750.8





ICAM3**
UCAUGUUCCUGAAGGUGUCC
498
chr19
ENST00000160262.9





ICAM3**
CAUGUUCCUGAAGGUGUCCA
499
chr19
ENST00000160262.9





ICAM3**
AUGUUCCUGAAGGUGUCCAG
500
chr19
ENST00000160262.9





ICAM3**
UCCUGAAGGUGUCCAGGGGC
501
chr19
ENST00000160262.9





ICOS**
UUUUAUGCAGGAGAAAUCAA
502
ch2
ENST00000316386.10





ICOS**
GAGAUGUUUAUAUUUCACAA
503
ch2
ENST00000316386.10





ICOS**
AUGUUUAUAUUUCACAACGG
504
ch2
ENST00000316386.10





ICOS**
UUUAAAAUGCAGUUGCUGAA
505
ch2
ENST00000316386.10





IFNA10
CAAACUCCUCCUGGGGGAUU
506
chr9
ENST00000357374.2





IFNA10
CAAACUCCUCCUGGGGGAUU
507
chr9
ENST00000357374.2





IFNA10
CAAACUCCUCCUGGGGGAUU
508
chr9
ENST00000357374.2





IFNA14
CAGGAGGACUUUGAUGCUCA
509
chr9
ENST00000380222.3





IFNA14
AAGCAAAGGGCAAUGCCAUU
510
chr9
ENST00000380222.3





IFNA14
AAGCAAAGGGCAAUGCCAUU
511
chr9
ENST00000380222.3





IFNA16
CAUCUCAUGGAAGGCAGAGA
512
chr9
ENST00000380216.1





IFNA16
AUAUGAUUUCGGAUUCCCCC
513
chr9
ENST00000380216.1





IFNA16
AUAUGAUUUCGGAUUCCCCC
514
chr9
ENST00000380216.1





IFNA2
UCAAGGUCCUCCUGCUACCC
515
chr9
ENST00000380206.3





IFNA2
CACCAGUAAAGCAAAGGUCA
516
chr9
ENST00000380206.3





IFNA2
CACCAGUAAAGCAAAGGUCA
517
chr9
ENST00000380206.3





IFNA5
UUGAUGAUAAUGGCACAAAU
518
chr9
ENST00000610521.1





IFNA5
CUUGCCCUUUGUUUUACUGA
519
chr9
ENST00000610521.1





IFNA5
CUUGCCCUUUGUUUUACUGA
520
chr9
ENST00000610521.1





IFNA6
AGGUCUGCUGAAUCACCUCA
521
chr9
ENST00000380210.1





IFNA6
ACAUGACUUCAGAUUUCCCC
522
chr9
ENST00000380210.1





IFNA6
UCAUGGUCCUCCUGUGACCC
523
chr9
ENST00000380210.1





IFNA8
UACAAGUCAUUCAGCUCUCU
524
chr9
ENST00000380205.1





IFNA8
GUUUAUCAUCAAACUCCUCC
525
chr9
ENST00000380205.1





IFNA8
GUUUAUCAUCAAACUCCUCC
526
chr9
ENST00000380205.1





IFNAR1
UUUACUUUAAAGAACUGGGA
527
chr21
ENST00000270139.7





IFNAR1
GAGUGAAGAAAAGUUGCAUU
528
chr21
ENST00000270139.7





IFNAR1
AAACACUUCUUCAUGGUAUG
529
chr21
ENST00000270139.7





IFNAR2
UAAACCAGAAGAUUUGAAGG
530
chr21
ENST00000342136.8





IFNAR2
UGAGUGGAGAAGCACACACG
531
chr21
ENST00000342136.8





IFNAR2
UCAGUUGCUCACACAAUUUC
532
chr21
ENST00000342136.8





IFNB1
UUGAAUACUGCCUCAAGGAC
533
chr9
ENST00000380232.3





IFNB1
GAAAAUUGCUGCUUCUUUGU
534
chr9
ENST00000380232.3





IFNB1
AGCACAACAGGAGAGCAAUU
535
chr9
ENST00000380232.3





IFNE
UGGCCAGAGUGUGUCCUUUU
536
chr9
ENST00000448696.4





IFNE
UAGACACUGCUGAAUUGACA
537
chr9
ENST00000448696.4





IFNE
CUUGAUUCACUUGUCUUUGC
538
chr9
ENST00000448696.4





IFNG*
UUUUUAAUAGUACUUGUUUG
539
chr12
ENST00000229135.3





IFNG*
CUUCUUUUACAUAUGGGUCC
540
chr12
ENST00000229135.3





IFNG*
GAAAUAUACAAGUUAUAUCU
541
chr12
ENST00000229135.3





IFNGR1**
AUUGUACACCCUAAUGUAAC
542
chr6
ENST00000367739.8





IFNGR1**
CUCCAUUUACAAAAACUGAA
543
chr6
ENST00000367739.8





IFNGR1**
UUUCUGAUAUCCAGUUUAGG
544
chr6
ENST00000367739.8





IFNK
UUGUGGCUUGAGAUCCUUAU
545
chr9
ENST00000276943.2





IFNK
CGUUCAGUAAGUUACAGUCC
546
chr9
ENST00000276943.2





IFNK
UUUUCUCGUAGACAUUCUAC
547
chr9
ENST00000276943.2





IFNL1
AUUUAGCCAUGGCUGCAGCU
548
chr19
ENST00000333625.2





IFNL1
GUGGCAGCCCUUCCCAGUUG
549
chr19
ENST00000333625.2





IFNL1
UAGCGAGCUUCAAGAAGGCC
550
chr19
ENST00000333625.2





IFNL3
CGGGCCUGUGUGAGUCGUCA
551
chr19
ENST0000041385L2





IFNL3
CCAAGACAUCCCCCAGGGCU
552
chr19
ENST0000041385L2





IFNL3
CCAAGACAUCCCCCAGGGCU
553
chr19
ENST0000041385L2





IFNW1
CUCCCUUUUACCAUCUCCUG
554
chr9
ENST00000380229.3





IFNW1
CAUUUGGUGCAGAAGCACCA
555
chr9
ENST00000380229.3





IFNW1
AGAUCCAACAGGGCUAUAGC
556
chr9
ENST00000380229.3





IL10*
CGGAGAUCUCGAAGCAUGUU
557
chr1
ENST00000423557.1





IL10*
UCAGACUGGGUGCCCUGGCC
558
chr1
ENST00000423557.1





IL10*
CUCAGCACUGCUCUGUUGCC
559
chr1
ENST00000423557.1





IL10RA**
GGUCUGGCUACAGUUGGAGA
560
chr11
ENST00000227752.7





IL10RA**
GGUAGCCAUUGCUGUGGUAC
561
chr11
ENST00000227752.7





IL10RA**
GGUGUUGGUGACGGUCCAGU
562
chr11
ENST00000227752.7





IL11
CGUGCCGCAGGUAGGACAGU
563
chr19
ENST00000264563.6





IL11
CGGCUCUAUCCCCAGCUCCC
564
chr19
ENST00000264563.6





IL11
CACUGGGAGCUCUACAGGUA
565
chr19
ENST00000264563.6





IL11RA
AGUGUCCUGGUUUCGGGAUG
566
chr9
ENST00000318041.13





IL11RA
GCUAGGGCAUGAACUGGUCC
567
chr9
ENST00000318041.13





IL11RA
UGCCCCCAAGUGCACCAUCC
568
chr9
ENST00000318041.13





IL12A
CCAGGGUAGCCACAAGGAGG
569
chr3
ENST00000305579.6





IL12A
GGUCUGGAGUGGCCACGGGG
570
chr3
ENST00000305579.6





IL12A
UGACGGCCCUCAGCAGGUUU
571
chr3
ENST00000305579.6





IL12B
ACUCUUUGACUUGGAUGGUC
572
chr5
ENST00000231228.2





IL12B
GGUGAUACCAUCUUCUUCAG
573
chr5
ENST00000231228.2





IL12B
UUUAUGUCGUAGAAUUGGAU
574
chr5
ENST00000231228.2





IL12RB1{circumflex over ( )}
CACUUCCUGCGGUGUUGGUG
575
chr19
ENST00000593993.6





IL12RB1{circumflex over ( )}
GGAGCACUCGUAACGAUCAC
576
chr19
ENST00000593993.6





IL12RB1{circumflex over ( )}
AUCUCUUCCAGGCUCGGCCU
577
chr19
ENST00000593993.6





IL13*
CAUGGCGCUUUUGUUGACCA
578
chr5
ENST00000304506.7





IL13*
GCCUCCCUCUACAGCCCUCA
579
chr5
ENST00000304506.7





IL13*
CGACACUCACCUUCUGGUUC
580
chr5
ENST00000304506.7





IL15
AUGGAAAUACUUCUCAAAUG
581
chr4
ENST00000296545.11





IL15
GGCAUUCAUGUCUUCAUUUU
582
chr4
ENST00000296545.11





IL15
AUGGUCAUGAUGAUUGUCCU
583
chr4
ENST00000296545.11





IL16
CAAAAUUUCGGUCCAUCUCC
584
chr15
ENST00000302987.8





IL16
AGGGCUAGAGCCAUCAUCAC
585
chr15
ENST00000302987.8





IL16
GGAAAAUUCCCUCCUUGCCC
586
chr15
ENST00000302987.8





IL17A{circumflex over ( )}
GCUACUGCUGCUGAGCCUGG
587
chr6
ENST00000340057.1





IL17A{circumflex over ( )}
UCAACCUGAACAUCCAUAAC
588
chr6
ENST00000340057.1





IL17A{circumflex over ( )}
GGAUCGGUUGUAGUAAUCUG
589
chr6
ENST00000340057.1





IL17B{circumflex over ( )}
GCUCUGAGCUGUUCCUCAGC
590
chr5
ENST00000261796.3





IL17B{circumflex over ( )}
GAAACCGUAUGCCCGCAUGG
591
chr5
ENST00000261796.3





IL17B{circumflex over ( )}
CUCUUGCUUUUGGGGCUCCU
592
chr5
ENST00000261796.3





IL17C{circumflex over ( )}
UCAGAAACAGGAGGCCGGGG
593
chr16
ENST00000244241.4





IL17C{circumflex over ( )}
CGGUACCCCACACUGCUACU
594
chr16
ENST00000244241.4





IL17C{circumflex over ( )}
GCACCUCGAGCCAGCAGGUG
595
chr16
ENST00000244241.4





IL17D{circumflex over ( )}
CGGCAGCGCCAGCAGGAAGC
596
chr13
ENST00000304920.3





IL17D{circumflex over ( )}
GGACCGGCCGGAGGAGCUAC
597
chr13
ENST00000304920.3





IL17D{circumflex over ( )}
GUGGAAGGCACUGAGCACGC
598
chr13
ENST00000304920.3





IL17F{circumflex over ( )}
GGUAGUAUGAAGCUUGACAU
599
chr6
ENST00000336123.4





IL17F{circumflex over ( )}
GAAAAAAGUAUGUCCUACUU
600
chr6
ENST00000336123.4





IL17F{circumflex over ( )}
UACUUGCUGCUGUCGAUAUU
601
chr6
ENST00000336123.4





IL18
GGAAAUAAUUUUGUUCUCAC
602
chr11
ENST00000280357.11





IL18
UUACAGCCAUACCUCUAGGC
603
chr11
ENST00000280357.11





IL18
UUAUUUCAGAUAAUGCACCC
604
chr11
ENST00000280357.11





IL18BP
GCAGGACCCACAAAGGGCUG
605
chr11
ENST00000260049.9





IL18BP
UGUGCUUCUAACUGAGGCAG
606
chr11
ENST00000260049.9





IL18BP
GCUGGACACUGCUUAGCUGC
607
chr11
ENST00000260049.9





IL19
AGUUACAGUGUGUUUCCCUU
608
chr1
ENST00000270218.10





IL19
CAGUAGACAACCACGGUCUC
609
chr1
ENST00000270218.10





IL19
CUUGGAAACUCUCUUCUAUA
610
chr1
ENST00000270218.10





IL1A
GGAAGGUUCUGAAGAAGAGA
611
chr2
ENST00000263339.3





IL1A
GGUAAGCUUGGAUGUUUUAG
612
chr2
ENST00000263339.3





IL1A
GCCAUAGCUUACAUGAUAGA
613
chr2
ENST00000263339.3





IL1B*
CAUUCUCCUGGAAGGUCUGU
614
chr2
ENST00000263341.6





IL1B*
CAUGGCCACAACAACUGACG
615
chr2
ENST00000263341.6





IL1B*
CUCUCCGCAGUGCUCCUUCC
616
chr2
ENST00000263341.6





IL1F10
AUCUGCAUACUUCCUAACAG
617
chr2
ENST00000341010.6





IL1F10
CCAGGCAGCGGCUCCCUCCC
618
chr2
ENST00000341010.6





IL1F10
GGCCUCUCACCUCCAGCUGU
619
chr2
ENST00000341010.6





IL1R1**
UCCUUUUAAAGAUAAAUGCA
620
chr2
ENST00000410023.5





IL1R1**
CUUAACCCAAAUGAACACAA
621
chr2
ENST00000410023.5





IL1R1**
UAUCUACAGAACAAGCCUCC
622
chr2
ENST00000410023.5





IL1R2**
CCAGAAGCUGCCGGUUUCGU
623
chr2
ENST00000332549.7





IL1R2**
UGUAGCCCUGAGGUGCCCCC
624
chr2
ENST00000332549.7





IL1R2**
UAAAAAUGACUCUGCUAGGA
625
chr2
ENST00000332549.7





IL1RAPL1**
AAUUCUUAAGGAGCAUCUUG
626
chrX
ENST00000378993.5





IL1RAPL1**
CUCUGUGGAAGAAGGUGACU
627
chrX
ENST00000378993.5





IL1RAPL1**
UACACUCACCUCGUUUAUGA
628
chrX
ENST00000378993.5





IL1RL1**
UUUCUUAUUUCAGAAUUGUC
629
chr2
ENST00000233954.5





IL1RL1**
AAUGUGAUGACUGAGGACGC
630
chr2
ENST00000233954.5





IL1RL1**
ACCCUUGACCGUGAAGGACC
631
chr2
ENST00000233954.5





IL1RN**
UUCCCCAGAAAAGAUAGAUG
632
chr2
ENST00000409930.3





IL1RN**
ACAGGCACAUCUUCCCUCCA
633
chr2
ENST00000409930.3





IL1RN**
AGCAUGUUUUUACCUCCAGC
634
chr2
ENST00000409930.3





IL2
UGUAAUAAUUUUAGUAAGAA
635
chr4
ENST00000226730.4





IL2
ACAACUGGAGCAUUUACUGC
636
chr4
ENST00000226730.4





IL2
GACUUAGUGCAAUGCAAGAC
637
chr4
ENST00000226730.4





IL20
AAGGCUGAAGGCAAGACUAG
638
chr1
ENST00000367096.7





IL20
UGGACUGAAGACACUCAAUU
639
chr1
ENST00000367096.7





IL20
UUCUGAGAUACGGGGCAGUG
640
chr1
ENST00000367096.7





IL20RA
CAUCCCCGACCCGCACCUGG
641
chr6
ENST00000316649.9





IL20RA
UGUUGCUCCUGGCGGCGCCU
642
chr6
ENST00000316649.9





IL20RA
CCGCCGCCCAUGCGGGCUCC
643
chr6
ENST00000316649.9





IL20RB
AGUGAGUGAGCACCAGCUGC
644
chr3
ENST00000329582.8





IL20RB
UGCCAUACAACCUUCGUGUC
645
chr3
ENST00000329582.8





IL20RB
UUCUAUUAAAGGGAUGCUUC
646
chr3
ENST00000329582.8





IL21
GAAAAAUUAUGUGAAUGACU
647
chr4
ENST00000264497.7





IL21
UCAUGUGGCGAUCUUGACCU
648
chr4
ENST00000264497.7





IL21
ACAAUCCUCUCCAUGUUGCC
649
chr4
ENST00000264497.7





IL22{circumflex over ( )}
UGGACUUGUCAAGCCUGCAG
650
chr12
ENST00000328087.5





IL22{circumflex over ( )}
UCUCUUGGCCCUCUUGGUAC
651
chr12
ENST00000328087.5





IL22{circumflex over ( )}
AAGAGCUCACAGAUUUCUGC
652
chr12
ENST00000328087.5





IL22RA1{circumflex over ( )}
GAUGUCUGGCUGCCCUAAGU
653
chr1
ENST00000270800.1





IL22RA1{circumflex over ( )}
UCUGGACCCAGGGAGCCCGC
654
chr1
ENST00000270800.1





IL22RA1{circumflex over ( )}
CAGGGAUCAGGACGUGCUCC
655
chr1
ENST00000270800.1





IL22RA2{circumflex over ( )}
UUCUGAGUAGCUCCCAGCCG
656
chr6
ENST00000349184.8





IL22RA2{circumflex over ( )}
CUGUAUGUCUGAGGUUUCAC
657
chr6
ENST00000349184.8





IL22RA2{circumflex over ( )}
ACAGAUAUGGACAGAGACAA
658
chr6
ENST00000349184.8





IL23A*
CUGCUGCCCUGGACAGCUCA
659
chr12
ENST00000228534.5





IL23A*
AAGCUGCUGGCACUGAGUCC
660
chr12
ENST00000228534.5





IL23A*
CUCACCAUGUGUCCCACUAG
661
chr12
ENST00000228534.5





IL23R**
CUGUUGCACAUAUGUAAAAU
662
chr1
ENST00000347310.9





IL23R**
GAUGUCAAGAAACAGGCAAA
663
chr1
ENST00000347310.9





IL23R**
UAAGUACACUCACCUGUUUC
664
chr1
ENST00000347310.9





IL24
UGAGAGGCUGUCGCCAGCAA
665
chr1
ENST00000294984.6





IL24
CUUCUCUGGAGCCAGGUAUC
666
chr1
ENST00000294984.6





IL24
ACAACCCCCUUCACUUGGCA
667
chr1
ENST00000294984.6





IL25
CCUACAGGUGGUUGCAUUCU
668
chr14
ENST00000329715.2





IL25
GAGGUGUCCUGCCCUUUGCU
669
chr14
ENST00000329715.2





IL25
UGCCUCCCCUAGAGCCUGCU
670
chr14
ENST00000329715.2





IL26
AUAGAGAGCGUCAACAGCUU
671
chr12
ENST00000229134.4





IL26
AGAUUGCUUGUGCUUGGCAA
672
chr12
ENST00000229134.4





IL26
UGCUGGUGAAUUUCAUUUUG
673
chr12
ENST00000229134.4





IL27
CAUUAGGGGGACUUACAAAG
674
chr16
ENST00000356897.1





IL27
GCAGCUCCUGCAGGCUCAGC
675
chr16
ENST00000356897.1





IL27
ACCAGCUUGAACCAGGAGCA
676
chr16
ENST00000356897.1





IL2RA
CAGAGCUUGUGCAUUGACAU
677
chr10
ENST00000379959.7





IL2RA
GUGACCCGCUUUUUAUUCUG
678
chr10
ENST00000379959.7





IL2RA
AUGGCUUUGAAUGUGGCGUG
679
chr10
ENST00000379959.7





IL2RB
UGUGGCCUGGGGACAGCGUC
680
chr22
ENST00000216223.9





IL2RB
CCCACAGAUGCAACAUAAGC
681
chr22
ENST00000216223.9





IL2RB
GUUGUCUCCAGUUCGCCUGA
682
chr22
ENST00000216223.9





IL2RG
CAUACCAAUAAUGCAGAGUG
683
chrX
ENST00000374202.6





IL2RG
UCGAGUACAUGAAUUGCACU
684
chrX
ENST00000374202.6





IL2RG
GAAACACUGAGGGAGUCAGU
685
chrX
ENST00000374202.6





IL3*
GCAGGAGCAGGACGGGCAGG
686
chr5
ENST00000296870.2





IL3*
CAACGCCCUUGAAGACAAGC
687
chr5
ENST00000296870.2





IL3*
CUACUCACCAGCAAAGGCAA
688
chr5
ENST00000296870.2





IL31
CUUUCAAAAGCAUCUUCGAG
689
chr12
ENST00000377035.1





IL31
GUAAACGGACGGGCAACGUG
690
chr12
ENST00000377035.1





IL31
AGAAAGAGCACAGACGUCGA
691
chr12
ENST00000377035.1





IL31RA
CAACUUCGCUAAGAACCGUA
692
chr5
ENST00000447346.6





IL31RA
AGCUCUGCGAUGUGCGGUCA
693
chr5
ENST00000447346.6





IL31RA
CAGUCAUUCCCAUUUUUUCU
694
chr5
ENST00000447346.6





IL32
CUGCCUCUCUUCACAGCACC
695
chr16
ENST00000325568.9





IL32
AGAAUCAGGACGUGGACAGG
696
chr16
ENST00000325568.9





IL32
UCAGGGUGAGAAGGAUGAAG
697
chr16
ENST00000325568.9





IL33
UUGUUUAGAAUCCCAACAGA
698
chr9
ENST00000381434.7





IL33
CUCUGGCCUUAUGAUAAAAA
699
chr9
ENST00000381434.7





IL33
UUUCAGUGAAGGCCUUUUGG
700
chr9
ENST00000381434.7





IL34
UCUUGGGAUCUUCCUUGGCG
701
chr16
ENST00000288098.6





IL34
GCACUCCUCAUUCUGCGUCA
702
chr16
ENST00000288098.6





IL34
CAGGAGCCGACUUCAGUACA
703
chr16
ENST00000288098.6





IL36A
CACAUGUCGGCAUGAGAUUA
704
chr2
ENST00000259211.6





IL36A
GAUUGAGUCCAUUCAGGCCC
705
chr2
ENST00000259211.6





IL36A
ACUCACCUUCAGCUGCAGUG
706
chr2
ENST00000259211.6





IL36B
CAAGCCUACUUUGCAGCUUA
707
chr2
ENST00000327407.2





IL36B
UAUGGUUUACCUGGGAAUCA
708
chr2
ENST00000327407.2





IL36B
GCUAUUAAAUGAAGAGUGAC
709
chr2
ENST00000327407.2





IL36G
UCAGUGUGUAAACCUAUUAC
710
chr2
ENST00000259205.4





IL36G
CACAAGGUUCUGACCCUGAA
711
chr2
ENST00000259205.4





IL36G
CUGUUUGCACUGCUGUGGCU
712
chr2
ENST00000259205.4





IL36RN
CACAGGUGAAGAGAUCAGCG
713
chr2
ENST00000346807.7





IL36RN
CACCCAGGAUGACGGGGGAC
714
chr2
ENST00000346807.7





IL36RN
GGAGCCGACUCUAACACUAG
715
chr2
ENST00000346807.7





IL37
GCUAAUGCAAAGAAGAUCUC
716
chr2
ENST00000263326.7





IL37
AUUCUCCUGGGGGUCUCUAA
717
chr2
ENST00000263326.7





IL37
AACUCUCACCUUCAGCUGAA
718
chr2
ENST00000263326.7





IL4*
CAGAGGGGGAAGCAGUUGGG
719
chr5
ENST00000231449.6





IL4*
UGAUAUCGCACUUGUGUCCG
720
chr5
ENST00000231449.6





IL4*
UACUCACCUUCUGCUCUGUG
721
chr5
ENST00000231449.6





IL5*
CAUAAAGAAAAUUACCUCAU
722
chr5
ENST00000231454.5





IL5*
UGCAUUGGUGAAAGAGACCU
723
chr5
ENST00000231454.5





IL5*
CAUUUGAGUUUGCUAGCUCU
724
chr5
ENST00000231454.5





IL6*
CCCUCCGGCACAGGCGCCUU
725
chr7
ENST00000258743.9





IL6*
ACAUCUUUGGAAUCUUCUCC
726
chr7
ENST00000258743.9





IL6*
AACGAAUUGACAAACAAAUU
727
chr7
ENST00000258743.9





IL6R**
CAGUCCGGCCGAAGACUUCC
728
chr1
ENST00000368485.7





IL6R**
UAACUGGCAGGAGAACUUCU
729
chr1
ENST00000368485.7





IL6R**
CAGCAAAACUCAAACCUUUC
730
chr1
ENST00000368485.7





IL6ST
AUUUACCUGGCUCCAAGUUG
731
chr5
ENST00000336909.9





IL6ST
UUAUAACACUCUUAAUACUU
732
chr5
ENST00000336909.9





IL6ST
UGAUAAAUUAUGUGGCGGAU
733
chr5
ENST00000336909.9





IL7
GGUCAGCAUCGAUCAAUUAU
734
chr8
ENST00000263851.8





IL7
AUGCUACUGGCAACAGAACA
735
chr8
ENST00000263851.8





IL7
UGUCAAAUUUAGUUUCUUUU
736
chr8
ENST00000263851.8





IL9{circumflex over ( )}
CUACCUGCAUCUUGUUGAUG
737
chr5
ENST00000274520.1





IL9{circumflex over ( )}
UGGACACCCCUGGCCUGCCA
738
chr5
ENST00000274520.1





IL9{circumflex over ( )}
UGUCAAGAUGCUUCUGGCCA
739
chr5
ENST00000274520.1





INHA
UCAGCAGCAAGAAGAGCAGU
740
chr2
ENST00000243786.2





INHA
CCGGGAACUUGUUCUGGCCA
741
chr2
ENST00000243786.2





INHA
CCGCGGUGACCAGGGAAGGU
742
chr2
ENST00000243786.2





INHBA
UGAACUUAUGGAGCAGACCU
743
chr7
ENST00000242208.4





INHBA
GAACGGGUAUGUGGAGAUAG
744
chr7
ENST00000242208.4





INHBA
GGGUACCGGCUGGGUGACAU
745
chr7
ENST00000242208.4





INHBB
GGCGGCGGCGCGGCAGGCGU
746
chr2
ENST00000295228.3





INHBB
GUACGUCGUGCGGCGGCUUC
747
chr2
ENST00000295228.3





INHBB
UGUGCCGCUUCACCGCCUCC
748
chr2
ENST00000295228.3





INHBC
AAAGGCCAGAAGCAAUGAGG
749
chr12
ENST00000309668.2





INHBC
UGGACACUGACCGCCAGCUC
750
chr12
ENST00000309668.2





INHBC
GCGGGAGCUGCUUCUUGAUC
751
chr12
ENST00000309668.2





INHBE
AAAAGUGAGCAGGGAGCUGU
752
chr12
ENST00000266646.2





INHBE
CAAGCAAAGAGUGCCAGGAA
753
chr12
ENST00000266646.2





INHBE
CCAGGAGAGUGCGGGACCCU
754
chr12
ENST00000266646.2





ITGA4**
CCACCGAGAGCGCAUGGCUU
755
chr2
ENST00000397033.6





ITGA4**
GACCGGCCGCCCCUACAACG
756
chr2
ENST00000397033.6





ITGA4**
AGCCGAACAGCGUGUUGUGG
757
chr2
ENST00000397033.6





ITGAV**
CGGCGAUGGCUUUUCCGCCG
758
chr2
ENST00000261023.7





ITGAV**
GUCCCGAGAGAAGAAGCGGG
759
chr2
ENST00000261023.7





ITGAV**
GACAGUCCUGCCGAGUACUC
760
chr2
ENST00000261023.7





ITGB1**
UGCUGUUCCUUUGCUACGGU
761
chr10
ENST00000302278.7





ITGB1**
GAUGACAUAGAAAAUCCCAG
762
chr10
ENST00000302278.7





ITGB1**
AUUUAGACAUUUUUACAGGA
763
chr10
ENST00000302278.7





ITGB3**
GCGAGGUGAGCCCAGAGGCA
764
chr17
ENST00000559488.5





ITGB3**
AGGCCCGAGUACUAGAGGAC
765
chr17
ENST00000559488.5





ITGB3**
GGGGACUGACUUGAGUGACC
766
chr17
ENST00000559488.5





KIT**
CUCAACCAUCUGUGAGUCCA
767
chr4
ENST00000288135.5





KIT**
AUCAGACUUAAUAGUCCGCG
768
chr4
ENST00000288135.5





KIT**
AAAGUCCAUUUGACAAAGCC
769
chr4
ENST00000288135.5





KITLG
UUUCUUUCACGCACUCCACA
770
chr12
ENST00000228280.9





KITLG
AAGUUUUCAAAUAUUUCUGA
771
chr12
ENST00000228280.9





KITLG
UCUUGCAGCCAAGUCAUUGU
772
chr12
ENST00000228280.9





KLHL20
GCACCGGGAGCUAUGUGAUG
773
chr1
ENST00000209884.4





KLHL20
UACUUCCGAGCUAUGUUUAC
774
chr1
ENST00000209884.4





KLHL20
UAGCCCUCUCGUCAAUGUCU
775
chr1
ENST00000209884.4





LEFTY1
GCUGCAGCAGGGCCACGUAC
776
chr1
ENST00000272134.5





LEFTY1
CCUCCAUGUCGGCCCUGUCC
777
chr1
ENST00000272134.5





LEFTY1
GUGUUGCCCCUGGCCAGCCC
778
chr1
ENST00000272134.5





LEFTY2
AUGUCGGCCCUGUCCAGUAC
779
chr1
ENST00000366820.9





LEFTY2
CUCCUCGGUCAGGGCCGCCC
780
chr1
ENST00000366820.9





LEFTY2
GCAGCACCAUGUGGCCCCUG
781
chr1
ENST00000366820.9





LIF
GUCCCGGGUGAUGUUGCCCA
782
chr22
ENST00000249075.3





LIF
UGCCGUUGGCGUGGAAGGGC
783
chr22
ENST00000249075.3





LIF
UGUUGGGGAACGGCUCCCCC
784
chr22
ENST00000249075.3





LIFR
UGAUGCACUUACCCUUUUUC
785
chr5
ENST00000263409.8





LIFR
UAAAUGUUGAUAACAGCCAC
786
chr5
ENST00000263409.8





LIFR
GAAACGACCAUCCUGGAUGG
787
chr5
ENST00000263409.8





LTA
CCCUAGGGGCUCCCUGGUGU
788
chr6
ENST00000418386.2





LTA
GAUGCAUCUUGGGGUGCUGA
789
chr6
ENST00000418386.2





LTA
CAGGUGGAUGUUUACCAAUG
790
chr6
ENST00000418386.2





LTB
UAUCACUGUCCUGGCUGUGC
791
chr6
ENST00000429299.2





LTB
UCCCUCCUGCUAGCUGUGGC
792
chr6
ENST00000429299.2





LTB
UUCAGUCUCAAUGGGGGCAC
793
chr6
ENST00000429299.2





LTBP1
AUGGUGGCCAGUGCAGUUCA
794
chr2
ENST00000407925.5





LTBP1
GUUUAGGCACGCUGGCACCA
795
chr2
ENST00000407925.5





LTBP1
GAGGCAAGGUAUGUGUUGAA
796
chr2
ENST00000407925.5





LTBP3
GCCCUCACCCACGCGGAAGC
797
chr11
ENST00000322147.8





LTBP3
AUGACGCUCAUCGGAGAGAA
798
chr11
ENST00000322147.8





LTBP3
CUGCAAGCGGACCUGUCUCA
799
chr11
ENST00000322147.8





LTBP4
CCCUUGAUCUGUCACAAUGG
800
chr19
ENST00000308370.11





LTBP4
GGCCGGGGGCCGGGCGCCCG
801
chr19
ENST00000308370.11





LTBP4
GUGCUCGUCGUCGCGGUGGU
802
chr19
ENST00000308370.11






MAF


CAGGAGAAUGGCAUCAGAAC


803


chr16


ENST00000326043.4







MAF


CAGGGGACUGGUGGGCAGGU


804


chr16


ENST00000326043.4







MAF


UCCAUGGCCAGGGGACUGGU


805


chr16


ENST00000326043.4







MAF


CGACCUGCCCACCAGUCCCC


806


chr16


ENST00000326043.4






MIF
UUGGUGUUUACGAUGAACAU
807
chr22
ENST00000215754.7





MIF
CAGCAGCUGGCGCAGGCCAC
808
chr22
ENST00000215754.7





MIF
GCGCGGAACCCCUCGUCCGG
809
chr22
ENST00000215754.7





MINOS1-
GGCCACGGGCAUGAUGCUUC
810
chr1
ENST00000602662.1





MINOS1-
GAACAGUGCCAGCUUGUUGA
811
chr1
ENST00000602662.1





MINOS1-
AGCCGCUGUGGCCCACGAUC
812
chr1
ENST00000602662.1





MSTN
UGACGAUUAUCACGCUACAA
813
chr2
ENST00000260950.4





MSTN
UUCCCGGAGUGGAGGAGCUU
814
chr2
ENST00000260950.4





MSTN
AAUCCUCAGUAAACUUCGUC
815
chr2
ENST00000260950.4





NAMPT
GCUGGGGACGAGCGCGCGGC
816
chr7
ENST00000222553.7





NAMPT
CUUUACCUUGUAGGAGUCGG
817
chr7
ENST00000222553.7





NAMPT
CCGGCCCGAGAUGAAUCCUG
818
chr7
ENST00000222553.7





NBL1
GGCCACGGGCAUGAUGCUUC
819
chr1
ENST00000375136.7





NBL1
GAACAGUGCCAGCUUGUUGA
820
chr1
ENST00000375136.7





NBL1
AGCCGCUGUGGCCCACGAUC
821
chr1
ENST00000375136.7





NDP
UUGAGCUACACUUGUACAAU
822
chrX
ENST00000378062.5





NDP
CAGCUCAUUCAUAAUGGACU
823
chrX
ENST00000378062.5





NDP
CAGCAGGGAGAGCAUAGAAA
824
chrX
ENST00000378062.5





NLRP7
GCCUCUUCCACCUCAGACCA
825
chr19
ENST00000328092.9





NLRP7
GAGUUUCAAAUCCCUUUUAU
826
chr19
ENST00000328092.9





NLRP7
CUGCAGAGUCCACUCUAGCU
827
chr19
ENST00000328092.9





NODAL
UGACCAGGCCUCUCUCCAAG
828
chr10
ENST00000287139.7





NODAL
GGGACAAAGUGACAGUGAAU
829
chr10
ENST00000287139.7





NODAL
CUGUGUCGGGCUUUGGCUGG
830
chr10
ENST00000287139.7





NOG
GACCACCACCAGGGCGUAGA
831
chr17
ENST00000332822.4





NOG
GGCGGAUGUGGAGAUAGUGC
832
chr17
ENST00000332822.4





NOG
UUGGGGUCAAAGAUAGGGUC
833
chr17
ENST00000332822.4





NRG1
GCUACAUCUACAUCCACCAC
834
chr8
ENST00000287842.7





NRG1
AAACUUUCUGUGUGAAUGGA
835
chr8
ENST00000287842.7





NRG1
UUACUUGCACAAGUAUCUCG
836
chr8
ENST00000287842.7





NRP1
UUGCAGAGCAGUGUCUCAGA
837
chr10
ENST00000265371.8





NRP1
GCUGUCGGUGUAAAAAACCA
838
chr10
ENST00000265371.8





NRP1
CCACAGUAACGCCCAAUGUG
839
chr10
ENST00000265371.8





NRP2
CUUUCAGACCCACCGUGCGG
840
chr2
ENST00000357785.9





NRP2
UCUGGUGGGAGGGGUAGUCC
841
chr2
ENST00000357785.9





NRP2
UUGAGGACAAUCUUCUGGUU
842
chr2
ENST00000357785.9





OSM
AGGGCCCAGGUGCUUACAUA
843
chr22
ENST00000215781.2





OSM
GAUCUGUCUGCUUCUGGAGC
844
chr22
ENST00000215781.2





OSM
GUUUCCAAGCAUGGCGAGCA
845
chr22
ENST00000215781.2





OSMR
AUUACAGCACCACUGUGAAG
846
chr5
ENST00000274276.7





OSMR
CUUUGUAAGAAUAAAGAGUU
847
chr5
ENST00000274276.7





OSMR
GAGCAACUGGAGUUCCUGGG
848
chr5
ENST00000274276.7





PARK7
UAGAUUAAGGUCACCGUUGC
849
chr1
ENST00000338639.9





PARK7
CAGGACAAAUGACCACAUCA
850
chr1
ENST00000338639.9





PARK7
CAUGGAGUUAUUCCUUCAUA
851
chr1
ENST00000338639.9





PDPN
CAACUGCAAAGUUUGCUGUC
852
chr1
ENST00000294489.10





PDPN
CCCCACCGUCGCGCUCCUCC
853
chr1
ENST00000294489.10





PDPN
CAGCUCAGAAUCUUGCUGCU
854
chr1
ENST00000294489.10





PF4
UGUGAGGGCUGGCAGCGGCG
855
chr4
ENST00000296029.3





PF4
GUGGCAGGAGCAGCAACCCC
856
chr4
ENST00000296029.3





PF4
CGCAGCAUGAGCUCCGCAGC
857
chr4
ENST00000296029.3





PF4V1
GGCGCGGGUGAGGCGGGACC
858
chr4
ENST00000226524.3





PF4V1
AGAGCAGAAACCAGGCUGGG
859
chr4
ENST00000226524.3





PF4V1
AGAGCAGAAACCAGGCUGGG
860
chr4
ENST00000226524.3





PGLYRP1
CGAUACCACCACAUAGCGUA
861
chr19
ENST00000008938.4





PGLYRP1
CAGGGCCUUCCACUCGUUCC
862
chr19
ENST00000008938.4





PGLYRP1
GCCGCUCUAUGCUGCUUGCC
863
chr19
ENST00000008938.4





PLP2
ACCCUUCCAGAUAUUAUGCC
864
chrX
ENST00000376327.5





PLP2
ACCGACAGGGAGGAGUAGCC
865
chrX
ENST00000376327.5





PLP2
GGACCCCUUCUCACACUCCA
866
chrX
ENST00000376327.5





PPBP
UUGGCGAAAGGCAAAGGUAG
867
chr4
ENST00000296028.3





PPBP
GUCAUUGCUGCUGACUGCUC
868
chr4
ENST00000296028.3





PPBP
AAGGGGUGGUAUCAAGUCUG
869
chr4
ENST00000296028.3





PXDN
ACCACAGUUCAGCUCUUCCG
870
chr2
ENST00000252804.8





PXDN
GCGUCUGGGAUAUUCACAGA
871
chr2
ENST00000252804.8





PXDN
ACUGCGACUGUGAAAUCCUG
872
chr2
ENST00000252804.8






RORC


CCGCGUUACAGCGCUGGCUC


873


chr1


ENST00000318247.6







RORC


AGUAGGCCGCGUUACAGCGC


874


chr1


ENST00000318247.6







RORC


CUGCUGACGGGUGCAGGAGU


875


chr1


ENST00000318247.6







RORC


GCAGUUCUGCUGACGGGUGC


876


chr1


ENST00000318247.6






SCG2
AGCCCUGAUCAUUUCAGGAC
877
chr2
ENST00000305409.2





SCG2
CUGGUUCUUUCUGAAGCAGC
878
chr2
ENST00000305409.2





SCG2
UGGCUGAAGCAAAGACCCAC
879
chr2
ENST00000305409.2





SCGB3A1
CCUCUAUGAGGUGGUUCACG
880
chr5
ENST0000029264L3





SCGB3A1
GGUGCCGAGGGGGUUGGCCA
881
chr5
ENST0000029264L3





SCGB3A1
UGCAGCUGCUGCUUUCUUAG
882
chr5
ENST0000029264L3





SECTM1
ACAGCCCAGCAUGGGAGUCC
883
chr17
ENST00000269389.7





SECTM1
CUGGAGCUGCCAGCCGUCCC
884
chr17
ENST00000269389.7





SECTM1
CUUGAUGUUGACAUGGGAGA
885
chr17
ENST00000269389.7





SLURP1
CCCUGACUCCAGUGCACCCA
886
chr8
ENST00000246515.1





SLURP1
CGUCACCAGCGUGGUCAUGC
887
chr8
ENST00000246515.1





SLURP1
CUUGCAGGUGUAGCACUUGA
888
chr8
ENST00000246515.1





SOSTDC1
AUCAAGCCAGAAAUGGAGGC
889
chr7
ENST00000307068.4





SOSTDC1
AGAAAUCCUUUAUUCACAUG
890
chr7
ENST00000307068.4





SOSTDC1
UUAGGAUGCAUGCAAGGGGA
891
chr7
ENST00000307068.4





SP100
UCUGAGAGUCCAUUUUCAGG
892
chr2
ENST00000340126.8





SP100
AGUGACAAAGAUGAUUCGCU
893
chr2
ENST00000340126.8





SP100
ACCCAGUCAGUCUUACCUGU
894
chr2
ENST00000340126.8






SP11


GUCCCCCACAGACCAUUACU


895


chr11


ENST00000378538.7







SP11


GGGGGUGGAAGUCCCAGUAA


896


chr11


ENST00000378538.7







SP11


CGCUGUGCACGUGGUGGGGG


897


chr11


ENST00000378538.7







SP11


ACUCGCUGUGCACGUGGUGG


898


chr11


ENST00000378538.7






SPP1
AAUGGUGAGACUCAUCAGAC
899
chr4
ENST00000237623.11





SPP1
UCGGUUGCUGGCAGGUCCGU
900
chr4
ENST00000237623.11





SPP1
UGAUGGCCGAGGUGAUAGUG
901
chr4
ENST00000237623.11






TBX21


CCCGCCCCGGAUGGGCAUCG


902


ch17


ENST00000177694.1







TBX21


GGCAUCGUGGAGCCGGGUUG


903


ch17


ENST00000177694.1







TBX21


GGGUUGCGGAGACAUGCUGA


904


ch17


ENST00000177694.1







TBX21


GUCGCUCCCCGGCAUCGGCU


905


ch17


ENST00000177694.1






TCAP
GCACUGCCCCUGCUGGUGGU
906
chr17
ENST00000309889.2





TCAP
AGUACCAGCUGCCCUACCAG
907
chr17
ENST00000309889.2





TCAP
CUCCUUGGUGGCGCCCAUCU
908
chr17
ENST00000309889.2





TGFB1*
GCCGCAGCUUGGACAGGAUC
909
chr19
ENST00000221930.5





TGFB1*
CUCCAUGUCGAUAGUCUUGC
910
chr19
ENST00000221930.5





TGFB1*
AGCAGCCGCAGCCCGGAGGG
911
chr19
ENST00000221930.5





TGFB2*
UACCUGCAGCACACUCGAUA
912
chr1
ENST00000366930.8





TGFB2*
UCGGGCUCAGGAUAGUCUUC
913
chr1
ENST00000366930.8





TGFB2*
CUUCUCCUGGAGCAAGUCCC
914
chr1
ENST00000366930.8





TGFB3*
GACAGAUCUUGAGCAAGCUC
915
chr14
ENST00000238682.7





TGFB3*
UGGUGCAAGUGGACAGAGAG
916
chr14
ENST00000238682.7





TGFB3*
GCACUUGCAAAGGGCUCUGG
917
chr14
ENST00000238682.7





TGFBR1**
UUUUCAGUAAAGUCAUCACC
918
chr9
ENST00000374994.8





TGFBR1**
CUGCAUCUCACUCAUGUUGA
919
chr9
ENST00000374994.8





TGFBR1**
CCUCUUCAUUUGGCACUCGA
920
chr9
ENST00000374994.8





TGFBR2**
UCUACUGCUACCGCGUUAAC
921
chr3
ENST00000295754.9





TGFBR2**
CGAGCACUGUGCCAUCAUCC
922
chr3
ENST00000295754.9





TGFBR2**
GUUGAUGUUGUUGGCACACG
923
chr3
ENST00000295754.9





TGFBR3**
CAGCCCAGUUGUGCCUCUGC
924
chr1
ENST00000212355.8





TGFBR3**
CAUCAAGGCCUGGACAGGAU
925
chr1
ENST00000212355.8





TGFBR3**
CCCUUGGCAGGUCCAGAGCC
926
chr1
ENST00000212355.8





THBS1
UCCUGUCUAACAGAGUCUGG
927
chr15
ENST00000260356.5





THBS1
GUCGGCGCCCAGACCCCUUG
928
chr15
ENST00000260356.5





THBS1
UCAGGUUGGCAUCCUCGAUG
929
chr15
ENST00000260356.5





THNSL2
GAGAAGUGGUCCAUCUGUCC
930
chr2
ENST00000324166.6





THNSL2
UGGCGUCACAUAUGCAUUUA
931
chr2
ENST00000324166.6





THNSL2
UCUUCUCCAGGAAGUACUGC
932
chr2
ENST00000324166.6





THPO
CCUGUCCAGAAAGCUGCCCC
933
chr3
ENST00000204615.11





THPO
CCAUCACUCCCUCCAGCAGA
934
chr3
ENST00000204615.11





THPO
UCUUUCUCAGGAGGAGACCA
935
chr3
ENST00000204615.11





TIMP1
CAGGGGCUCAAAGGGGGCCA
936
chrX
ENST00000218388.8





TIMP1
GACACAGGUGCAGGCCCUGC
937
chrX
ENST00000218388.8





TIMP1
GGGUGAGGACUCACCGAGGU
938
chrX
ENST00000218388.8





TNF*
GAUCAUGCUUUCAGUGCUCA
939
chr6
ENST00000449264.2





TNF*
CAAGAAGACAGGGGGGCCCC
940
chr6
ENST00000449264.2





TNF*
GGCGCCUGCCACGAUCAGGA
941
chr6
ENST00000449264.2





TNFRSF4**
CCGGCCCCUGCUCAGGAAGU
942
chr1
ENST00000379236.3





TNFRSF4**
UGAGCGGAAGCAGCUGUGCA
943
chr1
ENST00000379236.3





TNFRSF4**
GCAGCUGUGCACGGCCACAC
944
chr1
ENST00000379236.3





TNFRSF4**
GCGGCAGACUGUGUCCUGUG
945
chr1
ENST00000379236.3





TNFRSF11**
GUCUCCCGCAGGCAAGGCCC
946
chr18
ENST00000586569.2





TNFRSF11**
CCCCCGGCGCUGCGCGUGCA
947
chr18
ENST00000586569.2





TNFRSF11**
CCGCAACACCGAGUGCGCGC
948
chr18
ENST00000586569.2





TNFRSF11**
GCCAGCUGUCUGUGUAGUAG
949
chr8
ENST00000297350.8





TNFRSF11**
AACAACACUGUACAGCAAAG
950
chr8
ENST00000297350.8





TNFRSF11**
ACUUUGGAGGAAACGUUUCC
951
chr8
ENST00000297350.8





TNFRSF1A
AGCAAAUCGAAUUAUUUUGA
952
chr12
ENST00000162749.6





TNFRSF1A
UUCUCCCUGUCCCCUAGGUG
953
chr12
ENST00000162749.6





TNFRSF1A
CCAGGUGCUCCUGGAGCUGU
954
chr12
ENST00000162749.6





TNFRSF9**
UGUGAACAGGAUUGUAAACA
955
chr1
ENST00000377507.7





TNFRSF9**
CAGAGUGUGACUGCACUCCA
956
chr1
ENST00000377507.7





TNFRSF9**
GGGACUUUGUAGGUGUUUUC
957
chr1
ENST00000377507.7





TNFSF9***
GGGGUCCAGUGAAGCGUCAG
958
chr19
ENST00000245817.4





TNFSF9***
AUACGCCUCUGACGCUUCAC
959
chr19
ENST00000245817.4





TNFSF9***
CGGGAGGCCACGGGGCUUCG
960
chr19
ENST00000245817.4





TNFSF9***
CACUGGACCCCGAAGCCCCG
961
chr19
ENST00000245817.4





TNFSF10
CUACCUUUCUAACGAGCUGA
962
chr3
ENST00000241261.6





TNFSF10
GUUCAUACUCUCUUCGUCAU
963
chr3
ENST00000241261.6





TNFSF10
GAAACAAGCAAUGCCACUUU
964
chr3
ENST00000241261.6





TNFSF11
CCAUCUUCUGAUAUUCUAUU
965
chr13
ENST00000398795.6





TNFSF11
AGAUUUUCAAGACACAACUC
966
chr13
ENST00000398795.6





TNFSF11
UUCAUGUAGGAGAAUUAAAC
967
chr13
ENST00000398795.6





TNFSF12
CCGCCCCCAUGGCCGCCCGU
968
chr17
ENST00000293825.10





TNFSF12
CCUGCUGGUCCCGCUCGCGC
969
chr17
ENST00000293825.10





TNFSF12
CCGGGCAUCGCUGUCCGCCC
970
chr17
ENST00000293825.10





TNFSF12-
CCGCCCCCAUGGCCGCCCGU
971
chr17
ENST00000293826.4





TNFSF12-
CCUGCUGGUCCCGCUCGCGC
972
chr17
ENST00000293826.4





TNFSF12-
CCGGGCAUCGCUGUCCGCCC
973
chr17
ENST00000293826.4





TNFSF13
GGGGGGCCCAGUCAGAGAGC
974
chr17
ENST00000338784.8





TNFSF13
CAGAGCCAUGGCACAAGCCA
975
chr17
ENST00000338784.8





TNFSF13
AGAGAGGUGAGCCGGCUGCA
976
chr17
ENST00000338784.8





TNFSF13B
UCAUUUCUUCUCUUUUCUUA
977
chr13
ENST00000375887.8





TNFSF13B
CUCCAAAGACGGAAAGCUGC
978
chr13
ENST00000375887.8





TNFSF13B
CACGGUGGUGUCUUUCUACC
979
chr13
ENST00000375887.8





TNFSF14
CACACCGCCCAGCUGCACCU
980
chr19
ENST00000599359.1





TNFSF14
GGGCCCCAUCGUGGUAGCUG
981
chr19
ENST00000599359.1





TNFSF14
GCUGCCGGUCAAGCUGGAGU
982
chr19
ENST00000599359.1





TNFSF15
UCAUCCCACGGAAUGUGACC
983
chr9
ENST00000374045.4





TNFSF15
AUUUGUUGGUAUAGUUCAUU
984
chr9
ENST00000374045.4





TNFSF15
UGAUUUUUAAAGUGCUGUGU
985
chr9
ENST00000374045.4





TNFSF18
AAUAAAGAUUAGCCAACUGA
986
chr1
ENST00000404377.3





TNFSF18
GCAAAAGAGCCACAGCUUCC
987
chr1
ENST00000404377.3





TNFSF18
AAGUGGCUCAAACACAUUUU
988
chr1
ENST00000404377.3





TNFSF4
CCUCAUCCUUCUGGUAAUGA
989
chr1
ENST00000281834.3





TNFSF4
GUUUUAUCUCAUCUCCCUGA
990
chr1
ENST00000281834.3





TNFSF4
CUAGAAUAUAAGAAGGAGAA
991
chr1
ENST00000281834.3





TNFSF8
UCUGUGCCUUGUCUUCACGG
992
chr9
ENST00000223795.2





TNFSF8
GCUCGUGGUCCCCAGGUGGC
993
chr9
ENST00000223795.2





TNFSF8
UGAGUGCUUGCUGCAGCCCU
994
chr9
ENST00000223795.2





TNFSF9
AUACGCCUCUGACGCUUCAC
995
chr19
ENST00000245817.4





TNFSF9
ACGCGGCAGGCGCGAGCGCG
996
chr19
ENST00000245817.4





TNFSF9
CGUCUUCCUCGCCUGCCCCU
997
chr19
ENST00000245817.4





TRIM16
CUCCUCGGAGAAGCUUGGCA
998
chr17
ENST00000336708.11





TRIM16
GGUCACCCAGCCCAGAUUCU
999
chr17
ENST00000336708.11





TRIM16
AGCCUCCCAGAUGGCUGAGU
1000
chr17
ENST00000336708.11





TSLP
UGGAUUUCAGUAAGGCAAUG
1001
chr5
ENST00000344895.3





TSLP
AAUGUUCGCCAUGAAAACUA
1002
chr5
ENST00000344895.3





TSLP
CAAGGUACAGCAAACGUCUG
1003
chr5
ENST00000344895.3





TWSG1
GUGAGUGCCCGGAAGAGAGA
1004
chr18
ENST00000262120.9





TWSG1
UGUGAAAGUUCUUCUGCAAC
1005
chr18
ENST00000262120.9





TWSG1
ACAGACACAUUCUGGUGGUG
1006
chr18
ENST00000262120.9





TXLNA
UAACCAGGGGGGCCCCGGCG
1007
chr1
ENST00000373609.1





TXLNA
AGAGAAGUCCCGGACCUAUG
1008
chr1
ENST00000373609.1





TXLNA
UGUGUUUGGAUCCCCCUUGG
1009
chr1
ENST00000373609.1





VASN
GGCAGCUUUGCCGGCCUGCC
1010
chr16
ENST00000304735.3





VASN
ACUCGCCAACCUCAGCAACC
1011
chr16
ENST00000304735.3





VASN
AUGAGACCUUCCGUGGCCUG
1012
chr16
ENST00000304735.3





VEGFA
CAGGGGCCGGAGCCCGCGCC
1013
chr6
ENST00000425836.6





VEGFA
UUCUCGCUUCGGAGGAGCCG
1014
chr6
ENST00000425836.6





VEGFA
AAGUGCUAGCUCGGGCCGGG
1015
chr6
ENST00000425836.6





VSTM1
CGAAGCUGAAUUCCCCUUCA
1016
chr19
ENST00000338372.6





VSTM1
UGCGCAAGGUGAACGACUCU
1017
chr19
ENST00000338372.6





VSTM1
UUCAACCACCGAGCUGGGCC
1018
chr19
ENST00000338372.6





WFIKKN1
GCUCCUUCUCACAGCGGUCG
1019
chr16
ENST00000319070.2





WFIKKN1
CCGCUGCUAUAUGGACGCCG
1020
chr16
ENST00000319070.2





WFIKKN1
CCUGCAAGCACGUGCUCAGC
1021
chr16
ENST00000319070.2





WFIKKN2
GUGCAAAGACCGCUGUGAGA
1022
chr17
ENST00000311378.4





WFIKKN2
CCGCUGCUACAUGGAUGCCG
1023
chr17
ENST00000311378.4





WFIKKN2
CCUGCCGCUAUCACUUCACC
1024
chr17
ENST00000311378.4





WNT1
AGACUCCAAGAGUCUGCAAC
1025
chr12
ENST00000293549.3





WNT1
GCGUCUGAUACGCCAAAAUC
1026
chr12
ENST00000293549.3





WNT1
GGCAGUUCCGGAAUCGCCGC
1027
chr12
ENST00000293549.3





WNT2
UGUUGCAAUUCCAGCGGUGC
1028
chr7
ENST00000265441.7





WNT2
CUAAUGGCACGCAUCACAUC
1029
chr7
ENST00000265441.7





WNT2
GAGCUACAGGUGGCUCCUCC
1030
chr7
ENST00000265441.7





WNT5A
UGCAGUUCCACCUUCGAUGU
1031
chr3
ENST00000264634.8





WNT5A
CUUCUCCGAUGUACUGCAUG
1032
chr3
ENST00000264634.8





WNT5A
CCUGCCAGUUGGCUGCAGAG
1033
chr3
ENST00000264634.8





WNT7A
AGAGCGCACCGUCUUCGGGA
1034
chr3
ENST00000285018.4





WNT7A
ACUGAAACUGACACUCGUCC
1035
chr3
ENST00000285018.4





WNT7A
UGGCAGAUCGCCCGCUGUCU
1036
chr3
ENST00000285018.4





XCL1*
GGAGUGAAGUCUCAGAUAAG
1037
chr1
ENST00000367818.3





XCL1*
GGAGUGAAGUCUCAGAUAAG
1038
chr1
ENST00000367818.3





XCL1*
GGAGUGAAGUCUCAGAUAAG
1039
chr1
ENST00000367818.3





XCL2*
GAGUGUAUUUCUAUACCUCU
1040
chr1
ENST00000367819.2





XCL2*
UAGGGAGUGAAGUCUCACAU
1041
chr1
ENST00000367819.2





XCL2*
UAGGGAGUGAAGUCUCACAU
1042
chr1
ENST00000367819.2





ZFP36
CCUCCAUGGUCGGAUGGCAC
1043
chr19
ENST00000597629.1





ZFP36
UCGGAGGGGCUCAGGCUCCA
1044
chr19
ENST00000597629.1





ZFP36
GGAGGGCCGCAGCUGUGGCU
1045
chr19
ENST00000597629.1





*indicates cytokines/chemokines produced by T cells that activate or localize myeloid cells


**indicates T cell surface receptor genes that activate myeloid or CAR-T cells


***indicates T cell surface receptors that arc integrated into CAR-T cell signaling thus endogenous receptors are redundant


{circumflex over ( )}indicates cytokines that drive T cell/CAR-T cell differentiation


Bold rows indicate transcription factors that drive T cell/CAR-T cell differentiation






Example 6—Protocol—Nucleofection Using Nucleofector 4D

4×106 cells per reaction are used. EO-115-100 μl transfection volume is programmed, and the entire supplement added to the Nucleofector™ Solution P3. Cell culture plates are prepared by filling appropriate number of wells with desired volume of recommended culture media (2 ml in 6 well plate) and pre-incubating/equilibrating plates in a humidified 37° C./5% CO2 incubator. Beads are magnetically removed (twice to ensure complete removal), then cells counted and cell density determined. The required number of cells are centrifuged at 90×g for 10 minutes at room temperature, the supernatant removed completely. Cells are then resuspended in PBS (1 ml) and transferred to a microcentrifuge tube, and the required number of cells centrifuged at 90×g for 10 minutes at room temperature. The supernatant is removed completely, and the cell pellet resuspended carefully in complete room temperature 4D Nucleofector™ Solution P3, 4×106 per 100 μl). Twenty μg of gRNA (gGM-CSF) is added to each tube of 15 μg Cas9 mRNA. Then 100 μl of cells is added to each tube of Cas9/gRNA, gently mixed and everything transferred into the Nucleocuvette™. The cuvette is gently tapped to remove bubbles. Electroporation is carried out using program (Human T cell stim EO-115). After run completion, the Nucleocuvette™ is carefully removed from the vessel from the retainer using a specialized tool. Cells are resuspended with pre-warmed medium. The media is then taken up from destination well, added to cuvette, and gently pipetted up and down two to three times. This is then transferred to well. This procedure is repeated with media from same well and incubated at 37° C.


CAR Transduction (Day 2).


Genome-edited CAR-T cells may then be transduced with one or more CARs targeted to (i.e., that recognizes) one or more targets, for example with a lentivirus containing a CAR construct. Any other suitable method of transduction may be used.


Protocol—To transduce T cells with lentivirus: One μl of polybrene is added for each ml media (8 mg/ml stock). The required amount of virus is then added to give required M.O.I. Cells and virus are mixed and placed back in incubator at 37° C.


Assessment of Transduction Efficiency (Day 10).


Samples of cells may then be assessed for transduction efficiency by taking a 5×105 cells from each sample and analyzing by flow cytometry. Samples are washed with RB, and 3 μl of anti-CD34 PE antibody added (to detect the CAR as the construct contains human truncated CD34). Thereafter, 5 ul of CD3 APC is added, the cells washed, and flow cytometry performed. CAR-T cells should be CD3ε positive, CD34 positive.


Assessment of Genetic Deletion.


To assess genetic deletion, 5×105 cells are harvested from each sample and their DNA extracted. Gene editing efficiency is assessed using target sequencing of the target loci using TIDE-analysis or deep sequencing.


Assessment of tumor burden and cytokine level.


T cells are harvested (Day 11). Tumor burden may be imaged in mouse using bioluminescent imaging. 3×107 CAR-T are injected per mouse I.P.


Serum cytokine levels (Day 12) are measured, e.g., using the Luminex multiplex cytokine profiling assay to check for elevations in CRS-related cytokines. A 4-hr chromium release assay against targets cells (Raji) may be performed to assess in vitro activity (Day 11).


Example 7—Additional Examples of CAR-Bearing Immune Effector Cells

Several types of chimeric antigen receptor (CAR)-bearing immune effector cells that are deficient in a cytokine may be made using the methods above. Tables 11-13 below give Examples.















TABLE 11







Mono,


TCR
Gene




Dual, or
CAR
Antigen
Modifica-
Deleted/


Example
Cell
Tandem
Target(s)
Deletion(s)
tions
Suppressed





















1
T-Cell
Mono
CD19


GM-CSF


2
T-Cell
Mono
CD19


CCL2


3
T-Cell
Mono
CD19


MCP-2


4
T-Cell
Mono
CD19


G-CSF


5
T-Cell
Mono
CD19


M-CSF


6
T-Cell
Mono
CD19


Il-4


7
T-Cell
Mono
CD19


IFNγ


8
T-Cell
Mono
CD19

TRAC
GM-CSF


9
T-Cell
Mono
CD19

TRAC
CCL2


10
T-Cell
Mono
CD19

TRAC
MCP-2


11
T-Cell
Mono
CD19

TRAC
G-CSF


12
T-Cell
Mono
CD19

TRAC
M-CSF


13
T-Cell
Mono
CD19

TRAC
Il-4


14
T-Cell
Mono
CD19

TRAC
IFNγ


15
T-Cell
Mono
CD20


GM-CSF


16
T-Cell
Mono
CD20


CCL2


17
T-Cell
Mono
CD20


MCP-2


18
T-Cell
Mono
CD20


G-CSF


19
T-Cell
Mono
CD20


M-CSF


20
T-Cell
Mono
CD20


Il-4


21
T-Cell
Mono
CD20


IFNγ


22
T-Cell
Mono
CD20

TRAC
GM-CSF


23
T-Cell
Mono
CD20

TRAC
CCL2


24
T-Cell
Mono
CD20

TRAC
MCP-2


25
T-Cell
Mono
CD20

TRAC
G-CSF


26
T-Cell
Mono
CD20

TRAC
M-CSF


27
T-Cell
Mono
CD20

TRAC
Il-4


28
T-Cell
Mono
CD20

TRAC
IFNγ


29
T-Cell
Mono
BCMA


GM-CSF


30
T-Cell
Mono
BCMA


CCL2


31
T-Cell
Mono
BCMA


MCP-2


32
T-Cell
Mono
BCMA


G-CSF


33
T-Cell
Mono
BCMA


M-CSF


34
T-Cell
Mono
BCMA


Il-4


35
T-Cell
Mono
BCMA


IFNγ


36
T-Cell
Mono
BCMA

TRAC
GM-CSF


37
T-Cell
Mono
BCMA

TRAC
CCL2


38
T-Cell
Mono
BCMA

TRAC
MCP-2


39
T-Cell
Mono
BCMA

TRAC
G-CSF


40
T-Cell
Mono
BCMA

TRAC
M-CSF


41
T-Cell
Mono
BCMA

TRAC
Il-4


42
T-Cell
Mono
BCMA

TRAC
IFNγ


43
T-Cell
Mono
Mesothelin


GM-CSF


44
T-Cell
Mono
Mesothelin


CCL2


45
T-Cell
Mono
Mesothelin


MCP-2


46
T-Cell
Mono
Mesothelin


G-CSF


47
T-Cell
Mono
Mesothelin


M-CSF


48
T-Cell
Mono
Mesothelin


Il-4


49
T-Cell
Mono
Mesothelin


IFNγ


50
T-Cell
Mono
Mesothelin

TRAC
GM-CSF


51
T-Cell
Mono
Mesothelin

TRAC
CCL2


52
T-Cell
Mono
Mesothelin

TRAC
MCP-2


53
T-Cell
Mono
Mesothelin

TRAC
G-CSF


54
T-Cell
Mono
Mesothelin

TRAC
M-CSF


55
T-Cell
Mono
Mesothelin

TRAC
Il-4


56
T-Cell
Mono
Mesothelin

TRAC
IFNγ


57
T-Cell
Mono
CD7
CD7
TRAC
GM-CSF


58
T-Cell
Mono
CD7
CD7
TRAC
CCL2


59
T-Cell
Mono
CD7
CD7
TRAC
MCP-2


60
T-Cell
Mono
CD7
CD7
TRAC
G-CSF


61
T-Cell
Mono
CD7
CD7
TRAC
M-CSF


62
T-Cell
Mono
CD7
CD7
TRAC
Il-4


63
T-Cell
Mono
CD7
CD7
TRAC
IFNγ


64
T-Cell
Mono
CD3ε
CD3ε

GM-CSF


65
T-Cell
Mono
CD3ε
CD3ε

CCL2


66
T-Cell
Mono
CD3ε
CD3ε

MCP-2


67
T-Cell
Mono
CD3ε
CD3ε

G-CSF


68
T-Cell
Mono
CD3ε
CD3ε

M-CSF


69
T-Cell
Mono
CD3ε
CD3ε

Il-4


70
T-Cell
Mono
CD3ε
CD3ε

IFNγ


71
T-Cell
Mono
CD2
CD2
TRAC
GM-CSF


72
T-Cell
Mono
CD2
CD2
TRAC
CCL2


73
T-Cell
Mono
CD2
CD2
TRAC
MCP-2


74
T-Cell
Mono
CD2
CD2
TRAC
G-CSF


75
T-Cell
Mono
CD2
CD2
TRAC
M-CSF


76
T-Cell
Mono
CD2
CD2
TRAC
Il-4


77
T-Cell
Mono
CD2
CD2
TRAC
IFNγ


78
T-Cell
Mono
CD5

TRAC
GM-CSF


79
T-Cell
Mono
CD5

TRAC
CCL2


80
T-Cell
Mono
CD5

TRAC
MCP-2


81
T-Cell
Mono
CD5

TRAC
G-CSF


82
T-Cell
Mono
CD5

TRAC
M-CSF


83
T-Cell
Mono
CD5

TRAC
Il-4


84
T-Cell
Mono
CD5

TRAC
IFNγ


85
T-Cell
Mono
CD5
CD5
TRAC
GM-CSF


86
T-Cell
Mono
CD5
CD5
TRAC
CCL2


87
T-Cell
Mono
CD5
CD5
TRAC
MCP-2


88
T-Cell
Mono
CD5
CD5
TRAC
G-CSF


89
T-Cell
Mono
CD5
CD5
TRAC
M-CSF


90
T-Cell
Mono
CD5
CD5
TRAC
Il-4


91
T-Cell
Mono
CD5
CD5
TRAC
IFNγ


92
T-Cell
Tandem
CD7 × CD2
CD7 & CD2
TRAC
GM-CSF


93
T-Cell
Tandem
CD7 × CD2
CD7 & CD2
TRAC
CCL2


94
T-Cell
Tandem
CD7 × CD2
CD7 & CD2
TRAC
MCP-2


95
T-Cell
Tandem
CD7 × CD2
CD7 & CD2
TRAC
G-CSF


96
T-Cell
Tandem
CD7 × CD2
CD7 & CD2
TRAC
M-CSF


97
T-Cell
Tandem
CD7 × CD2
CD7 & CD2
TRAC
Il-4


98
T-Cell
Tandem
CD7 × CD2
CD7 & CD2
TRAC
IFNγ


99
T-Cell
Tandem
CD3ε × CD2
CD3 & CD2

GM-CSF


100
T-Cell
Tandem
CD3ε × CD2
CD3 & CD2

CCL2


101
T-Cell
Tandem
CD3ε × CD2
CD3 & CD2

MCP-2


102
T-Cell
Tandem
CD3ε × CD2
CD3 & CD2

G-CSF


103
T-Cell
Tandem
CD3ε × CD2
CD3 & CD2

M-CSF


104
T-Cell
Tandem
CD3ε × CD2
CD3 & CD2

Il-4


105
T-Cell
Tandem
CD3ε × CD2
CD3 & CD2

IFNγ


106
T-Cell
Tandem
CD19 × CD20

TRAC
GM-CSF


107
T-Cell
Tandem
CD19 × CD20

TRAC
CCL2


108
T-Cell
Tandem
CD19 × CD20

TRAC
MCP-2


109
T-Cell
Tandem
CD19 × CD20

TRAC
G-CSF


110
T-Cell
Tandem
CD19 × CD20

TRAC
M-CSF


111
T-Cell
Tandem
CD19 × CD20

TRAC
Il-4


112
T-Cell
Tandem
CD19 × CD20

TRAC
IFNγ


113
T-Cell
Dual
CD7 × CD2
CD7 & CD2
TRAC
GM-CSF


114
T-Cell
Dual
CD7 × CD2
CD7 & CD2
TRAC
CCL2


115
T-Cell
Dual
CD7 × CD2
CD7 & CD2
TRAC
MCP-2


116
T-Cell
Dual
CD7 × CD2
CD7 & CD2
TRAC
G-CSF


117
T-Cell
Dual
CD7 × CD2
CD7 & CD2
TRAC
M-CSF


118
T-Cell
Dual
CD7 × CD2
CD7 & CD2
TRAC
Il-4


119
T-Cell
Dual
CD7 × CD2
CD7 & CD2
TRAC
IFNγ


120
T-Cell
Dual
CD3ε × CD2
CD3 & CD2

GM-CSF


121
T-Cell
Dual
CD3ε × CD2
CD3 & CD2

CCL2


122
T-Cell
Dual
CD3ε × CD2
CD3 & CD2

MCP-2


123
T-Cell
Dual
CD3ε × CD2
CD3 & CD2

G-CSF


124
T-Cell
Dual
CD3ε × CD2
CD3 & CD2

M-CSF


125
T-Cell
Dual
CD3ε × CD2
CD3 & CD2

Il-4


126
T-Cell
Dual
CD3ε × CD2
CD3 & CD2

IFNγ


127
T-Cell
Dual
CD19 × CD20

TRAC
GM-CSF


128
T-Cell
Dual
CD19 × CD20

TRAC
CCL2


129
T-Cell
Dual
CD19 × CD20

TRAC
MCP-2


130
T-Cell
Dual
CD19 × CD20

TRAC
G-CSF


131
T-Cell
Dual
CD19 × CD20

TRAC
M-CSF


132
T-Cell
Dual
CD19 × CD20

TRAC
Il-4


133
T-Cell
Dual
CD19 × CD20

TRAC
IFNγ





















TABLE 12







Mono,


Gene




Dual, or
CAR
Antigen
Deleted/


Example
Cell
Tandem
Target(s)
Deletion(s)
Suppressed







134
NK Cell
Mono
CD19

GM-CSF


135
NK Cell
Mono
CD19

CCL2


136
NK Cell
Mono
CD19

MCP-2


137
NK Cell
Mono
CD19

G-CSF


138
NK Cell
Mono
CD19

M-CSF


139
NK Cell
Mono
CD19

Il-4


140
NK Cell
Mono
CD19

IFNγ


141
NK Cell
Mono
CD19

GM-CSF


142
NK Cell
Mono
CD19

CCL2


143
NK Cell
Mono
CD19

MCP-2


144
NK Cell
Mono
CD19

G-CSF


145
NK Cell
Mono
CD19

M-CSF


146
NK Cell
Mono
CD19

Il-4


147
NK Cell
Mono
CD19

IFNγ


148
NK Cell
Mono
CD20

GM-CSF


149
NK Cell
Mono
CD20

CCL2


150
NK Cell
Mono
CD20

MCP-2


151
NK Cell
Mono
CD20

G-CSF


152
NK Cell
Mono
CD20

M-CSF


153
NK Cell
Mono
CD20

Il-4


154
NK Cell
Mono
CD20

IFNγ


155
NK Cell
Mono
CD20

GM-CSF


156
NK Cell
Mono
CD20

CCL2


157
NK Cell
Mono
CD20

MCP-2


158
NK Cell
Mono
CD20

G-CSF


159
NK Cell
Mono
CD20

M-CSF


160
NK Cell
Mono
CD20

Il-4


161
NK Cell
Mono
CD20

IFNγ


162
NK Cell
Mono
BCMA

GM-CSF


163
NK Cell
Mono
BCMA

CCL2


164
NK Cell
Mono
BCMA

MCP-2


165
NK Cell
Mono
BCMA

G-CSF


166
NK Cell
Mono
BCMA

M-CSF


167
NK Cell
Mono
BCMA

Il-4


168
NK Cell
Mono
BCMA

IFNγ


169
NK Cell
Mono
BCMA

GM-CSF


170
NK Cell
Mono
BCMA

CCL2


171
NK Cell
Mono
BCMA

MCP-2


172
NK Cell
Mono
BCMA

G-CSF


173
NK Cell
Mono
BCMA

M-CSF


174
NK Cell
Mono
BCMA

Il-4


175
NK Cell
Mono
BCMA

IFNγ


176
NK Cell
Mono
Mesothelin

GM-CSF


177
NK Cell
Mono
Mesothelin

CCL2


178
NK Cell
Mono
Mesothelin

MCP-2


179
NK Cell
Mono
Mesothelin

G-CSF


180
NK Cell
Mono
Mesothelin

M-CSF


181
NK Cell
Mono
Mesothelin

Il-4


182
NK Cell
Mono
Mesothelin

IFNγ


183
NK Cell
Mono
Mesothelin

GM-CSF


184
NK Cell
Mono
Mesothelin

CCL2


185
NK Cell
Mono
Mesothelin

MCP-2


186
NK Cell
Mono
Mesothelin

G-CSF


187
NK Cell
Mono
Mesothelin

M-CSF


188
NK Cell
Mono
Mesothelin

Il-4


189
NK Cell
Mono
Mesothelin

IFNγ


190
NK Cell
Mono
CD7
CD7
GM-CSF


191
NK Cell
Mono
CD7
CD7
CCL2


192
NK Cell
Mono
CD7
CD7
MCP-2


193
NK Cell
Mono
CD7
CD7
G-CSF


194
NK Cell
Mono
CD7
CD7
M-CSF


195
NK Cell
Mono
CD7
CD7
Il-4


196
NK Cell
Mono
CD7
CD7
IFNγ


197
NK Cell
Mono
CD3ε
CD3ε
GM-CSF


198
NK Cell
Mono
CD3ε
CD3ε
CCL2


199
NK Cell
Mono
CD3ε
CD3ε
MCP-2


200
NK Cell
Mono
CD3ε
CD3ε
G-CSF


201
NK Cell
Mono
CD3ε
CD3ε
M-CSF


202
NK Cell
Mono
CD3ε
CD3ε
Il-4


203
NK Cell
Mono
CD3ε
CD3ε
IFNγ


204
NK Cell
Mono
CD2
CD2
GM-CSF


205
NK Cell
Mono
CD2
CD2
CCL2


206
NK Cell
Mono
CD2
CD2
MCP-2


207
NK Cell
Mono
CD2
CD2
G-CSF


208
NK Cell
Mono
CD2
CD2
M-CSF


209
NK Cell
Mono
CD2
CD2
Il-4


210
NK Cell
Mono
CD2
CD2
IFNγ


211
NK Cell
Mono
CD5

GM-CSF


212
NK Cell
Mono
CD5

CCL2


213
NK Cell
Mono
CD5

MCP-2


214
NK Cell
Mono
CD5

G-CSF


215
NK Cell
Mono
CD5

M-CSF


216
NK Cell
Mono
CD5

Il-4


217
NK Cell
Mono
CD5

IFNγ


218
NK Cell
Tandem
CD7xCD2
CD7 & CD2
GM-CSF


219
NK Cell
Tandem
CD7xCD2
CD7 & CD2
CCL2


220
NK Cell
Tandem
CD7xCD2
CD7 & CD2
MCP-2


221
NK Cell
Tandem
CD7xCD2
CD7 & CD2
G-CSF


222
NK Cell
Tandem
CD7xCD2
CD7 & CD2
M-CSF


223
NK Cell
Tandem
CD7xCD2
CD7 & CD2
Il-4


224
NK Cell
Tandem
CD7xCD2
CD7 & CD2
IFNγ


225
NK Cell
Tandem
CD3εxCD2
CD3 & CD2
GM-CSF


226
NK Cell
Tandem
CD3εxCD2
CD3 & CD2
CCL2


227
NK Cell
Tandem
CD3εxCD2
CD3 & CD2
MCP-2


228
NK Cell
Tandem
CD3εxCD2
CD3 & CD2
G-CSF


229
NK Cell
Tandem
CD3εxCD2
CD3 & CD2
M-CSF


230
NK Cell
Tandem
CD3εxCD2
CD3 & CD2
Il-4


231
NK Cell
Tandem
CD3εxCD2
CD3 & CD2
IFNγ


232
NK Cell
Tandem
CD19xCD20

GM-CSF


233
NK Cell
Tandem
CD19xCD20

CCL2


234
NK Cell
Tandem
CD19xCD20

MCP-2


235
NK Cell
Tandem
CD19xCD20

G-CSF


236
NK Cell
Tandem
CD19xCD20

M-CSF


237
NK Cell
Tandem
CD19xCD20

Il-4


238
NK Cell
Tandem
CD19xCD20

IFNγ


239
NK Cell
Dual
CD7xCD2
CD7 & CD2
GM-CSF


240
NK Cell
Dual
CD7xCD2
CD7 & CD2
CCL2


241
NK Cell
Dual
CD7xCD2
CD7 & CD2
MCP-2


242
NK Cell
Dual
CD7xCD2
CD7 & CD2
G-CSF


243
NK Cell
Dual
CD7xCD2
CD7 & CD2
M-CSF


244
NK Cell
Dual
CD7xCD2
CD7 & CD2
Il-4


245
NK Cell
Dual
CD7xCD2
CD7 & CD2
IFNγ


246
NK Cell
Dual
CD3εxCD2
CD3 & CD2
GM-CSF


247
NK Cell
Dual
CD3εxCD2
CD3 & CD2
CCL2


248
NK Cell
Dual
CD3εxCD2
CD3 & CD2
MCP-2


249
NK Cell
Dual
CD3εxCD2
CD3 & CD2
G-CSF


250
NK Cell
Dual
CD3εxCD2
CD3 & CD2
M-CSF


251
NK Cell
Dual
CD3εxCD2
CD3 & CD2
Il-4


252
NK Cell
Dual
CD3εxCD2
CD3 & CD2
IFNγ


253
NK Cell
Dual
CD19xCD20

GM-CSF


254
NK Cell
Dual
CD19xCD20

CCL2


255
NK Cell
Dual
CD19xCD20

MCP-2


256
NK Cell
Dual
CD19xCD20

G-CSF


257
NK Cell
Dual
CD19xCD20

M-CSF


258
NK Cell
Dual
CD19xCD20

Il-4


259
NK Cell
Dual
CD19xCD20

IFNγ





















TABLE 13







Mono,


Gene




Dual, or
CAR
Antigen
Deleted/


Example
Cell
Tandem
Target(s)
Deletion(s)
Suppressed







260
iNKT
Mono
CD19

GM-CSF


261
iNKT
Mono
CD19

CCL2


262
iNKT
Mono
CD19

MCP-2


263
iNKT
Mono
CD19

G-CSF


264
iNKT
Mono
CD19

M-CSF


265
iNKT
Mono
CD19

Il-4


266
iNKT
Mono
CD19

IFNγ


267
iNKT
Mono
CD19

GM-CSF


268
iNKT
Mono
CD19

CCL2


269
iNKT
Mono
CD19

MCP-2


270
iNKT
Mono
CD19

G-CSF


271
iNKT
Mono
CD19

M-CSF


272
iNKT
Mono
CD19

Il-4


273
iNKT
Mono
CD19

IFNγ


274
iNKT
Mono
CD20

GM-CSF


275
iNKT
Mono
CD20

CCL2


276
iNKT
Mono
CD20

MCP-2


277
iNKT
Mono
CD20

G-CSF


278
iNKT
Mono
CD20

M-CSF


279
iNKT
Mono
CD20

Il-4


280
iNKT
Mono
CD20

IFNγ


281
iNKT
Mono
CD20

GM-CSF


282
iNKT
Mono
CD20

CCL2


283
iNKT
Mono
CD20

MCP-2


284
iNKT
Mono
CD20

G-CSF


285
iNKT
Mono
CD20

M-CSF


286
iNKT
Mono
CD20

Il-4


287
iNKT
Mono
CD20

IFNγ


288
iNKT
Mono
BCMA

GM-CSF


289
iNKT
Mono
BCMA

CCL2


290
iNKT
Mono
BCMA

MCP-2


291
iNKT
Mono
BCMA

G-CSF


292
iNKT
Mono
BCMA

M-CSF


293
iNKT
Mono
BCMA

Il-4


294
iNKT
Mono
BCMA

IFNγ


295
iNKT
Mono
BCMA

GM-CSF


296
iNKT
Mono
BCMA

CCL2


297
iNKT
Mono
BCMA

MCP-2


298
iNKT
Mono
BCMA

G-CSF


299
iNKT
Mono
BCMA

M-CSF


300
iNKT
Mono
BCMA

Il-4


301
iNKT
Mono
BCMA

IFNγ


302
iNKT
Mono
Mesothelin

GM-CSF


303
iNKT
Mono
Mesothelin

CCL2


304
iNKT
Mono
Mesothelin

MCP-2


305
iNKT
Mono
Mesothelin

G-CSF


306
iNKT
Mono
Mesothelin

M-CSF


307
iNKT
Mono
Mesothelin

Il-4


308
iNKT
Mono
Mesothelin

IFNγ


309
iNKT
Mono
Mesothelin

GM-CSF


310
iNKT
Mono
Mesothelin

CCL2


311
iNKT
Mono
Mesothelin

MCP-2


312
iNKT
Mono
Mesothelin

G-CSF


313
iNKT
Mono
Mesothelin

M-CSF


314
iNKT
Mono
Mesothelin

Il-4


315
iNKT
Mono
Mesothelin

IFNγ


316
iNKT
Mono
CD7
CD7
GM-CSF


317
iNKT
Mono
CD7
CD7
CCL2


318
iNKT
Mono
CD7
CD7
MCP-2


319
iNKT
Mono
CD7
CD7
G-CSF


320
iNKT
Mono
CD7
CD7
M-CSF


321
iNKT
Mono
CD7
CD7
Il-4


322
iNKT
Mono
CD7
CD7
IFNγ


323
iNKT
Mono
CD3ε
CD3ε
GM-CSF


324
iNKT
Mono
CD3ε
CD3ε
CCL2


325
iNKT
Mono
CD3ε
CD3ε
MCP-2


326
iNKT
Mono
CD3ε
CD3ε
G-CSF


327
iNKT
Mono
CD3ε
CDε
M-CSF


328
iNKT
Mono
CD3ε
CD3ε
Il-4


329
iNKT
Mono
CD3ε
CD3ε
IFNγ


330
iNKT
Mono
CD2
CD2
GM-CSF


331
iNKT
Mono
CD2
CD2
CCL2


332
iNKT
Mono
CD2
CD2
MCP-2


333
iNKT
Mono
CD2
CD2
G-CSF


334
iNKT
Mono
CD2
CD2
M-CSF


335
iNKT
Mono
CD2
CD2
Il-4


336
iNKT
Mono
CD2
CD2
IFNγ


337
iNKT
Mono
CD5

GM-CSF


338
iNKT
Mono
CD5

CCL2


339
iNKT
Mono
CD5

MCP-2


340
iNKT
Mono
CD5

G-CSF


341
iNKT
Mono
CD5

M-CSF


342
iNKT
Mono
CD5

Il-4


343
iNKT
Mono
CD5

IFNγ


344
iNKT
Mono
CD5
CD5
GM-CSF


345
iNKT
Mono
CD5
CD5
CCL2


346
iNKT
Mono
CD5
CD5
MCP-2


347
iNKT
Mono
CD5
CD5
G-CSF


348
iNKT
Mono
CD5
CD5
M-CSF


349
iNKT
Mono
CD5
CD5
Il-4


350
iNKT
Mono
CD5
CD5
IFNγ


351
iNKT
Tandem
CD7xCD2
CD7 & CD2
GM-CSF


352
iNKT
Tandem
CD7xCD2
CD7 & CD2
CCL2


353
iNKT
Tandem
CD7xCD2
CD7 & CD2
MCP-2


354
iNKT
Tandem
CD7xCD2
CD7 & CD2
G-CSF


355
iNKT
Tandem
CD7xCD2
CD7 & CD2
M-CSF


356
iNKT
Tandem
CD7xCD2
CD7 & CD2
Il-4


357
iNKT
Tandem
CD7xCD2
CD7 & CD2
IFNγ


358
iNKT
Tandem
CD3εxCD2
CD3 & CD2
GM-CSF


359
iNKT
Tandem
CD3εxCD2
CD3 & CD2
CCL2


360
iNKT
Tandem
CD3εxCD2
CD3 & CD2
MCP-2


361
iNKT
Tandem
CD3εxCD2
CD3 & CD2
G-CSF


362
iNKT
Tandem
CD3εxCD2
CD3 & CD2
M-CSF


363
iNKT
Tandem
CD3εxCD2
CD3 & CD2
Il-4


364
iNKT
Tandem
CD3εxCD2
CD3 & CD2
IFNγ


365
iNKT
Tandem
CD19xCD20

GM-CSF


366
iNKT
Tandem
CD19xCD20

CCL2


367
iNKT
Tandem
CD19xCD20

MCP-2


368
iNKT
Tandem
CD19xCD20

G-CSF


369
iNKT
Tandem
CD19xCD20

M-CSF


370
iNKT
Tandem
CD19xCD20

Il-4


371
iNKT
Tandem
CD19xCD20

IFNγ


372
iNKT
Dual
CD7xCD2
CD7 & CD2
GM-CSF


373
iNKT
Dual
CD7xCD2
CD7 & CD2
CCL2


374
iNKT
Dual
CD7xCD2
CD7 & CD2
MCP-2


375
iNKT
Dual
CD7xCD2
CD7 & CD2
G-CSF


376
iNKT
Dual
CD7xCD2
CD7 & CD2
M-CSF


377
iNKT
Dual
CD7xCD2
CD7 & CD2
Il-4


378
iNKT
Dual
CD7xCD2
CD7 & CD2
IFNγ


379
iNKT
Dual
CD3εxCD2
CD3 & CD2
GM-CSF


380
iNKT
Dual
CD3εxCD2
CD3 & CD2
CCL2


381
iNKT
Dual
CD3εxCD2
CD3 & CD2
MCP-2


382
iNKT
Dual
CD3εxCD2
CD3 & CD2
G-CSF


383
iNKT
Dual
CD3εxCD2
CD3 & CD2
M-CSF


384
iNKT
Dual
CD3εxCD2
CD3 & CD2
Il-4


385
iNKT
Dual
CD3εxCD2
CD3 & CD2
IFNγ


386
iNKT
Dual
CD19xCD20

GM-CSF


387
iNKT
Dual
CD19xCD20

CCL2


388
iNKT
Dual
CD19xCD20

MCP-2


389
iNKT
Dual
CD19xCD20

G-CSF


390
iNKT
Dual
CD19xCD20

M-CSF


391
iNKT
Dual
CD19xCD20

Il-4


392
iNKT
Dual
CD19xCD20

IFNγ









Example 8—Biological Assays

The following assays, or variations thereon, may be used to assess efficacy of the cytokine-deficient chimeric antigen receptor (CAR)-bearing immune effector cells disclosed herein.


T-ALL. Testing efficacy of cells in a xenogeneic model of T-ALL: 1×105 Click Beetle Red luciferase (CBR) labeled CCRF-CEM T-ALL (99% CD7+ by FACS) cells will be injected I.V. into NSG recipients prior to infusion of 2×106 to 1×107 CAR7-bearing immune effector cells or non-targeting CAR19-bearing immune effector cells control cells i.v. on day +4. In contrast to mice receiving CART19-bearing immune effector cells or mice injected with tumor only, mice receiving CAR7-bearing immune effector cells will demonstrate significantly prolonged survival and reduced tumor burden as determined by bioluminescent imaging.


Multiple Myeloma. Testing efficacy of iNKT-CAR-CS1 in a xenogeneic model of multiple myeloma: 5×105 Click Beetle Red luciferase (CBR) labeled MM.1S (99% CS1+ by FACS) cells will be injected I.V. into NSG recipients prior to infusion of 2×106 to 1×107 iNKT-CAR-CS1 or non-targeting iNKT-CAR19 control cells i.v. on day +4, or +14 or +28. In contrast to mice receiving iNKT-CAR19 or mice injected with tumor only, mice receiving iNKT-CAR-CS1 will demonstrate significantly prolonged survival and reduced tumor burden as determined by bioluminescent imaging.


In Vivo Models of Cytokine Release Syndrome and Neurotoxicity.


An in vivo mouse model of CRS is disclosed in Giavridis et al., “CAR T cell-induced cytokine release syndrome is mediated by macrophages and abated by IL-1 blockade,” Nat Med 2018 May 28. For example, in order to initiate CRS as a model system, tumor cells are injected intraperitoneally into immune deficient mice and allowed to develop into a tumor. The mice are then given CAR-T cells targeting the cancerous cells and monitored for several days to induce the onset of CRS, after which mice are sacrificed and cells and tissue obtained for analysis. Mice may also be treated for CRS and monitored for success or failure of treatment (i.e., administering antibodies against cytokines produces as a result of administration of CAR-T cells).


Analyses appropriate for monitoring CRS in such a model may include monitoring of weight change in the mice after administration of CAR-T cells, percent survival of mice, serum levels of inflammatory factors, i.e., murine SAA3 (equivalent to human C-reactive protein), cytokine levels before and after administration of CAR-T cells, species of origin of pro-inflammatory cytokines (i.e., human versus murine cytokines; and/or percent survival of mice treated with CAR-T cells that received antibodies against specific cytokines.


Mice that eventually die from CRS symptoms or complications may be classified as having severe CRS, while mice that survive but suffer greater than 10% weight loss may be classified as having non-severe CRS. Using this model, it was discovered that monocytes and macrophages are the main source for IL-6 in CRS.


Another model useful for assessing both CRS and CAR-T associated neuropathy is disclosed in Norelli, “Monocyte-derived IL-1 and IL-6 are differentially required for cytokine-release syndrome and neurotoxicity due to CAR T cells,” Nat Med 2018 May 28. In this model, human stem cells, such as hematopoietic stem and progenitor cells (HSPCs), are injected into humanized NSG mice, which are immunocompromised transgenic mice expressing human stem cell factor, GM-CSF, and IL-3 to support and enhance hematopoiesis from the injected human stem cells. The mice are given tumor cells to serve as targets for subsequently administered CAR-T cells, and then monitored for CRS symptoms. Following injection of CAR-T cells, these mice exhibit typical symptoms of CRS, including high fever and elevated levels of certain cytokines implicated in CRS, such as IL-1 and/or IL-6. Control of CRS in these mice may be accomplished by blocking the receptor for the cytokine using, for example, antibodies, depletion of cell types expressing the cytokine, or administration of an antagonist of the cytokine. As above, it was determined that monocytes are the main source of the pro-CRS cytokine IL-6, and depletion of monocytes eliminated CRS and protected the mice from death from CRS.


Other CRS animal models are known in the art and may also be used as deemed appropriate.


Methods for Inducing and Testing Cytokine Release Syndrome for Control Purposes


In one model, IL-7 may be directly injected into a mouse or other animal model to induce or initiate CRS for control purposes, as described herein. In yet another example, recombinant or transgenic IL-7 may be expressed in a cell to result in increased IL-7 signaling.


In a still further example, a constitutively signaling cytokine receptor, such as an IL-7 receptor, may be engineered into an immune effector cell such that the immune effector cell itself triggers IL-7 signaling, but is unresponsive to extracellular IL-7, and initiation of IL-7 signaling of surrounding lymphocytes is avoided. Additionally, co-expressing a constitutively signaling IL-7 receptor with a CAR recognizing a specific disease or tumor antigen results in increased T-cell proliferation, survival, and antitumor activity. A constitutively expressing IL-7 receptor is able to transmit IL-7 signaling without the need for IL-7 ligand or the common gamma γc chain, a component of the native IL-7 receptor, along with IL-7Rα. This can be accomplished by engineering cysteine and/or proline into the transmembrane domain of the IL-7Rα chain, which results in homodimerization of IL-7Rα and subsequent phosphorylation of JAK1/JAK1, which then activates downstream signaling of IL-7.


An example of a method for making and testing a genome-edited CAR-T cell deficient at inducing CRS by insertion of selectable marker into gene edited locus is provided below.


Example 9—Insertion of CAR into T Cell Receptor Gene

A CAR or any protein of interest may be inserted into the gene for the T cell receptor. MacLeod et al. (“Integration of a CD19 CAR into the TCR Alpha Chain Locus Streamlines Production of Allogeneic Gene-Edited CAR T Cells,” Molec Therapy 25(4):P949-961, 2017) reports the generation of allogeneic CAR T cells by targeting the insertion of a CAR transgene directly into the native TCR locus using an engineered homing endonuclease and an AAV donor template. Anti-CD19 CAR T cells produced in this manner do not express the endogenous cell-surface TCR, exhibit potent effector functions in vitro, and mediate clearance of CD19+ tumors in an in vivo mouse model. The resulting gene-edited CAR T cells exhibit potent anti-tumor activity in vitro and in vivo in preclinical models, suggesting that these cells have potential for safe and efficacious use as adoptive cellular therapy in unrelated patients with CD19+ hematological malignancies.


Example 10—Anti-Cytokine/chemokineAntibodies to Reduce CRS

Introduction of immune effector cells to a patient or subject for treatment of cancer often results in CRS due to the production and secretion of cytokines and/or chemokines by the immune effector cells. One way to prevent CRS is to administer antibodies recognizing a specific CRS-inducing cytokine or chemokine to the patient or subject such that the amount of circulating cytokine/chemokine is reduced. For example, as reported by Sachdeva et al., (J Biol Chem 294(14):5430-5437, 2019), administration of anti-GMCSF antibodies reduced the GMCSF secretion thereby effectively reducing CRS. Therefore, administering antibodies recognizing a specific cytokine and/or chemokine can prevent or reduce the occurrence of CRS in a patient or subject.


Example 11—Insertion of CAR into GMCSF Gene

One exemplary way to prevent CRS is to insert a CAR into a gene for a cytokine or chemokine that is involved in initiation or prolonging of CRS. Disruption or ablation of a cytokine or chemokine gene in a CAR-T or other immune effector cell prevents the initiation of CRS by that particular cytokine or chemokine. One example of such a gene is GMCSF, although other genes involved in initiating or prolonging CRS may also be disrupted in this or a similar manner as described herein. Disruption of the GMCSF gene is described at least in Sachdeva et al. (J Biol Chem 294(14):5430-5437, 2019) and a description of making and testing a CAR-T cell inserted into the GM-CSF gene is provided below. Briefly, a TALEN or other gene-editing enzyme may be designed or engineered to target a cytokine or chemokine gene of choice in a CAR-T cell, and the expression of the gene monitored by methods known in the art. Significant reduction in the expression and secretion of the cytokine or chemokine gene by the CAR-T cell was reported, which implies that the occurrence of CRS would be reduced. Additionally, this approach was found to simultaneously maintain both the proliferation capability and the anti-tumor activity of the CAR-T cell, as assessed by transwell assays and serial killing assays.


In addition to the use of gene editing to delete a gene such as GM-CSF, antibodies to a cytokine or chemokine causing or contributing to CRS may also be used. For example, an antibody recognizing the GM-CSF protein may be administered to a patient or subject receiving CAR-T therapy. This practice is known in the art, particularly for reduction of CRS as a result of IL-6 signaling using the IL-6R antagonist tocilizumab, however other antibodies or antagonists recognizing cytokines or chemokines, or antagonists of their receptors, may be used as deemed appropriate by a clinician. Appropriate antibodies will bind to the circulating cytokine or chemokine, and antagonists will bind to the native receptors for the cytokine or chemokine, thus preventing downstream CRS-promoting activities in the subject. In some instances, both of these approaches may be employed in a subject, i.e., insertion of a CAR into the gene for a cytokine or chemokine to inactivate the gene, and also administering antibodies against the same cytokine or chemokine to the subject. This approach has been shown to be effective while conferring no negative effects on the activity of the CAR-T cells in vivo or in vitro (Sterner et al., “GM-CSF inhibition reduces cytokine release syndrome and neuroinflammation but enhances CAR-T cell function in xenografts,” Blood 133:697-709, 2019).


Example 12—Method of Making and Testing Genome-Edited CAR-T Cells Deficient at Inducing CRS by Insertion of Selectable Marker into Gene Edited Loci

Inject tumor in SCID-Beige mice (3e6 Raji containing Luciferase) if performing in vivo CRS experiment. This should be completed 3 weeks prior to infusion of CAR-T into mice.


The following steps may be taken to provide a genome-edited, CRS-resistant, CAR-T cell in which the CAR is expressed from the gene edited loci with a selectable marker disclosed herein. It is not possible to select and purify edited cells based on the deletion of genes that encode internal or secreted proteins, thus a selectable marker is required to enrich for this gene modification. This example describes the making of a CD19CARTΔGMCSFΔCD3ε cell, in which the deletion of GM-CSF mitigates risk of CRS and deletion of CD3ε prevents TCR signaling and graft vs host disease (GvHD). As those of skill in the art will recognize, certain of the steps may be conducted sequentially or out of the order listed below, though perhaps leading to different efficiency.


Step 1: T Cell Activation (Day 0)


Purify T cells from leukapheresis chamber using a Miltenyi human PanT isolation kit. Resuspend in media. Count cells. Determine number of human T cell activation CD3/CD28 beads required to obtain 3:1 bead:cell ratio. Wash beads 2× with T cell media. Dilute cells at 1.256 cells/mL in hXcyte media. Add human T cell activation CD3/CD28 beads. Aliquot 4 mL/well of 1.256 cell/mL solution into 6 well plate. Incubate cells at 37° C.


Step 2: CRISPR (Day 2)


The target gene is genetically deleted and the CAR inserted into the gene edited loci. The DNA double-stranded break can be repaired using homology directed repair using a donor template to repair the break and insert the desired sequence into the edited loci. Target deletion may be accomplished by electroporating with Cas9 mRNA and gRNA against the target(s). The donor template may be a DNA plasmid, or a double-stranded linear DNA, or a single stranded-stranded linear DNA containing homology to the DNA surrounding the double-stranded breaks electroplated with the Cas9/gRNA. In this example the homology arms align to either side of the double-stranded break induced by gRNA GM-CSF. Additionally, a viral vector such as AAV may be used as the source of the donor template. Other techniques, however, could be used to induce DNA double strand breaks. These include other genome editing techniques such as TALENs and mega-nucleases.


The sequences of the gRNAs for GM-CSF and CD3ε are as follows:









hGMCSF gRNA:


(SEQ ID NO: 50)


5′_2′OMe(U(ps)A(ps)C(ps))UCAGGUUCAGGAGACGCGUUUUA





GAGCUAGAAAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUGA





AAAAGUGGCACCGAGUCGGUGC2′OMe(U(ps)U(ps)U(ps)U_3′





CD3ϵ gRNA:


(SEQ ID NO: 47)


5′_2′OMe(A(ps)G(ps)G(ps))GCAUGUCAAUAUUACUGGUUUUA





GAGCUAGAAAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUGA





AAAAGUGGCACCGAGUCGGUGC2′OMe(U(ps)U(ps)U(ps)U 3′
























Nucleofection


Sample ID
gRNA#1
gRNA#2
Cas9
Buffer P3







UCART19
20 μg
20 μg gCD3ε
15 μg Cas9
100 μl



gGM-CSF

mRNA









Nucleofection Using Nucleofector 4D:

Perform nucleofection protocol as described above (Example 6), using 20 μg of each gRNA (gGM-CSF and gCD3ε) to each tube of 15 μg cas9 mRNA.


Step 3: Transduction of T Cells with AAV Vector Containing HDR Repair Construct to Introduce CAR and a Selectable Marker into GM-CSF Loci


Sequences for components of vectors described herein are provided in the sequence listing, such as including, but not limited to, SEQ ID NOs: 3048-3064. For example, recombinant AAV6 donor vector (FIG. 3, containing ITR-Right homology arm (SEQ ID NO:51), EF1a promotor (SEQ ID NO:52), CAR19 p2a trCD34 (SEQ ID NO:53), Left homology arm (SEQ ID NO:54), ITR (SEQ ID NO:55), is added to the cell culture 2-4 hrs after electroporation with a MOI between 1×104 and 1×106.


Step 4: Assessment of CRISPR Activity and Td Efficiency (Day 10)


Take 5×105 cells from each sample and analyze by flow cytometry. Wash samples with RB. Add 3 μl of anti-CD34 PE antibody (This detects the insertion TrCD34 tag into GM-CSF loci). Add 5 μl of CD3 APC and 2 μl of anti-FAB BV421 (detects CAR transduction). Wash. Perform Flow cytometry. Cells should be CD3ε-negative, CD34-positive and FAB+. Harvest T cells (Day 11).


Purification of CAR-T cells. TCR-negative cells can be purified using TCRa/b negative selection to remove TCR positive cells. GM-CSF deleted cells can be enriched using a CD34-positive selection on the Miltenyi Automacs. This enriches the GM-SCF− cells and removes TCR+ cells.


Step 5: Assessment of CAR-T Activity In Vivo


Inject 3×107 CAR-T per mouse I.P. Assess serum cytokine levels. (Day 12, Day 13, Day 14) Measure serum cytokine levels using Luminex multiplex cytokine profiling assay to check for elevations in CRS related cytokines. Perform a 4 hr chromium release assay against targets cells (Raji) to assess in vitro activity (Day 11).


Example 13—Confirmation of GFP Expression with EGFP Integration into CD3e Locus of Jurkats Under Control of CD3e Promotor

Direct insertion of GFP into the CD3e locus of a Jurkat cell line may be performed using the method details below.


Days prior: Digest plasmid with PST1 (FIG. 4). Two fragments (3.5 kb and 2.6 kb) are generated upon digestion. Purify using gel extraction kit and ethanol precipitate. Resuspend at 500 ng/μl. This generates donor template ‘DNA’ in table below


Make and titre AAV. The sequence of the AAV is shown in FIG. 5 and provided in SEQ ID NOs:______. This generates donor template ‘AAV’ in the table below.


Donor template ‘plasmid’ is shown in the table below.


Day 0:


Harvest jurkats and count. Spin cells down @100 g for 10 mins. Transfer cells to 1.5 m microcentrifuge tube and wash in PBS. Spin cells down @100×g for 10 mins. Resuspend in pre-warmed buffer SE. Add 100 μl to tube containing gRNA/Cas9. Transfer to nucleocuvette and zap using the Jurkat program on 4D. Transfer to pre-warmed media in 2 ml respective media in a 6-well plate. Return to incubator and expand for 4 days.

















TABLE 14








Nucleofector

Cell
Cas9

Donor



Name
Solution
Cell line
number
mRNA
gRNA
template























1
WT

Jurkat
4 × 106

20 μg CD3e



2

SE
Jurkat
4 × 106
15 μg
20 μg CD3e



3
P-1ug
SE
Jurkat
4 × 106
15 μg
20 μg CD3e
Plasmid 1ug


4
P-2ug
SE
Jurkat
4 × 106
15 μg
20 μg CD3e
Plasmid 2ug


5
P-2ug
SE
Jurkat
4 × 106
15 μg
20 μg CD3e
Plasmid 3ug


6
D-1ug
SE
Jurkat
4 × 106
15 μg
20 μg CD3e
DNA 1ug


7
D-2ug
SE
Jurkat
4 × 106
15 μg
20 μg CD3e
DNA 2ug


8
D-4ug
SE
Jurkat
4 × 106
15 μg
20 μg CD3e
DNA 3ug


9
AAV Td
SE
Jurkat
4 × 106
15 μg
20 μg CD3e
AAV 1e4


10
AAV Td
SE
Jurkat
4 × 106
15 μg
20 μg CD3e
AAV 1e5


11
AAV Td
SE
Jurkat
4 × 106
15 μg
20 μg CD3e
AAV 1e6









Day 4: FACS cells for GFP and CD3 APC. Assess editing efficiency by loss of CD3 and integration of the AAV-GFP donor by GFP fluorescence.


Example 14—Method of Making and Testing a Genome-Edited CAR-T Cells by Insertion of CAR into CD3e Loci

The following steps may be taken to provide a genome-edited CAR-T cell in which the car is expressed from the gene edited loci (CAR-T) disclosed herein. This example describes the making of a CD7CART ΔCD7 ΔCD3ε cell. As those of skill in the art will recognize, certain of the steps may be conducted sequentially or out of the order listed below, though perhaps leading to different efficiency.


Step 1: T Cell Activation (Day 0)


Purify T cells from leukapheresis chamber using Miltenyi human PanT isolation kit. Resuspend in media. Count cells. Determine number of human T cell activation CD3/CD28 beads required to obtain 3:1 bead:cell ratio. Wash beads 2× with T cell media. Dilute cells at 1.256 cells/mL in hXcyte media. Add human T cell activation CD3/CD28 beads. Aliquot 4 mL/well of 1.256 cell/mL solution into 6 well plate. Incubate cells at 37° C.


Step 2: CRISPR (Day 2)


The target gene is genetically deleted and the CAR inserted into the gene edited loci. The DNA double-stranded break can be repaired using homology-directed repair using a donor template to repair the break and insert the desired sequence into the edited loci. Target deletion may be accomplished by electroporating with Cas9 mRNA and gRNA against the target(s). The donor template may be a DNA plasmid, or double-stranded linear DNA, or single-stranded linear DNA containing homology to the DNA surrounding the double-stranded breaks electroporated with the Cas9/gRNA. Additionally, a viral vector such as AAV may be used as the source of the donor template. Other techniques, however, could be used to induce DNA double-stranded breaks. These include other genome editing techniques, such as TALENs and mega-nucleases.




















Nucleofection


Sample ID
gRNA#1
gRNA#2
Cas9
Buffer P3







UCART7
20 μg gCD7
20 μg gCD3ε
15 μg Cas9
100 μl





mRNA









Nucleofection Using Nucleofector 4D:

Perform nucleofection protocol as described above (Example 6), using 20 μg of each gRNA (gCD7 and gCD38) to each tube of 15 μg Cas9.


Step 3: Transduction of T Cells with AAV Vector Containing HDR Repair Construct


Recombinant AAV6 (or other serotypes of AAV) donor vector is added to the cell culture 2-4 hrs after electroporation with an MOI between 1×104 and 1×106.


Step 4: Assessment of CRISPR Activity and Td Efficiency (Day 10)


Take 5×105 cells from each sample and analyze by flow cytometry. Wash samples with RB. Add 3 μl of anti-CD34 PE antibody (This detects the CAR, as the construct contains human truncated CD34). Add 5 μl of CD3 APC and 2 μl of CD7 BV421. Wash. Perform Flow cytometry. Cells should be CD3ε-negative, CD7-negative and CD34-positive. Harvest T cells (Day 11).


Purification of CAR-T cells. CD34+ (CAR+) and TCR-negative cells can be purified in a single step using a positive selection of CD34+ cells on a Miltenyi Automacs. This enriches the CAR+ cells and removes and TCR+ cells (as CAR insertion disrupts TCR signaling).


Step 5: Assessment of CAR-T Activity In Vivo


Inject tumor in NSG mice (5×105 MOLT3 or HH: containing Luciferase) if performing in vivo imaging experiment (Day 7).


Image tumor burden in mouse using bioluminescent imaging. Inject 2×106 CD34+CAR-T per mouse I.V. via tail vein or perform a 4 hr chromium release assay against targets cell (MOLT3 or HH) (Day 11). Those of skill in the art will appreciate that some flexibility is possible in the specified time frames.


Example 15—Cytokine/Chemokine Gene Silencing Using shRNA

Similar to siRNAs, short hairpin RNA can be used to knock down or eliminate expression of a cytokine/chemokine/transcription factor gene in an immune effector cell.


Cherkassky et al. (“Human CAR T cells with cell-intrinsic PD-1 checkpoint blockade resist tumor-mediated inhibition,” J Clin Investig 2016; 126:3130-3144) reported use of shRNA to knock down expression of immune checkpoint PD-1 in CD28 mesothelin-specific CAR-T cells, which showed enhanced proliferative function upon antigen stimulation, augmented cytotoxicity, and enhanced cytokine secretion. In addition, a CD19-specific CAR with a PD-1 shRNA lentiviral cassette has been tested for use in CD19-positive B-cell lymphoma. A list of useful shRNAs and their sequences is provided in Tables 15 and 16.


Table 15. 5′ to 3′ (Forward) shRNA Sequences for Silencing of Cytokine/Chemokine GC Genes to Reduce the Incidence of CRS text missing or illegible when filed









TABLE 16







3′ to 5′ (Reverse) shRNA sequences for Silencing of Cytokine/Chemokine


Genes to Reduce the Incidence of CRS











Target

SEQ




Gene

ID
Lo-



Symbol
shRNA Reverse Oligo Sequence
NO:
cation
Gene ID No.





A2M
AATTCAAAAAAGGCGTCCCTATACCAAATAACTCGAGTTATTTGGTATAGGGACGCCT
2036
chr12
ENST00000318602.11





A2M
AATTCAAAAAAGACTGCATCAATCGTCATAACTCGAGTTATGACGATTGATGCAGTCT
2037
chr12
ENST00000318602.11





A2M
AATTCAAAAACCTCCAGACATCCTTGAAATACTCGAGTATTTCAAGGATGTCTGGAGG
2038
chr12
ENST00000318602.11





ACKR1
AATTCAAAAAATCCTGTGGGCCTGGTTTATTCTCGAGAATAAACCAGGCCCACAGGAT
2039
chr1
ENST00000368122.2





ACKR1
AATTCAAAAATGGCAGAAGCCCTGGCAATTTCTCGAGAAATTGCCAGGGCTTCTGCCA
2040
chr1
ENST00000368122.2





ACKR1
AATTCAAAAAGGCGGATGGTTGAGATCTATCCTCGAGGATAGATCTCAACCATCCGCC
2041
chr1
ENST00000368122.2





ACKR2
AATTCAAAAAGAGCACTCTTTATACTATTAACTCGAGTTAATAGTATAAAGAGTGCTC
2042
chr3
ENST00000422265.5





ACKR2
AATTCAAAAAGTGGAACTGCCACGCAGATTTCTCGAGAAATCTGCGTGGCAGTTCCAC
2043
chr3
ENST00000422265.5





ACKR2
AATTCAAAAACATTGGGTCTTCGGGAGTTTCCTCGAGGAAACTCCCGAAGACCCAATG
2044
chr3
ENST00000422265.5





ACKR3
AATTCAAAAACACTATTGGTGTACCTTATAACTCGAGTTATAAGGTACACCAATAGTG
2045
chr2
ENST00000272928.3





ACKR3
AATTCAAAAAGGCATAGTGCTGACATATATTCTCGAGAATATATGTCAGCACTATGCC
2046
chr2
ENST00000272928.3





ACKR3
AATTCAAAAAGCCGTTCCCTTCTCCATTATCCTCGAGGATAATGGAGAAGGGAACGGC
2047
chr2
ENST00000272928.3





ACVR1
AATTCAAAAATGATAATTCCCTCGACAAATTCTCGAGAATTTGTCGAGGGAATTATCA
2048
chr2
ENST00000263640.7





ACVR1
AATTCAAAAAGCAGAACGTATTTAGCCATTACTCGAGTAATGGCTAAATACGTTCTGC
2049
chr2
ENST00000263640.7





ACVR1
AATTCAAAAACTGGTCTGTCTTTGGATAATACTCGAGTATTATCCAAAGACAGACCAG
2050
chr2
ENST00000263640.7





ACVR2B
AATTCAAAAAATGTCACGAGGCCTCTCATACCTCGAGGTATGAGAGGCCTCGTGACAT
2051
chr3
ENST00000352511.4





ACVR2B
AATTCAAAAACCCAGCTCATGAATGACTTTGCTCGAGCAAAGTCATTCATGAGCTGGG
2052
chr3
ENST00000352511.4





ACVR2B
AATTCAAAAACTTTGGCTTGGCTGTTCGATTCTCGAGAATCGAACAGCCAAGCCAAAG
2053
chr3
ENST00000352511.4





ACVRL1
AATTCAAAAACCGGGAGACTGAGATCTATAACTCGAGTTATAGATCTCAGTCTCCCGG
2054
chr12
ENST00000388922.8





ACVRL1
AATTCAAAAACAGGAGCACCTGATTCCTTTCCTCGAGGAAAGGAATCAGGTGCTCCTG
2055
chr12
ENST00000388922.8





ACVRL1
AATTCAAAAACGTGGAGATCTTCGGTACACACTCGAGTGTGTACCGAAGATCTCCACG
2056
chr12
ENST00000388922.8





ADIPOQ
AATTCAAAAAGTTGGAGGCCTTTAGATATTACTCGAGTAATATCTAAAGGCCTCCAAC
2057
chr3
ENST00000320741.6





ADIPOQ
AATTCAAAAAATGCTCATATCAATCCTATAACTCGAGTTATAGGATTGATATGAGCAT
2058
chr3
ENST00000320741.6





ADIPOQ
AATTCAAAAAACGGTTAGGAAGTTGATTATTCTCGAGAATAATCAACTTCCTAACCGT
2059
chr3
ENST00000320741.6





AGER
AATTCAAAAACACACTGCAGTCGGAGCTAATCTCGAGATTAGCTCCGACTGCAGTGTG
2060
chr6
ENST00000375076.8





AGER
AATTCAAAAAGAAGCCAGAAATTGTAGATTCCTCGAGGAATCTACAATTTCTGGCTTC
2061
chr6
ENST00000375076.8





AGER
AATTCAAAAATGCTGATCCTCCCTGAGATAGCTCGAGCTATCTCAGGGAGGATCAGCA
2062
chr6
ENST00000375076.8





AGRN
AATTCAAAAAGAGTTCTGTGTGGAAGATAAACTCGAGTTTATCTTCCACACAGAACTC
2063
chr1
ENST00000379370.6





AGRN
AATTCAAAAAGCGCACGTATGACAGTGATTGCTCGAGCAATCACTGTCATACGTGCGC
2064
chr1
ENST00000379370.6





AGRN
AATTCAAAAAACGACGGAGTCACCTACGAAACTCGAGTTTCGTAGGTGACTCCGTCGT
2065
chr1
ENST00000379370.6





AHR
AATTCAAAAACGGCATAGAGACCGACTTAATCTCGAGATTAAGTCGGTCTCTATGCCG
2066
chr7
ENST00000242057.8





AHR
AATTCAAAAAGCGGCATAGAGACCGACTTAACTCGAGTTAAGTCGGTCTCTATGCCGC
2067
chr7
ENST00000242057.8





AHR
AATTCAAAAAATCCACAGTCAGCCATAATAACTCGAGTTATTATGGCTGACTGTGGAT
2068
chr7
ENST00000242057.8





AHR
AATTCAAAAAACTGCTTAAAGTTGGTATTAACTCGAGTTAATACCAACTTTAAGCAGT
2069
chr7
ENST00000242057.8





AIMP1
AATTCAAAAACAGCCTGATCTTCACACTAATCTCGAGATTAGTGTGAAGATCAGGCTG
2070
chr4
ENST00000358008.7





AIMP1
AATTCAAAAAAGAAGTAGATGTCGGAGAAATCTCGAGATTTCTCCGACATCTACTTCT
2071
chr4
ENST00000358008.7





AIMP1
AATTCAAAAAGCCAGAGTAACCCTGACTAATCTCGAGATTAGTCAGGGTTACTCTGGC
2072
chr4
ENST00000358008.7





AREG
AATTCAAAAACCTCTTTCCAGTGGATCATAACTCGAGTTATGATCCACTGGAAAGAGG
2073
chr4
ENST00000395748.7





AREG
AATTCAAAAATACTCGGCTCAGGCCATTATGCTCGAGCATAATGGCCTGAGCCGAGTA
2074
chr4
ENST00000395748.7





AREG
AATTCAAAAAATTCACGGAGAATGCAAATATCTCGAGATATTTGCATTCTCCGTGAAT
2075
chr4
ENST00000395748.7





BCL6
AATTCAAAAATGTGCCACAGCAATATCTATTCTCGAGAATAGATATTGCTGTGGCACA
2076
chr3
ENST00000406870.6





BCL6
AATTCAAAAAACTGCGTTAAAGGCTCGATTTCTCGAGAAATCGAGCCTTTAACGCAGT
2077
chr3
ENST00000406870.6





BCL6
AATTCAAAAACCGGCTCAATAACATCGTTAACTCGAGTTAACGATGTTATTGAGCCGG
2078
chr3
ENST00000406870.6





BCL6
AATTCAAAAAACAAGCCAGCCGGCTCAATAACTCGAGTTATTGAGCCGGCTGGCTTGT
2079
chr3
ENST00000406870.6





BMP1
AATTCAAAAAACTGACGAGGACAGCTATATTCTCGAGAATATAGCTGTCCTCGTCAGT
2080
chr8
ENST00000306385.9





BMP1
AATTCAAAAAGCTCGTAAGTCCTCCATCAAACTCGAGTTTGATGGAGGACTTACGAGC
2081
chr8
ENST00000306385.9





BMP1
AATTCAAAAAACAGCTGTGCCTACGACTATCCTCGAGGATAGTCGTAGGCACAGCTGT
2082
chr8
ENST00000306385.9





BMP10
AATTCAAAAAAGCAAGACGGTGTCGACTTTACTCGAGTAAAGTCGACACCGTCTTGCT
2083
chr2
ENST00000295379.1





BMP10
AATTCAAAAAGCGTCGTCACCTACAAGTTTACTCGAGTAAACTTGTAGGTGACGACGC
2084
chr2
ENST00000295379.1





BMP10
AATTCAAAAACATGGCTGAACTTAGGCTATACTCGAGTATAGCCTAAGTTCAGCCATG
2085
chr2
ENST00000295379.1





BMP15
AATTCAAAAATACTACGCGATGGTCTCAATTCTCGAGAATTGAGACCATCGCGTAGTA
2086
chrX
ENST00000252677.3





BMP15
AATTCAAAAAGCCTTCTTGTTACTCTATTTCCTCGAGGAAATAGAGTAACAAGAAGGC
2087
chrX
ENST00000252677.3





BMP15
AATTCAAAAAATGGAACACAGGGCCCAAATGCTCGAGCATTTGGGCCCTGTGTTCCAT
2088
chrX
ENST00000252677.3





BMP2
AATTCAAAAAGATCATCTGAACTCCACTAATCTCGAGATTAGTGGAGTTCAGATGATC
2089
chr20
ENST00000378827.4





BMP2
AATTCAAAAACCGGAGATTCTTCTTTAATTTCTCGAGAAATTAAAGAAGAATCTCCGG
2090
chr20
ENST00000378827.4





BMP2
AATTCAAAAACAAGATGCTTTAGGAAACAATCTCGAGATTGTTTCCTAAAGCATCTTG
2091
chr20
ENST00000378827.4





BMP3
AATTCAAAAACTTACAGGGACACCGGAATTTCTCGAGAAATTCCGGTGTCCCTGTAAG
2092
chr4
ENST00000282701.2





BMP3
AATTCAAAAAGGCCAAATCTCATCGAGATATCTCGAGATATCTCGATGAGATTTGGCC
2093
chr4
ENST00000282701.2





BMP3
AATTCAAAAAGTGGATTGAACCTCGGAATTGCTCGAGCAATTCCGAGGTTCAATCCAC
2094
chr4
ENST00000282701.2





BMP4
AATTCAAAAACCCTGGTCAATTCTGTCAATTCTCGAGAATTGACAGAATTGACCAGGG
2095
chr14
ENST00000245451.8





BMP4
AATTCAAAAATCCTTGAGGATAGACAGATATCTCGAGATATCTGTCTATCCTCAAGGA
2096
chr14
ENST00000245451.8





BMP4
AATTCAAAAAAGGGCCAGCATGTCAGGATTACTCGAGTAATCCTGACATGCTGGCCCT
2097
chr14
ENST00000245451.8





BMP5
AATTCAAAAATGGACGCAGTATCAACGTAAACTCGAGTTTACGTTGATACTGCGTCCA
2098
chr6
ENST00000370830.3





BMP5
AATTCAAAAACCAGAAGGATACGCTGCATTTCTCGAGAAATGCAGCGTATCCTTCTGG
2099
chr6
ENST00000370830.3





BMP5
AATTCAAAAAATGCCACCAACCACGCTATAGCTCGAGCTATAGCGTGGTTGGTGGCAT
2100
chr6
ENST00000370830.3





BMP6
AATTCAAAAAATACAGGAATATGGTTGTAAGCTCGAGCTTACAACCATATTCCTGTAT
2101
chr6
ENST00000283147.6





BMP6
AATTCAAAAACGCCGACAACAGAGTCGTAATCTCGAGATTACGACTCTGTTGTCGGCG
2102
chr6
ENST00000283147.6





BMP6
AATTCAAAAACTGTCTATCAAAGGTAGATTTCTCGAGAAATCTACCTTTGATAGACAG
2103
chr6
ENST00000283147.6





BMP7
AATTCAAAAAACGTTCCGGATCAGCGTTTATCTCGAGATAAACGCTGATCCGGAACGT
2104
chr20
ENST00000395863.7





BMP7
AATTCAAAAAACTCGTTTCCAGAGGTAATTACTCGAGTAATTACCTCTGGAAACGAGT
2105
chr20
ENST00000395863.7





BMP7
AATTCAAAAAACCATCGAGAGTTCCGGTTTGCTCGAGCAAACCGGAACTCTCGATGGT
2106
chr20
ENST00000395863.7





BMP8A
AATTCAAAAAAGGGTGCAGTTAGCATATTAGCTCGAGCTAATATGCTAACTGCACCCT
2107
chr1
ENST00000331593.5





BMP8A
AATTCAAAAAAGCACAGAAGTCCTATCTTAGCTCGAGCTAAGATAGGACTTCTGTGCT
2108
chr1
ENST00000331593.5





BMP8A
AATTCAAAAAAGGTACAACACTGGCCATTTCCTCGAGGAAATGGCCAGTGTTGTACCT
2109
chr1
ENST00000331593.5





BMP8B
AATTCAAAAACCAAGGCTACTCGGCCTATTACTCGAGTAATAGGCCGAGTAGCCTTGG
2110
chr1
ENST00000372827.7





BMP8B
AATTCAAAAAGACCCTCACAACCACGTACATCTCGAGATGTACGTGGTTGTGAGGGTC
2111
chr1
ENST00000372827.7





BMP8B
AATTCAAAAACGTTAACATGGTGGAGCGAGACTCGAGTCTCGCTCCACCATGTTAACG
2112
chr1
ENST00000372827.7





BMPR2
AATTCAAAAAGAACGGCTATGTGCGTTTAAACTCGAGTTTAAACGCACATAGCCGTTC
2113
chr2
ENST00000374580.8





BMPR2
AATTCAAAAAGCCTATGGAGTGAAATTATTTCTCGAGAAATAATTTCACTCCATAGGC
2114
chr2
ENST00000374580.8





BMPR2
AATTCAAAAAATTACCACGAGGAGATCATTACTCGAGTAATGATCTCCTCGTGGTAAT
2115
chr2
ENST00000374580.8





C10orf99
AATTCAAAAACATCATGTGAGGCTCTGTAAACTCGAGTTTACAGAGCCTCACATGATG
2116
chr10
ENST00000372126.3





C10orf99
AATTCAAAAACCCAACTCAACAAACCTGAAACTCGAGTTTCAGGTTTGTTGAGTTGGG
2117
chr10
ENST00000372126.3





C10orf99
AATTCAAAAAGCCATCAACTTTCAGAGCTATCTCGAGATAGCTCTGAAAGTTGATGGC
2118
chr10
ENST00000372126.3





C1QTNF4
AATTCAAAAAACACCGAGTTCGTCAACATTGCTCGAGCAATGTTGACGAACTCGGTGT
2119
chr11
ENST00000302514.3





C1QTNF4
AATTCAAAAAGCGTAAGACGCTGTCGGTTAACTCGAGTTAACCGACAGCGTCTTACGC
2120
chr11
ENST00000302514.3





C1QTNF4
AATTCAAAAACGAGGTGCAGGCCATGATTTACTCGAGTAAATCATGGCCTGCACCTCG
2121
chr11
ENST00000302514.3





C5
AATTCAAAAAACGATGGAGCCTGCGTTAATACTCGAGTATTAACGCAGGCTCCATCGT
2122
chr9
ENST00000223642.2





C5
AATTCAAAAATCCCGACTTCTGGTCTATTACCTCGAGGTAATAGACCAGAAGTCGGGA
2123
chr9
ENST00000223642.2





C5
AATTCAAAAAGCCCGAGAGAACAGCTTATATCTCGAGATATAAGCTGTTCTCTCGGGC
2124
chr9
ENST00000223642.2





CCL1
AATTCAAAAAGCAATCCTGTGTTACAGAAATCTCGAGATTTCTGTAACACAGGATTGC
2125
chr17
ENST00000225842.3





CCL1
AATTCAAAAACTGCTCCAATGAGGGCTTAATCTCGAGATTAAGCCCTCATTGGAGCAG
2126
chr17
ENST00000225842.3





CCL1
AATTCAAAAACCTGAGGGCAATCCTGTGTTACTCGAGTAACACAGGATTGCCCTCAGG
2127
chr17
ENST00000225842.3





CCL11
AATTCAAAAACCAACTCCAAAGCCATAAATACTCGAGTATTTATGGCTTTGGAGTTGG
2128
chr17
ENST00000305869.3





CCL11
AATTCAAAAACATTCTGAGGTAACCTCATTACTCGAGTAATGAGGTTACCTCAGAATG
2129
chr17
ENST00000305869.3





CCL11
AATTCAAAAATCCTCCATGAATATCAGTTATCTCGAGATAACTGATATTCATGGAGGA
2130
chr17
ENST00000305869.3





CCL13
AATTCAAAAATGGAATACTTCTACCATAATTCTCGAGAATTATGGTAGAAGTATTCCA
2131
chr17
ENST00000225844.6





CCL13
AATTCAAAAATCAAGCTGGAGTACGTGAAATCTCGAGATTTCACGTACTCCAGCTTGA
2132
chr17
ENST00000225844.6





CCL13
AATTCAAAAAACTCAACGTCCCATCTACTTGCTCGAGCAAGTAGATGGGACGTTGAGT
2133
chr17
ENST00000225844.6





CCL14
AATTCAAAAACCTAGGGACCAAGACTGAATCCTCGAGGATTCAGTCTTGGTCCCTAGG
2134
chr17
ENST00000618404.4





CCL14
AATTCAAAAACAGCGGATTATGGATTACTATCTCGAGATAGTAATCCATAATCCGCTG
2135
chr17
ENST00000618404.4





CCL14
AATTCAAAAACAAGCCCGGAATTGTCTTCATCTCGAGATGAAGACAATTCCGGGCTTG
2136
chr17
ENST00000618404.4





CCL15
AATTCAAAAAGCTGAAGCCCTACTCAATATACTCGAGTATATTGAGTAGGGCTTCAGC
2137
chr17
ENST00000617897.1





CCL15
AATTCAAAAAACAGAGTTAATGATGTCAAAGCTCGAGCTTTGACATCATTAACTCTGT
2138
chr17
ENST00000617897.1





CCL15
AATTCAAAAAATCCCGTGTTCACTCATGAAACTCGAGTTTCATGAGTGAACACGGGAT
2139
chr17
ENST00000617897.1





CCL16
AATTCAAAAAAGGAGAAGTATTTCGAATATTCTCGAGAATATTCGAAATACTTCTCCT
2140
chr17
ENST00000611905.1





CCL16
AATTCAAAAAAGGAACTTGTCCACGGTTAAACTCGAGTTTAACCGTGGACAAGTTCCT
2141
chr17
ENST00000611905.1





CCL16
AATTCAAAAACTGCCTGAAGTATTATGAGAACTCGAGTTCTCATAATACTTCAGGCAG
2142
chr17
ENST00000611905.1





CCL17
AATTCAAAAAGAGAGTGAAGAATGCAGTTAACTCGAGTTAACTGCATTCTTCACTCTC
2143
chr16
ENST00000219244.8





CCL17
AATTCAAAAAGAGTACTTCAAGGGAGCCATTCTCGAGAATGGCTCCCTTGAAGTACTC
2144
chr16
ENST00000219244.8





CCL17
AATTCAAAAAGCAAAGCCTTGAGAGGTCTTGCTCGAGCAAGACCTCTCAAGGCTTTGC
2145
chr16
ENST00000219244.8





CCL18
AATTCAAAAATCTATTGTTGAGCTGCATTATCTCGAGATAATGCAGCTCAACAATAGA
2146
chr17
ENST00000616054.1





CCL18
AATTCAAAAACCTTCAACTCTTCGTACATTCCTCGAGGAATGTACGAAGAGTTGAAGG
2147
chr17
ENST00000616054.1





CCL18
AATTCAAAAAGTCTATACCTCCTGGCAGATTCTCGAGAATCTGCCAGGAGGTATAGAC
2148
chr17
ENST00000616054.1





CCL19
AATTCAAAAAGAGTCAAGCATTGTGAATTATCTCGAGATAATTCACAATGCTTGACTC
2149
chr9
ENST00000311925.6





CCL19
AATTCAAAAAGCCGCAGCAGTTAACCTATGACTCGAGTCATAGGTTAACTGCTGCGGC
2150
chr9
ENST00000311925.6





CCL19
AATTCAAAAACCAACTCTGAGTGGCACCAATCTCGAGATTGGTGCCACTCAGAGTTGG
2151
chr9
ENST00000311925.6





CCL2
AATTCAAAAACCCAGTCACCTGCTGTTATAACTCGAGTTATAACAGCAGGTGACTGGG
2152
chr17
ENST00000225831.4





CCL2
AATTCAAAAACCCAGTCACCTGCTGTTATAACTCGAGTTATAACAGCAGGTGACTGGG
2153
chr17
ENST00000225831.4





CCL2
AATTCAAAAAGCTCGCGAGCTATAGAAGAATCTCGAGATTCTTCTATAGCTCGCGAGC
2154
chr17
ENST00000225831.4





CCL20
AATTCAAAAAAGTTGTCTGTGTGCGCAAATCCTCGAGGATTTGCGCACACAGACAACT
2155
chr2
ENST00000358813.4





CCL20
AATTCAAAAAGACCGTATTCTTCATCCTAAACTCGAGTTTAGGATGAAGAATACGGTC
2156
chr2
ENST00000358813.4





CCL20
AATTCAAAAAAGGGTTTAGTGCTTATCTAATCTCGAGATTAGATAAGCACTAAACCCT
2157
chr2
ENST00000358813.4





CCL21
AATTCAAAAACACTCTTTCTCCTGCTTTAACCTCGAGGTTAAAGCAGGAGAAAGAGTG
2158
chr9
ENST00000259607.6





CCL21
AATTCAAAAAGAGCTATGTGCAGACCCAAAGCTCGAGCTTTGGGTCTGCACATAGCTC
2159
chr9
ENST00000259607.6





CCL21
AATTCAAAAACCATCCCAGCTATCCTGTTCTCTCGAGAGAACAGGATAGCTGGGATGG
2160
chr9
ENST00000259607.6





CCL22
AATTCAAAAACCCTACCTCCCTGCCATTATACTCGAGTATAATGGCAGGGAGGTAGGG
2161
chr16
ENST00000219235.4





CCL22
AATTCAAAAAGCGTGGTGAAACACTTCTACTCTCGAGAGTAGAAGTGTTTCACCACGC
2162
chr16
ENST00000219235.4





CCL22
AATTCAAAAAATGTTGCTGACACCCAGAAAGCTCGAGCTTTCTGGGTGTCAGCAACAT
2163
chr16
ENST00000219235.4





CCL23
AATTCAAAAACACTCCTGGAGAGTTACTTTGCTCGAGCAAAGTAACTCTCCAGGAGTG
2164
chr17
ENST00000615050.1





CCL23
AATTCAAAAACCTTTCTCATGCTGCAGGATTCTCGAGAATCCTGCAGCATGAGAAAGG
2165
chr17
ENST00000615050.1





CCL23
AATTCAAAAAGAAGCTGGACACACGGATCAACTCGAGTTGATCCGTGTGTCCAGCTTC
2166
chr17
ENST00000615050.1





CCL24
AATTCAAAAAAGTGATCTTCACCACCAAGAACTCGAGTTCTTGGTGGTGAAGATCACT
2167
chr7
ENST00000222902.6





CCL24
AATTCAAAAAGTTCTTTGTTTCCAAGAGAATCTCGAGATTCTCTTGGAAACAAAGAAC
2168
chr7
ENST00000222902.6





CCL24
AATTCAAAAACCTGCTGCATGTTCTTTGTTTCTCGAGAAACAAAGAACATGCAGCAGG
2169
chr7
ENST00000222902.6





CCL25
AATTCAAAAAGCCGGATCTTTCTCCGATAAACTCGAGTTTATCGGAGAAAGATCCGGC
2170
chr19
ENST00000390669.7





CCL25
AATTCAAAAAGCTCCTGGATGCTCGAAATAACTCGAGTTATTTCGAGCATCCAGGAGC
2171
chr19
ENST00000390669.7





CCL25
AATTCAAAAACCCTCCTGATATCAGCTAATTCTCGAGAATTAGCTGATATCAGGAGGG
2172
chr19
ENST00000390669.7





CCL26
AATTCAAAAACACACGTGGGAGTGACATATCCTCGAGGATATGTCACTCCCACGTGTG
2173
chr7
ENST00000005180.8





CCL26
AATTCAAAAATCCGAAACAATTGTGACTCAGCTCGAGCTGAGTCACAATTGTTTCGGA
2174
chr7
ENST00000005180.8





CCL26
AATTCAAAAAGCTGTGATATTCACTACCAAACTCGAGTTTGGTAGTGAATATCACAGC
2175
chr7
ENST00000005180.8





CCL27
AATTCAAAAATTCGTGCTTCACCTGGCTCAACTCGAGTTGAGCCAGGTGAAGCACGAA
2176
chr9
ENST00000259631.4





CCL27
AATTCAAAAACCGAAAGCCACTCTCAGACAACTCGAGTTGTCTGAGAGTGGCTTTCGG
2177
chr9
ENST00000259631.4





CCL27
AATTCAAAAACAAGCTACTGAGGAAGGTCATCTCGAGATGACCTTCCTCAGTAGCTTG
2178
chr9
ENST00000259631.4





CCL28
AATTCAAAAACGGAGGTTTCACATCATATTTCTCGAGAAATATGATGTGAAACCTCCG
2179
chr5
ENST00000361115.4





CCL28
AATTCAAAAACACGAAACATACGGCCATAAACTCGAGTTTATGGCCGTATGTTTCGTG
2180
chr5
ENST00000361115.4





CCL28
AATTCAAAAATTAGAGAGTCTACAGATAAATCTCGAGATTTATCTGTAGACTCTCTAA
2181
chr5
ENST00000361115.4





CCL3
AATTCAAAAACCGGCAGATTCCACAGAATTTCTCGAGAAATTCTGTGGAATCTGCCGG
2182
chr17
ENST00000613922.1





CCL3
AATTCAAAAATTCGATTTCACAGTGTGTTTGCTCGAGCAAACACACTGTGAAATCGAA
2183
chr17
ENST00000613922.1





CCL3
AATTCAAAAACAACCAGTTCTCTGCATCACTCTCGAGAGTGATGCAGAGAACTGGTTG
2184
chr17
ENST00000613922.1





CCL4
AATTCAAAAAATGTGCCGTGTTATTGTATTACTCGAGTAATACAATAACACGGCACAT
2185
chr17
ENST00000615863.1





CCL4
AATTCAAAAATCCTGTCCCTTCTCTTAATTTCTCGAGAAATTAAGAGAAGGGACAGGA
2186
chr17
ENST00000615863.1





CCL4
AATTCAAAAACTGTGCTGATCCCAGTGAATCCTCGAGGATTCACTGGGATCAGCACAG
2187
chr17
ENST00000615863.1





CCL5
AATTCAAAAATGAACCTGAACTTACACAAATCTCGAGATTTGTGTAAGTTCAGGTTCA
2188
chr17
ENST00000603197.5





CCL5
AATTCAAAAACCTGCTGCTTTGCCTACATTGCTCGAGCAATGTAGGCAAAGCAGCAGG
2189
chr17
ENST00000603197.5





CCL5
AATTCAAAAACTACCACACAGCAGCAGTTACCTCGAGGTAACTGCTGCTGTGTGGTAG
2190
chr17
ENST00000603197.5





CCL7
AATTCAAAAACATAAAGCCTTGGATGTATATCTCGAGATATACATCCAAGGCTTTATG
2191
chr17
ENST00000378569.2





CCL7
AATTCAAAAAGCTGCTACAGATTTATCAATACTCGAGTATTGATAAATCTGTAGCAGC
2192
chr17
ENST00000378569.2





CCL7
AATTCAAAAATCTAAGGAATATGAGCTTTATCTCGAGATAAAGCTCATATTCCTTAGA
2193
chr17
ENST00000378569.2





CCL8
AATTCAAAAAGCTGCTTTAACGTGATCAATACTCGAGTATTGATCACGTTAAAGCAGC
2194
chr17
ENST00000394620.1





CCL8
AATTCAAAAACAGGTGCAGTGTGACATTATTCTCGAGAATAATGTCACACTGCACCTG
2195
chr17
ENST00000394620.1





CCL8
AATTCAAAAATTGTACTGCTGTTGTTGAAATCTCGAGATTTCAACAACAGCAGTACAA
2196
chr17
ENST00000394620.1





CCR1
AATTCAAAAAATTCTGCTAAGACGACCAAATCTCGAGATTTGGTCGTCTTAGCAGAAT
2197
chr3
ENST00000296140.3





CCR1
AATTCAAAAAATCTGCTACACAGGGATTATACTCGAGTATAATCCCTGTGTAGCAGAT
2198
chr3
ENST00000296140.3





CCR1
AATTCAAAAACCTCTGTACTCCTTGGTATTTCTCGAGAAATACCAAGGAGTACAGAGG
2199
chr3
ENST00000296140.3





CCR2
AATTCAAAAAACGGTGCTCCCTGTCATAAATCTCGAGATTTATGACAGGGAGCACCGT
2200
chr3
ENST00000445132.2





CCR2
AATTCAAAAACTTCTGGACTCCCTATAATATCTCGAGATATTATAGGGAGTCCAGAAG
2201
chr3
ENST00000445132.2





CCR2
AATTCAAAAAGCTGTATCACATCGGTTATTTCTCGAGAAATAACCGATGTGATACAGC
2202
chr3
ENST00000445132.2





CCR3
AATTCAAAAAATACAGGAGGCTCCGAATTATCTCGAGATAATTCGGAGCCTCCTGTAT
2203
chr3
ENST00000357422.2





CCR3
AATTCAAAAACCCAGAGGATACAGTATATAGCTCGAGCTATATACTGTATCCTCTGGG
2204
chr3
ENST00000357422.2





CCR3
AATTCAAAAATAGCAGCTCTTCCTGAATTTACTCGAGTAAATTCAGGAAGAGCTGCTA
2205
chr3
ENST00000357422.2





CCR5
AATTCAAAAAACTCTTGACAGGGCTCTATTTCTCGAGAAATAGAGCCCTGTCAAGAGT
2206
chr3
ENST00000292303.4





CCR5
AATTCAAAAACGAGCGAGCAAGCTCAGTTTACTCGAGTAAACTGAGCTTGCTCGCTCG
2207
chr3
ENST00000292303.4





CCR5
AATTCAAAAATCCATACAGTCAGTATCAATTCTCGAGAATTGATACTGACTGTATGGA
2208
chr3
ENST00000292303.4





CCR6
AATTCAAAAAGGTCTATGACAGACGTCTATCCTCGAGGATAGACGTCTGTCATAGACC
2209
chr6
ENST00000341935.9





CCR6
AATTCAAAAACGACTCCAGTGAAGATTATTTCTCGAGAAATAATCTTCACTGGAGTCG
2210
chr6
ENST00000341935.9





CCR6
AATTCAAAAATCGACTCCAGTGAAGATTATTCTCGAGAATAATCTTCACTGGAGTCGA
2211
chr6
ENST00000341935.9





CCR7
AATTCAAAAAGATGAGGTCACGGACGATTACCTCGAGGTAATCGTCCGTGACCTCATC
2212
chr17
ENST00000246657.2





CCR7
AATTCAAAAAGCTGGTCGTGTTGACCTATATCTCGAGATATAGGTCAACACGACCAGC
2213
chr17
ENST00000246657.2





CCR7
AATTCAAAAACGTGTTGACCTATATCTATTTCTCGAGAAATAGATATAGGTCAACACG
2214
chr17
ENST00000246657.2





CD109
AATTCAAAAAGCCGATCCTTACATAGATATTCTCGAGAATATCTATGTAAGGATCGGC
2215
chr6
ENST00000287097.5





CD109
AATTCAAAAACCCGGAGGAAATGTGACTATTCTCGAGAATAGTCACATTTCCTCCGGG
2216
chr6
ENST00000287097.5





CD109
AATTCAAAAACCGCTTATCATTTGAGACCAACTCGAGTTGGTCTCAAATGATAAGCGG
2217
chr6
ENST00000287097.5





CD27
AATTCAAAAACTTACCTTATGTCAGTGAGATCTCGAGATCTCACTGACATAAGGTAAG
2218
chr12
ENST00000266557.3





CD27
AATTCAAAAAGCACTGTAACTCTGGTCTTCTCTCGAGAGAAGACCAGAGTTACAGTGC
2219
chr12
ENST00000266557.3





CD27
AATTCAAAAACCACCCACTTACCTTATGTCACTCGAGTGACATAAGGTAAGTGGGTGG
2220
chr12
ENST00000266557.3





CD27
AATTCAAAAAGCCAGATGTGTGAGCCAGGAACTCGAGTTCCTGGCTCACACATCTGGC
2221
chr12
ENST00000266557.3





CD28
AATTCAAAAACCTCCAGAATTTGTATGTTAACTCGAGTTAACATACAAATTCTGGAGG
2222
ch2
ENST00000458610.6





CD28
AATTCAAAAACAACCTTAGCTGCAAGTATTCCTCGAGGAATACTTGCAGCTAAGGTTG
2223
ch2
ENST00000458610.6





CD28
AATTCAAAAATGGAGTCCTGGCTTGCTATAGCTCGAGCTATAGCAAGCCAGGACTCCA
2224
ch2
ENST00000458610.6





CD28
AATTCAAAAACCTCCTCCTTACCTAGACAATCTCGAGATTGTCTAGGTAAGGAGGAGG
2225
ch2
ENST00000458610.6





CD36
AATTCAAAAAAGAACCTATTGATGGATTAAACTCGAGTTTAATCCATCAATAGGTTCT
2226
chr7
ENST00000309881.11





CD36
AATTCAAAAAGCCATAATCGACACATATAAACTCGAGTTTATATGTGTCGATTATGGC
2227
chr7
ENST00000309881.11





CD36
AATTCAAAAAACGGCTGCAGGTCAACCTATTCTCGAGAATAGGTTGACCTGCAGCCGT
2228
chr7
ENST00000309881.11





CD4
AATTCAAAAACCTGATCATCAAGAATCTTAACTCGAGTTAAGATTCTTGATGATCAGG
2229
chr12
ENST00000011653.8





CD4
AATTCAAAAAAGAGCGGATGTCTCAGATCAACTCGAGTTGATCTGAGACATCCGCTCT
2230
chr12
ENST00000011653.8





CD4
AATTCAAAAACCACTCGCCTTTACAGTTGAACTCGAGTTCAACTGTAAAGGCGAGTGG
2231
chr12
ENST00000011653.8





CD40LG
AATTCAAAAAGGGAAACAGCTGACCGTTAAACTCGAGTTTAACGGTCAGCTGTTTCCC
2232
chrX
ENST00000370629.6





CD40LG
AATTCAAAAATTCGAGTCAAGCTCCATTTATCTCGAGATAAATGGAGCTTGACTCGAA
2233
chrX
ENST00000370629.6





CD40LG
AATTCAAAAATGACGCTGGGAGTCTTCATAACTCGAGTTATGAAGACTCCCAGCGTCA
2234
chrX
ENST00000370629.6





CD70
AATTCAAAAACCATCGTGATGGCATCTACATCTCGAGATGTAGATGCCATCACGATGG
2235
chr19
ENST00000245903.3





CD70
AATTCAAAAACAGCTACGTATCCATCGTGATCTCGAGATCACGATGGATACGTAGCTG
2236
chr19
ENST00000245903.3





CD70
AATTCAAAAACCAACCTCACTGGGACACTTTCTCGAGAAAGTGTCCCAGTGAGGTTGG
2237
chr19
ENST00000245903.3





CD74
AATTCAAAAACAAGTCGGAACAGCAGATAACCTCGAGGTTATCTGCTGTTCCGACTTG
2238
chr5
ENST00000009530.11





CD74
AATTCAAAAAGAGAACCTGAGACACCTTAAGCTCGAGCTTAAGGTGTCTCAGGTTCTC
2239
chr5
ENST00000009530.11





CD74
AATTCAAAAACCACACAGCTACAGCTTTCTTCTCGAGAAGAAAGCTGTAGCTGTGTGG
2240
chr5
ENST00000009530.11





CD8A
AATTCAAAAAGCTGGACTTCGCCTGTGATATCTCGAGATATCACAGGCGAAGTCCAGC
2241
ch2
ENST00000409511.6





CD8A
AATTCAAAAAGTGTATTCATTCTCATGATTACTCGAGTAATCATGAGAATGAATACAC
2242
ch2
ENST00000409511.6





CD8A
AATTCAAAAACCAGAGACAGCTTGATCAAAGCTCGAGCTTTGATCAAGCTGTCTCTGG
2243
ch2
ENST00000409511.6





CD8A
AATTCAAAAACCTTCTCCTGTCACTGGTTATCTCGAGATAACCAGTGACAGGAGAAGG
2244
ch2
ENST00000409511.6





CER1
AATTCAAAAAGAGAAGATGCTGTCCAGATTTCTCGAGAAATCTGGACAGCATCTTCTC
2245
chr9
ENST00000380911.3





CER1
AATTCAAAAACCAGCCGATAGATGGAATGAACTCGAGTTCATTCCATCTATCGGCTGG
2246
chr9
ENST00000380911.3





CER1
AATTCAAAAACAAGAAATTCTGGCACCACTTCTCGAGAAGTGGTGCCAGAATTTCTTG
2247
chr9
ENST00000380911.3





CHRD
AATTCAAAAAACGTCCTGCAAAGTGTCCTTTCTCGAGAAAGGACACTTTGCAGGACGT
2248
chr3
ENST00000204604.5





CHRD
AATTCAAAAAACTGATCCAGAGCTGGAGAAACTCGAGTTTCTCCAGCTCTGGATCAGT
2249
chr3
ENST00000204604.5





CHRD
AATTCAAAAACGGCTGCTGAAGGGATTCTATCTCGAGATAGAATCCCTTCAGCAGCCG
2250
chr3
ENST00000204604.5





CKLF
AATTCAAAAACCTTTGCTTGTGTTTGCACTTCTCGAGAAGTGCAAACACAAGCAAAGG
2251
chr16
ENST00000264001.8





CKLF
AATTCAAAAACCCTGAACCATATATTGTTATCTCGAGATAACAATATATGGTTCAGGG
2252
chr16
ENST00000264001.8





CKLF
AATTCAAAAATGGATTTGAAGTCACCGTTATCTCGAGATAACGGTGACTTCAAATCCA
2253
chr16
ENST00000264001.8





CLCF1
AATTCAAAAAGAAACAAACATGGTGGCAATTCTCGAGAATTGCCACCATGTTTGTTTC
2254
chr11
ENST00000312438.7





CLCF1
AATTCAAAAACCACAGGATTTCCTGAAAGTTCTCGAGAACTTTCAGGAAATCCTGTGG
2255
chr11
ENST00000312438.7





CLCF1
AATTCAAAAACATGGTGGCAATTCTACACAACTCGAGTTGTGTAGAATTGCCACCATG
2256
chr11
ENST00000312438.7





CMTM1
AATTCAAAAACACCACTTGCTGACCTATTTACTCGAGTAAATAGGTCAGCAAGTGGTG
2257
chr16
ENST00000379500.6





CMTM1
AATTCAAAAAGACCTATTTACATTGGCCCTTCTCGAGAAGGGCCAATGTAAATAGGTC
2258
chr16
ENST00000379500.6





CMTM1
AATTCAAAAAGCTGTGTTCCTTTCAGTAGTTCTCGAGAACTACTGAAAGGAACACAGC
2259
chr16
ENST00000379500.6





CMTM2
AATTCAAAAAGTAAGGAGCCATCGGACAAACCTCGAGGTTTGTCCGATGGCTCCTTAC
2260
chr16
ENST00000268595.2





CMTM2
AATTCAAAAACTTACTTGCTGTGATCCTTATCTCGAGATAAGGATCACAGCAAGTAAG
2261
chr16
ENST00000268595.2





CMTM2
AATTCAAAAACATAAGGAGCCATCGGACAAACTCGAGTTTGTCCGATGGCTCCTTATG
2262
chr16
ENST00000268595.2





CMTM3
AATTCAAAAACATCGTGTTTGCAACTGATTTCTCGAGAAATCAGTTGCAAACACGATG
2263
chr16
ENST00000361909.8





CMTM3
AATTCAAAAAGCTTCTTAACAGATGGCATTTCTCGAGAAATGCCATCTGTTAAGAAGC
2264
chr16
ENST00000361909.8





CMTM3
AATTCAAAAAGCTTCTTAACAGATGGCATTTCTCGAGAAATGCCATCTGTTAAGAAGC
2265
chr16
ENST00000361909.8





CMTM4
AATTCAAAAACAACTGGAATCTGACAGATTTCTCGAGAAATCTGTCAGATTCCAGTTG
2266
chr16
ENST00000394106.6





CMTM4
AATTCAAAAAGACTGGCGTCTTGCTGATTATCTCGAGATAATCAGCAAGACGCCAGTC
2267
chr16
ENST00000394106.6





CMTM4
AATTCAAAAAGCAGAAATTGCTGCCGTGATACTCGAGTATCACGGCAGCAATTTCTGC
2268
chr16
ENST00000394106.6





CMTM5
AATTCAAAAACAAGATCTACCGGACTGAGATCTCGAGATCTCAGTCCGGTAGATCTTG
2269
chr14
ENST00000359320.7





CMTM5
AATTCAAAAACCTGACCCTCATCATCTTCATCTCGAGATGAAGATGATGAGGGTCAGG
2270
chr14
ENST00000359320.7





CMTM5
AATTCAAAAACTTCCTCTATGCCACCCAGTACTCGAGTACTGGGTGGCATAGAGGAAG
2271
chr14
ENST00000359320.7





CMTM6
AATTCAAAAAGCCCTCACTGAGCCACTTAATCTCGAGATTAAGTGGCTCAGTGAGGGC
2272
chr3
ENST00000205636.3





CMTM6
AATTCAAAAACCCAAGACAGTGAAAGTAATTCTCGAGAATTACTTTCACTGTCTTGGG
2273
chr3
ENST00000205636.3





CMTM6
AATTCAAAAACCCAAGACAGTGAAAGTAATTCTCGAGAATTACTTTCACTGTCTTGGG
2274
chr3
ENST00000205636.3





CMTM7
AATTCAAAAACTGCTGAAAGTGGCGCAAATGCTCGAGCATTTGCGCCACTTTCAGCAG
2275
chr3
ENST00000334983.9





CMTM7
AATTCAAAAACAAAGCCCTGTCCTAATTTATCTCGAGATAAATTAGGACAGGGCTTTG
2276
chr3
ENST00000334983.9





CMTM7
AATTCAAAAACCTGTCGATCTTTGGTTTCATCTCGAGATGAAACCAAAGATCGACAGG
2277
chr3
ENST00000334983.9





CMTM8
AATTCAAAAATGCTGGTATGGACGCTTATTGCTCGAGCAATAAGCGTCCATACCAGCA
2278
chr3
ENST00000307526.3





CMTM8
AATTCAAAAAGCAAAGTGTTGTAGCTTATAACTCGAGTTATAAGCTACAACACTTTGC
2279
chr3
ENST00000307526.3





CMTM8
AATTCAAAAAATATTCCCAGAGAATTGTATTCTCGAGAATACAATTCTCTGGGAATAT
2280
chr3
ENST00000307526.3





CNTF
AATTCAAAAAGTAACCTCTACAGGCATTTAACTCGAGTTAAATGCCTGTAGAGGTTAC
2281
chr11
ENST00000361987.5





CNTF
AATTCAAAAAGTTGAAGGACTACAGGTATTTCTCGAGAAATACCTGTAGTCCTTCAAC
2282
chr11
ENST00000361987.5





CNTF
AATTCAAAAAGGTGACTTCCATCAAGCTATACTCGAGTATAGCTTGATGGAAGTCACC
2283
chr11
ENST00000361987.5





CNTFR
AATTCAAAAACATTCTCTTCAGACACAATTTCTCGAGAAATTGTGTCTGAAGAGAATG
2284
chr9
ENST00000351266.8





CNTFR
AATTCAAAAAGCATTCTCTTCAGACACAATTCTCGAGAATTGTGTCTGAAGAGAATGC
2285
chr9
ENST00000351266.8





CNTFR
AATTCAAAAAGCCGGGAAGGAGTACATTATCCTCGAGGATAATGTACTCCTTCCCGGC
2286
chr9
ENST00000351266.8





COPS5
AATTCAAAAACTAAGGATCACCATTACTTTACTCGAGTAAAGTAATGGTGATCCTTAG
2287
chr8
ENST00000357849.8





COPS5
AATTCAAAAACCAGACTATTCCACTTAATAACTCGAGTTATTAAGTGGAATAGTCTGG
2288
chr8
ENST00000357849.8





COPS5
AATTCAAAAACAGTCTCTGAGAAGTACTTTACTCGAGTAAAGTACTTCTCAGAGACTG
2289
chr8
ENST00000357849.8





CRLF1
AATTCAAAAACGATGTACTCACGCTGGATATCTCGAGATATCCAGCGTGAGTACATCG
2290
chr19
ENST00000392386.7





CRLF1
AATTCAAAAACGATGTACTCACGCTGGATATCTCGAGATATCCAGCGTGAGTACATCG
2291
chr19
ENST00000392386.7





CRLF1
AATTCAAAAACCCAGAGAAACCCGTCAACATCTCGAGATGTTGACGGGTTTCTCTGGG
2292
chr19
ENST00000392386.7





CSF1
AATTCAAAAATCTCCTGGTACAAGACATAATCTCGAGATTATGTCTTGTACCAGGAGA
2293
chr1
ENST00000329608.10





CSF1
AATTCAAAAAGTCGGCCTGATTTCCCGTAAACTCGAGTTTACGGGAAATCAGGCCGAC
2294
chr1
ENST00000329608.10





CSF1
AATTCAAAAATTGACAAGGACTGGAATATTTCTCGAGAAATATTCCAGTCCTTGTCAA
2295
chr1
ENST00000329608.10





CSF1R
AATTCAAAAAGTGAACAGCAAGTTCTATAAACTCGAGTTTATAGAACTTGCTGTTCAC
2296
chr5
ENST00000286301.7





CSF1R
AATTCAAAAAACAGGAGAGAGCGGGACTATACTCGAGTATAGTCCCGCTCTCTCCTGT
2297
chr5
ENST00000286301.7





CSF1R
AATTCAAAAAGCTGCTATTGTACAAGTATAACTCGAGTTATACTTGTACAATAGCAGC
2298
chr5
ENST00000286301.7





CSF2
AATTCAAAAAGGAGCTGCTCTCTCATGAAACCTCGAGGTTTCATGAGAGAGCAGCTCC
2299
chr5
ENST00000296871.3





CSF2
AATTCAAAAACCCAGATTATCACCTTTGAAACTCGAGTTTCAAAGGTGATAATCTGGG
2300
chr5
ENST00000296871.3





CSF2
AATTCAAAAAGAAGTCATCTCAGAAATGTTTCTCGAGAAACATTTCTGAGATGACTTC
2301
chr5
ENST00000296871.3





CSF3
AATTCAAAAAGTCTATTTAAGCCTCATATTTCTCGAGAAATATGAGGCTTAAATAGAC
2302
chr17
ENST00000225474.6





CSF3
AATTCAAAAAGATAGGTAAATACCAAGTATTCTCGAGAATACTTGGTATTTACCTATC
2303
chr17
ENST00000225474.6





CSF3
AATTCAAAAAGCTTAGAGCAAGTGAGGAAGACTCGAGTCTTCCTCACTTGCTCTAAGC
2304
chr17
ENST00000225474.6





CSF3R
AATTCAAAAACAGCCAGGCCTGCACATAAATCTCGAGATTTATGTGCAGGCCTGGCTG
2305
chr1
ENST00000361632.8





CSF3R
AATTCAAAAACTATGCCTACTCTCAAGAAATCTCGAGATTTCTTGAGAGTAGGCATAG
2306
chr1
ENST00000361632.8





CSF3R
AATTCAAAAACCAGCTTCACTCTGAAGAGTTCTCGAGAACTCTTCAGAGTGAAGCTGG
2307
chr1
ENST00000361632.8





CTF1
AATTCAAAAAGAGCAGCTGCTCCAGGAATATCTCGAGATATTCCTGGAGCAGCTGCTC
2308
chr16
ENST00000279804.2





CTF1
AATTCAAAAAGTCTCTCCTTCCGCTTCTTTGCTCGAGCAAAGAAGCGGAAGGAGAGAC
2309
chr16
ENST00000279804.2





CTF1
AATTCAAAAATGTCTGTCTGTCTGCTCTTAGCTCGAGCTAAGAGCAGACAGACAGACA
2310
chr16
ENST00000279804.2





CX3CL1
AATTCAAAAACCCGGAGCTGTGGTAGTAATTCTCGAGAATTACTACCACAGCTCCGGG
2311
chr16
ENST00000006053.6





CX3CL1
AATTCAAAAAGCTGCTGCCCTAACTCGAAATCTCGAGATTTCGAGTTAGGGCAGCAGC
2312
chr16
ENST00000006053.6





CX3CL1
AATTCAAAAACGGTGTGACGAAATGCAACATCTCGAGATGTTGCATTTCGTCACACCG
2313
chr16
ENST00000006053.6





CX3CR1
AATTCAAAAATGGCCTGTGTCTAGTTGTTTGCTCGAGCAAACAACTAGACACAGGCCA
2314
chr3
ENST00000399220.2





CX3CR1
AATTCAAAAATGGGATCCCTCATCCTCATACCTCGAGGTATGAGGATGAGGGATCCCA
2315
chr3
ENST00000399220.2





CX3CR1
AATTCAAAAAGCTTTGCTCATCCACTATCAACTCGAGTTGATAGTGGATGAGCAAAGC
2316
chr3
ENST00000399220.2





CXCL1
AATTCAAAAAACCTGCACACTGTCCTATTATCTCGAGATAATAGGACAGTGTGCAGGT
2317
chr4
ENST00000395761.3





CXCL1
AATTCAAAAAAGATGCTGAACAGTGACAAATCTCGAGATTTGTCACTGTTCAGCATCT
2318
chr4
ENST00000395761.3





CXCL1
AATTCAAAAAGTTCTCCAGTCATTATGTTAACTCGAGTTAACATAATGACTGGAGAAC
2319
chr4
ENST00000395761.3





CXCL10
AATTCAAAAAGTATATGTCAAGCCATAATTGCTCGAGCAATTATGGCTTGACATATAC
2320
chr4
ENST00000306602.2





CXCL10
AATTCAAAAAACTCTACCCTGGCACTATAATCTCGAGATTATAGTGCCAGGGTAGAGT
2321
chr4
ENST00000306602.2





CXCL10
AATTCAAAAACCTGTTAATCCAAGGTCTTTACTCGAGTAAAGACCTTGGATTAACAGG
2322
chr4
ENST00000306602.2





CXCL11
AATTCAAAAAGCTGGTTACCATCGGAGTTTACTCGAGTAAACTCCGATGGTAACCAGC
2323
chr4
ENST00000306621.7





CXCL11
AATTCAAAAAGCAGTGAAAGTGGCAGATATTCTCGAGAATATCTGCCACTTTCACTGC
2324
chr4
ENST00000306621.7





CXCL11
AATTCAAAAAGAAGCAAGCAAGGCTTATAATCTCGAGATTATAAGCCTTGCTTGCTTC
2325
chr4
ENST00000306621.7





CXCL12
AATTCAAAAAGCTTAGACTAAGGCCATTATTCTCGAGAATAATGGCCTTAGTCTAAGC
2326
chr10
ENST00000343575.10





CXCL12
AATTCAAAAACTCTCACTATACCAGTATAATCTCGAGATTATACTGGTATAGTGAGAG
2327
chr10
ENST00000343575.10





CXCL12
AATTCAAAAACAAACTGTGCCCTTCAGATTGCTCGAGCAATCTGAAGGGCACAGTTTG
2328
chr10
ENST00000343575.10





CXCL13
AATTCAAAAAGATTCCCTGATGCTGATATTTCTCGAGAAATATCAGCATCAGGGAATC
2329
chr4
ENST00000286758.4





CXCL13
AATTCAAAAAAGGAATCCATGTAGTAGATATCTCGAGATATCTACTACATGGATTCCT
2330
chr4
ENST00000286758.4





CXCL13
AATTCAAAAAAGGTGTTCTGGAGGTCTATTACTCGAGTAATAGACCTCCAGAACACCT
2331
chr4
ENST00000286758.4





CXCL14
AATTCAAAAAATTTGTCCATACGTCACTATACTCGAGTATAGTGACGTATGGACAAAT
2332
chr5
ENST00000337225.5





CXCL14
AATTCAAAAACAAAGGACTTTGCAGATTAAACTCGAGTTTAATCTGCAAAGTCCTTTG
2333
chr5
ENST00000337225.5





CXCL14
AATTCAAAAAGCGCAGGGTCTACGAAGAATACTCGAGTATTCTTCGTAGACCCTGCGC
2334
chr5
ENST00000337225.5





CXCL16
AATTCAAAAAACATCCAGCCTACACGTATTTCTCGAGAAATACGTGTAGGCTGGATGT
2335
chr17
ENST00000293778.10





CXCL16
AATTCAAAAATCTGAAGGTGCGAGGATTATACTCGAGTATAATCCTCGCACCTTCAGA
2336
chr17
ENST00000293778.10





CXCL16
AATTCAAAAATCCAGATCTGCCGGTTCATTACTCGAGTAATGAACCGGCAGATCTGGA
2337
chr17
ENST00000293778.10





CXCL17
AATTCAAAAAGCGCCCACTCTTCCAATTAAACTCGAGTTTAATTGGAAGAGTGGGCGC
2338
chr19
ENST00000601181.5





CXCL17
AATTCAAAAATCCAGAGCCTGCCAGCAATTTCTCGAGAAATTGCTGGCAGGCTCTGGA
2339
chr19
ENST00000601181.5





CXCL17
AATTCAAAAAAGAATGTGAGTGCAAAGATTGCTCGAGCAATCTTTGCACTCACATTCT
2340
chr19
ENST00000601181.5





CXCL2
AATTCAAAAACTTGCACACTCTCCCATTATACTCGAGTATAATGGGAGAGTGTGCAAG
2341
chr4
ENST00000508487.2





CXCL2
AATTCAAAAAGCAGATATTCTCTAGTCATTTCTCGAGAAATGACTAGAGAATATCTGC
2342
chr4
ENST00000508487.2





CXCL2
AATTCAAAAAATTTCTTCGTGATGACATATCCTCGAGGATATGTCATCACGAAGAAAT
2343
chr4
ENST00000508487.2





CXCL3
AATTCAAAAATTACGAGGGTTCTACTTATTTCTCGAGAAATAAGTAGAACCCTCGTAA
2344
chr4
ENST00000296026.4





CXCL3
AATTCAAAAAATTTAGTGGGAGACCATAATGCTCGAGCATTATGGTCTCCCACTAAAT
2345
chr4
ENST00000296026.4





CXCL3
AATTCAAAAAACTGACAGGAGAGAAGTAAGACTCGAGTCTTACTTCTCTCCTGTCAGT
2346
chr4
ENST00000296026.4





CXCL5
AATTCAAAAATGAATTGTAGGTGACTATTATCTCGAGATAATAGTCACCTACAATTCA
2347
chr4
ENST00000296027.4





CXCL5
AATTCAAAAACAGACCACGCAAGGAGTTCATCTCGAGATGAACTCCTTGCGTGGTCTG
2348
chr4
ENST00000296027.4





CXCL5
AATTCAAAAACGGGAAGGAAATTTGTCTTGACTCGAGTCAAGACAAATTTCCTTCCCG
2349
chr4
ENST00000296027.4





CXCL6
AATTCAAAAATTTACCCTAGGATGCTATTTACTCGAGTAAATAGCATCCTAGGGTAAA
2350
chr4
ENST00000226317.9





CXCL6
AATTCAAAAAGTTGCACTTGTTTACGCGTTACTCGAGTAACGCGTAAACAAGTGCAAC
2351
chr4
ENST00000226317.9





CXCL6
AATTCAAAAAGCTGTGGATTTCGTATGGAAACTCGAGTTTCCATACGAAATCCACAGC
2352
chr4
ENST00000226317.9





CXCL8
AATTCAAAAATGCGCCAACACAGAAATTATTCTCGAGAATAATTTCTGTGTTGGCGCA
2353
chr4
ENST00000307407.7





CXCL8
AATTCAAAAACAAGAGAATATCCGAACTTTACTCGAGTAAAGTTCGGATATTCTCTTG
2354
chr4
ENST00000307407.7





CXCL8
AATTCAAAAATGCACGGGAGAATATACAAATCTCGAGATTTGTATATTCTCCCGTGCA
2355
chr4
ENST00000307407.7





CXCL9
AATTCAAAAACCAAAGGAGGATGGCATATAACTCGAGTTATATGCCATCCTCCTTTGG
2356
chr4
ENST00000264888.5





CXCL9
AATTCAAAAACCAAACGTTAAGAATTGTTAACTCGAGTTAACAATTCTTAACGTTTGG
2357
chr4
ENST00000264888.5





CXCL9
AATTCAAAAAGATGTGAAGGAACTGATTAAACTCGAGTTTAATCAGTTCCTTCACATC
2358
chr4
ENST00000264888.5





CXCR1
AATTCAAAAACTTGGCACGTCATCGTGTTACCTCGAGGTAACACGATGACGTGCCAAG
2359
chr2
ENST00000295683.2





CXCR1
AATTCAAAAAACCCACTAACTGGCTAATTAGCTCGAGCTAATTAGCCAGTTAGTGGGT
2360
chr2
ENST00000295683.2





CXCR1
AATTCAAAAAGAGACACTCAACAAGTATGTTCTCGAGAACATACTTGTTGAGTGTCTC
2361
chr2
ENST00000295683.2





CXCR2
AATTCAAAAAGAAGCGCTACTTGGTCAAATTCTCGAGAATTTGACCAAGTAGCGCTTC
2362
chr2
ENST00000318507.6





CXCR2
AATTCAAAAAGCCACTAAATTGACACTTAAACTCGAGTTTAAGTGTCAATTTAGTGGC
2363
chr2
ENST00000318507.6





CXCR2
AATTCAAAAACCCTGGAAATCAACAAGTATTCTCGAGAATACTTGTTGATTTCCAGGG
2364
chr2
ENST00000318507.6





CXCR3
AATTCAAAAACCTTCTCATTTGGAAACTAAACTCGAGTTTAGTTTCCAAATGAGAAGG
2365
chrX
ENST00000373693.3





CXCR3
AATTCAAAAACGCTACCTGAACATAGTTCATCTCGAGATGAACTATGTTCAGGTAGCG
2366
chrX
ENST00000373693.3





CXCR3
AATTCAAAAAGAGTACAAGGCATGGCGTAGACTCGAGTCTACGCCATGCCTTGTACTC
2367
chrX
ENST00000373693.3





CXCR4
AATTCAAAAACTATTCCCGACTTCATCTTTGCTCGAGCAAAGATGAAGTCGGGAATAG
2368
chr2
ENST00000409817.1





CXCR4
AATTCAAAAAGCGTGTAGTGAATCACGTAAACTCGAGTTTACGTGATTCACTACACGC
2369
chr2
ENST00000409817.1





CXCR4
AATTCAAAAACCTGTTCTTAAGACGTGATTTCTCGAGAAATCACGTCTTAAGAACAGG
2370
chr2
ENST00000409817.1





CXCR6
AATTCAAAAAGCTTGCTCATCTGGGTGATATCTCGAGATATCACCCAGATGAGCAAGC
2371
chr3
ENST00000304552.4





CXCR6
AATTCAAAAATACTGGGCATCTACACTATTACTCGAGTAATAGTGTAGATGCCCAGTA
2372
chr3
ENST00000304552.4





CXCR6
AATTCAAAAATTATCTATGGCAATGTCTTTACTCGAGTAAAGACATTGCCATAGATAA
2373
chr3
ENST00000304552.4





EBI3
AATTCAAAAATGAACTGTCACTGTGAGATATCTCGAGATATCTCACAGTGACAGTTCA
2374
chr19
ENST00000221847.5





EBI3
AATTCAAAAAATGTACTACTCTCTCCTTTACCTCGAGGTAAAGGAGAGAGTAGTACAT
2375
chr19
ENST00000221847.5





EBI3
AATTCAAAAAGCCTTTCATAACAGAGCACATCTCGAGATGTGCTCTGTTATGAAAGGC
2376
chr19
ENST00000221847.5





EDN1
AATTCAAAAACCATGAGAAACAGCGTCAAATCTCGAGATTTGACGCTGTTTCTCATGG
2377
chr6
ENST00000379375.5





EDN1
AATTCAAAAAGCTCGTCCCTGATGGATAAAGCTCGAGCTTTATCCATCAGGGACGAGC
2378
chr6
ENST00000379375.5





EDN1
AATTCAAAAAAGACAAGAAGTGCTGGAATTTCTCGAGAAATTCCAGCACTTCTTGTCT
2379
chr6
ENST00000379375.5





ELANE
AATTCAAAAAGCACTGCGTGGCGAATGTAAACTCGAGTTTACATTCGCCACGCAGTGC
2380
chr19
ENST00000263621.1





ELANE
AATTCAAAAATGCTCAACGACATCGTGATTCCTCGAGGAATCACGATGTCGTTGAGCA
2381
chr19
ENST00000263621.1





ELANE
AATTCAAAAACAACGGGCTAATCCACGGAATCTCGAGATTCCGTGGATTAGCCCGTTG
2382
chr19
ENST00000263621.1





ENG
AATTCAAAAAGCAGGTGTCAGCAAGTATGATCTCGAGATCATACTTGCTGACACCTGC
2383
chr9
ENST00000344849.4





ENG
AATTCAAAAAGCAGGTGTCAGCAAGTATGATCTCGAGATCATACTTGCTGACACCTGC
2384
chr9
ENST00000344849.4





ENG
AATTCAAAAAGTCTTGCAGAAACAGTCCATTCTCGAGAATGGACTGTTTCTGCAAGAC
2385
chr9
ENST00000344849.4





EPO
AATTCAAAAACCCAGACACCAAAGTTAATTTCTCGAGAAATTAACTTTGGTGTCTGGG
2386
chr7
ENST00000252723.2





EPO
AATTCAAAAATGCAGCTGCATGTGGATAAAGCTCGAGCTTTATCCACATGCAGCTGCA
2387
chr7
ENST00000252723.2





EPO
AATTCAAAAAAGAGCAACTCTGAGATCTAAGCTCGAGCTTAGATCTCAGAGTTGCTCT
2388
chr7
ENST00000252723.2





FAM3B
AATTCAAAAATTGGAACTCCCTTCCGAAATTCTCGAGAATTTCGGAAGGGAGTTCCAA
2389
chr21
ENST00000357985.6





FAM3B
AATTCAAAAATAAATCCAACAGCCCATATTTCTCGAGAAATATGGGCTGTTGGATTTA
2390
chr21
ENST00000357985.6





FAM3B
AATTCAAAAATCAGGTCTAGCTGGGTATTTACTCGAGTAAATACCCAGCTAGACCTGA
2391
chr21
ENST00000357985.6





FAM3C
AATTCAAAAACTTGGTGTGTGCATGAGTATTCTCGAGAATACTCATGCACACACCAAG
2392
chr7
ENST00000359943.7





FAM3C
AATTCAAAAAGAGGAGATGTGGCACCATTTACTCGAGTAAATGGTGCCACATCTCCTC
2393
chr7
ENST00000359943.7





FAM3C
AATTCAAAAAGCCATACAAGATGGAACAATACTCGAGTATTGTTCCATCTTGTATGGC
2394
chr7
ENST00000359943.7





FAM3D
AATTCAAAAACACCTAGTGAAATTCCTTAAACTCGAGTTTAAGGAATTTCACTAGGTG
2395
chr3
ENST00000358781.6





FAM3D
AATTCAAAAACATGTACTCTGGAGATGTTATCTCGAGATAACATCTCCAGAGTACATG
2396
chr3
ENST00000358781.6





FAM3D
AATTCAAAAACAATGTGGGCAGAGGCCTAAACTCGAGTTTAGGCCTCTGCCCACATTG
2397
chr3
ENST00000358781.6





FAS
AATTCAAAAAGCGTATGACACATTGATTAAACTCGAGTTTAATCAATGTGTCATACGC
2398
chr10
ENST00000355740.6





FAS
AATTCAAAAACCTGAAACAGTGGCAATAAATCTCGAGATTTATTGCCACTGTTTCAGG
2399
chr10
ENST00000355740.6





FAS
AATTCAAAAACTATCATCCTCAAGGACATTACTCGAGTAATGTCCTTGAGGATGATAG
2400
chr10
ENST00000355740.6





FASLG
AATTCAAAAAGACTAGAGGCTTGCATAATAACTCGAGTTATTATGCAAGCCTCTAGTC
2401
chr1
ENST00000367721.2





FASLG
AATTCAAAAAACTGGGCTGTACTTTGTATATCTCGAGATATACAAAGTACAGCCCAGT
2402
chr1
ENST00000367721.2





FASLG
AATTCAAAAATGAGCTCTCTCTGGTCAATTTCTCGAGAAATTGACCAGAGAGAGCTCA
2403
chr1
ENST00000367721.2





FGF2
AATTCAAAAAGAAACGAACTGGGCAGTATAACTCGAGTTATACTGCCCAGTTCGTTTC
2404
chr4
ENST00000608478.1





FGF2
AATTCAAAAATATAGCTCAGTTTGGATAATTCTCGAGAATTATCCAAACTGAGCTATA
2405
chr4
ENST00000608478.1





FGF2
AATTCAAAAATGAACGATTGGAATCTAATAACTCGAGTTATTAGATTCCAATCGTTCA
2406
chr4
ENST00000608478.1





FLT3LG
AATTCAAAAACTGTCTGACTACCTGCTTCAACTCGAGTTGAAGCAGGTAGTCAGACAG
2407
chr19
ENST00000594009.5





FLT3LG
AATTCAAAAATCCTCCGACTTCGCTGTCAAACTCGAGTTTGACAGCGAAGTCGGAGGA
2408
chr19
ENST00000594009.5





FLT3LG
AATTCAAAAAGCTTCGTCCAGACCAACATCTCTCGAGAGATGTTGGTCTGGACGAAGC
2409
chr19
ENST00000594009.5





FOXP3
AATTCAAAAAAGCTGGAGTTCCGCAAGAAACCTCGAGGTTTCTTGCGGAACTCCAGCT
2410
chrX
ENST00000376207.8





FOXP3
AATTCAAAAATCCTACCCACTGCTGGCAAATCTCGAGATTTGCCAGCAGTGGGTAGGA
2411
chrX
ENST00000376207.8





FOXP3
AATTCAAAAATGTCCCTCACTCAACACAAACCTCGAGGTTTGTGTTGAGTGAGGGACA
2412
chrX
ENST00000376207.8





FOXP3
AATTCAAAAACACACGCATGTTTGCCTTCTTCTCGAGAAGAAGGCAAACATGCGTGTG
2413
chrX
ENST00000376207.8





FZD4
AATTCAAAAACGTGTGTGATTGCCTGTTATTCTCGAGAATAACAGGCAATCACACACG
2414
chr11
ENST00000531380.1





FZD4
AATTCAAAAATCTCAGTATGTGCTATAATATCTCGAGATATTATAGCACATACTGAGA
2415
chr11
ENST00000531380.1





FZD4
AATTCAAAAATTCTCAGTATGTGCTATAATACTCGAGTATTATAGCACATACTGAGAA
2416
chr11
ENST00000531380.1





GATA3
AATTCAAAAAAGCCTAAACGCGATGGATATACTCGAGTATATCCATCGCGTTTAGGCT
2417
chr10
ENST00000346208.4





GATA3
AATTCAAAAACCCAAGAACAGCTCGTTTAACCTCGAGGTTAAACGAGCTGTTCTTGGG
2418
chr10
ENST00000346208.4





GATA3
AATTCAAAAAGCCAAGAAGTTTAAGGAATATCTCGAGATATTCCTTAAACTTCTTGGC
2419
chr10
ENST00000346208.4





GATA3
AATTCAAAAACCCTGTAATTGTTGTTTGTATCTCGAGATACAAACAACAATTACAGGG
2420
chr10
ENST00000346208.4





GBP1
AATTCAAAAACCAGATGAGTACCTGACATACCTCGAGGTATGTCAGGTACTCATCTGG
2421
chr1
ENST00000370473.4





GBP1
AATTCAAAAACGACGAAAGGCATGTACCATACTCGAGTATGGTACATGCCTTTCGTCG
2422
chr1
ENST00000370473.4





GBP1
AATTCAAAAACGACGAAAGGCATGTACCATACTCGAGTATGGTACATGCCTTTCGTCG
2423
chr1
ENST00000370473.4





GDF1
AATTCAAAAAGTTCACCAAGCTCAACATTTACTCGAGTAAATGTTGAGCTTGGTGAAC
2424
chr19
ENST00000247005.7





GDF1
AATTCAAAAAGAGTTCACCAAGCTCAACATTCTCGAGAATGTTGAGCTTGGTGAACTC
2425
chr19
ENST00000247005.7





GDF1
AATTCAAAAACCCTTATGAACCTCTACTGGTCTCGAGACCAGTAGAGGTTCATAAGGG
2426
chr19
ENST00000247005.7





GDF10
AATTCAAAAACAGGATAATCGTGGTGTAAATCTCGAGATTTACACCACGATTATCCTG
2427
chr10
ENST00000580279.1





GDF10
AATTCAAAAACGACCAGAAGGCCGTGTATTTCTCGAGAAATACACGGCCTTCTGGTCG
2428
chr10
ENST00000580279.1





GDF10
AATTCAAAAACTGTCCGCCAGTGCATCATTACTCGAGTAATGATGCACTGGCGGACAG
2429
chr10
ENST00000580279.1





GDF11
AATTCAAAAACCTGCAGATCTTGCGACTAAACTCGAGTTTAGTCGCAAGATCTGCAGG
2430
chr12
ENST00000257868.9





GDF11
AATTCAAAAAGATCGCTGTGGCTGCTCTTAACTCGAGTTAAGAGCAGCCACAGCGATC
2431
chr12
ENST00000257868.9





GDF11
AATTCAAAAAGAGATGTAGAGACAGTGATAGCTCGAGCTATCACTGTCTCTACATCTC
2432
chr12
ENST00000257868.9





GDF15
AATTCAAAAAAGACTCCAGATTCCGAGAGTTCTCGAGAACTCTCGGAATCTGGAGTCT
2433
chr19
ENST00000252809.3





GDF15
AATTCAAAAAGCTCCAGACCTATGATGACTTCTCGAGAAGTCATCATAGGTCTGGAGC
2434
chr19
ENST00000252809.3





GDF15
AATTCAAAAAAGAGCTGGGAAGATTCGAACACTCGAGTGTTCGAATCTTCCCAGCTCT
2435
chr19
ENST00000252809.3





GDF2
AATTCAAAAACAACAGGTACACGTCCGATAACTCGAGTTATCGGACGTGTACCTGTTG
2436
chr10
ENST00000581492.2





GDF2
AATTCAAAAATGAAAGGAAGCGTGGTCATTTCTCGAGAAATGACCACGCTTCCTTTCA
2437
chr10
ENST00000581492.2





GDF2
AATTCAAAAATATGAAGCCTACGAGTGTAAGCTCGAGCTTACACTCGTAGGCTTCATA
2438
chr10
ENST00000581492.2





GDF3
AATTCAAAAAACCGTCACCAGCTATTCATTACTCGAGTAATGAATAGCTGGTGACGGT
2439
chr12
ENST00000329913.3





GDF3
AATTCAAAAACTCTCAACAGCTCCAATTATGCTCGAGCATAATTGGAGCTGTTGAGAG
2440
chr12
ENST00000329913.3





GDF3
AATTCAAAAATTGGGCCAGGCAGTCCAATTTCTCGAGAAATTGGACTGCCTGGCCCAA
2441
chr12
ENST00000329913.3





GDF5
AATTCAAAAACAACACCATCACCAGCTTTATCTCGAGATAAAGCTGGTGATGGTGTTG
2442
chr20
ENST00000374369.7





GDF5
AATTCAAAAAATGAGACTCAGCCCACCATTTCTCGAGAAATGGTGGGCTGAGTCTCAT
2443
chr20
ENST00000374369.7





GDF5
AATTCAAAAATGAGTGTGACTTGGGCTAAAGCTCGAGCTTTAGCCCAAGTCACACTCA
2444
chr20
ENST00000374369.7





GDF6
AATTCAAAAAGACTCCCATCAGCATTCTATACTCGAGTATAGAATGCTGATGGGAGTC
2445
chr8
ENST00000287020.6





GDF6
AATTCAAAAACGAGTACATGCTGTCAATCTACTCGAGTAGATTGACAGCATGTACTCG
2446
chr8
ENST00000287020.6





GDF6
AATTCAAAAACAGTCTTCCAAGTCGGCTAATCTCGAGATTAGCCGACTTGGAAGACTG
2447
chr8
ENST00000287020.6





GDF7
AATTCAAAAAGCAGAGGAAAGAGAGCTTATTCTCGAGAATAAGCTCTCTTTCCTCTGC
2448
chr2
ENST00000272224.4





GDF7
AATTCAAAAAGTTCGACGTGTCCAGCCTTAACTCGAGTTAAGGCTGGACACGTCGAAC
2449
chr2
ENST00000272224.4





GDF7
AATTCAAAAACCACTTCATGATGTCGCTTTACTCGAGTAAAGCGACATCATGAAGTGG
2450
chr2
ENST00000272224.4





GDF9
AATTCAAAAAGATGGCTCAATTGCCTATAAACTCGAGTTTATAGGCAATTGAGCCATC
2451
chr5
ENST00000378673.2





GDF9
AATTCAAAAACCATCAGTGGAACTGCTATTTCTCGAGAAATAGCAGTTCCACTGATGG
2452
chr5
ENST00000378673.2





GDF9
AATTCAAAAAGAGTGAATACTTCAGACAATTCTCGAGAATTGTCTGAAGTATTCACTC
2453
chr5
ENST00000378673.2





GPI
AATTCAAAAACGTCTGGTATGTCTCCAACATCTCGAGATGTTGGAGACATACCAGACG
2454
chr19
ENST00000356487.9





GPI
AATTCAAAAACGTCTGGTATGTCTCCAACATCTCGAGATGTTGGAGACATACCAGACG
2455
chr19
ENST00000356487.9





GPI
AATTCAAAAAGCGGATGTTCAATGGTGAGAACTCGAGTTCTCACCATTGAACATCCGC
2456
chr19
ENST00000356487.9





GREM1
AATTCAAAAAGCAGTGTCGTTGCATATCCATCTCGAGATGGATATGCAACGACACTGC
2457
chr15
ENST00000622074.1





GREM1
AATTCAAAAAACAGCCACCTACCAAGAAGAACTCGAGTTCTTCTTGGTAGGTGGCTGT
2458
chr15
ENST00000622074.1





GREM1
AATTCAAAAACAACAGTCGCACCATCATCAACTCGAGTTGATGATGGTGCGACTGTTG
2459
chr15
ENST00000622074.1





GREM2
AATTCAAAAAGCTGTGAAGGAAGGAAATTTACTCGAGTAAATTTCCTTCCTTCACAGC
2460
chr1
ENST00000318160.4





GREM2
AATTCAAAAACAAGGTGCATTTCTGTCATTTCTCGAGAAATGACAGAAATGCACCTTG
2461
chr1
ENST00000318160.4





GREM2
AATTCAAAAACGGAAGTAGACGTAACTTATTCTCGAGAATAAGTTACGTCTACTTCCG
2462
chr1
ENST00000318160.4





GRN
AATTCAAAAAGCCCTGATAGTCAGTTCGAATCTCGAGATTCGAACTGACTATCAGGGC
2463
chr17
ENST00000053867.7





GRN
AATTCAAAAAGCCCTGATAGTCAGTTCGAATCTCGAGATTCGAACTGACTATCAGGGC
2464
chr17
ENST00000053867.7





GRN
AATTCAAAAACTTCCAAAGATCAGGTAACAACTCGAGTTGTTACCTGATCTTTGGAAG
2465
chr17
ENST00000053867.7





HAX1
AATTCAAAAAACAGACACTTCGGGACTCAATCTCGAGATTGAGTCCCGAAGTGTCTGT
2466
chr1
ENST00000328703.11





HAX1
AATTCAAAAACCAGCCCAAATCCTATTTCAACPCGAGTTGAAATAGGATTTGGGCTGG
2467
chr1
ENST00000328703.11





HAX1
AATTCAAAAACCAGAGGCCATTTCATAGGTTCTCGAGAACCTATGAAATGGCCTCTGG
2468
chr1
ENST00000328703.11





HFE2
AATTCAAAAAGACATGATCATTAGCCATAAGCTCGAGCTTATGGCTAATGATCATGTC
2469
chr1
ENST00000336751.10





HFE2
AATTCAAAAAGCCTACATTGGCACAACTATACTCGAGTATAGTTGTGCCAATGTAGGC
2470
chr1
ENST00000336751.10





HFE2
AATTCAAAAAGAAGCTCACCATCATATTTAACTCGAGTTAAATATGATGGTGAGCTTC
2471
chr1
ENST00000336751.10





HMGB1
AATTCAAAAAGATGCAGCTTATACGAAATAACTCGAGTTATTTCGTATAAGCTGCATC
2472
chr13
ENST00000339872.8





HMGB1
AATTCAAAAAGTTGGTGCACAGCACAAATTACTCGAGTAATTTGTGCTGTGCACCAAC
2473
chr13
ENST00000339872.8





HMGB1
AATTCAAAAAAGAAGATGATGATGATGAATACTCGAGTATTCATCATCATCATCTTCT
2474
chr13
ENST00000339872.8





HYAL2
AATTCAAAAACCTGCCAGTACCTCAAAGATTCTCGAGAATCTTTGAGGTACTGGCAGG
2475
chr3
ENST00000357750.8





HYAL2
AATTCAAAAACCTGCCAGTACCTCAAAGATTCTCGAGAATCTTTGAGGTACTGGCAGG
2476
chr3
ENST00000357750.8





HYAL2
AATTCAAAAACTGGACCTGAATGCCTTTGATCTCGAGATCAAAGGCATTCAGGTCCAG
2477
chr3
ENST00000357750.8





ICAM3
AATTCAAAAAGTCCAGCTCACGAGGCAAATACTCGAGTATTTGCCTCGTGAGCTGGAC
2478
chr19
ENST00000160262.9





ICAM3
AATTCAAAAACCAGCTCAACTTCAGCTAAATCTCGAGATTTAGCTGAAGTTGAGCTGG
2479
chr19
ENST00000160262.9





ICAM3
AATTCAAAAAGCACTTGAAATGGAAAGATAACTCGAGTTATCTTTCCATTTCAAGTGC
2480
chr19
ENST00000I60262.9





ICAM3
AATTCAAAAAGAGCGGCAGTTACCATGTTAGCTCGAGCTAACATGGTAACTGCCGCTC
2481
chr19
ENST00000160262.9





ICOS
AATTCAAAAAGCACGACCCTAACGGTGAATACTCGAGTATTCACCGTTAGGGTCGTGC
2482
ch2
ENST00000316386.10





ICOS
AATTCAAAAAGTCCGCATTTCACTATCATACCTCGAGGTATGATAGTGAAATGCGGAC
2483
ch2
ENST00000316386.10





ICOS
AATTCAAAAACCATTCTCATGCCAACTATTACTCGAGTAATAGTTGGCATGAGAATGG
2484
ch2
ENST00000316386.10





ICOS
AATTCAAAAACACAGATGTGACCCTATAATACTCGAGTATTATAGGGTCACATCTGTG
2485
ch2
ENST00000316386.10





IFNA10
AATTCAAAAATGTAAAGAAGTGTCGTGTATACTCGAGTATACACGACACTTCTTTACA
2486
chr9
ENST00000357374.2





IFNA10
AATTCAAAAAATAACCACGACGCGTTGAATCCTCGAGGATTCAACGCGTCGTGGTTAT
2487
chr9
ENST00000357374.2





IFNA10
AATTCAAAAACCTGGGACAAATGGGAAGAATCTCGAGATTCTTCCCATTTGTCCCAGG
2488
chr9
ENST00000357374.2





IFNA14
AATTCAAAAACTGGGCTGTAATCTGTCTCAACTCGAGTTGAGACAGATTACAGCCCAG
2489
chr9
ENST00000380222.3





IFNA14
AATTCAAAAACCTGAATAACAGGAGGACTTTCTCGAGAAAGTCCTCCTGTTATTCAGG
2490
chr9
ENST00000380222.3





IFNA14
AATTCAAAAAGCAGCAGACCTTCAATCTCTTCTCGAGAAGAGATTGAAGGTCTGCTGC
2491
chr9
ENST00000380222.3





IFNA16
AATTCAAAAATGTAAAGAAGCATCGTGTTTACTCGAGTAAACACGATGCTTCTTTACA
2492
chr9
ENST00000380216.1





IFNA16
AATTCAAAAAATGATCCTCATTGATTAATACCTCGAGGTATTAATCAATGAGGATCAT
2493
chr9
ENST00000380216.1





IFNA16
AATTCAAAAACCTGAAGGACAGATATGATTTCTCGAGAAATCATATCTGTCCTTCAGG
2494
chr9
ENST00000380216.1





IFNA2
AATTCAAAAAGCACAGTGGTTAATGTAATAACTCGAGTTATTACATTAACCACTGTGC
2495
chr9
ENST00000380206.3





IFNA2
AATTCAAAAATATGACCATGACACGATTTAACTCGAGTTAAATCGTGTCATGGTCATA
2496
chr9
ENST00000380206.3





IFNA2
AATTCAAAAACCATGCTGACTGATCCATTATCTCGAGATAATGGATCAGTCAGCATGG
2497
chr9
ENST00000380206.3





IFNA5
AATTCAAAAAACTTGGGATGAGACACTTCTACTCGAGTAGAAGTGTCTCATCCCAAGT
2498
chr9
ENST00000610521.1





IFNA5
AATTCAAAAAAGTGGAAGACACTCCTCTGATCTCGAGATCAGAGGAGTGTCTTCCACT
2499
chr9
ENST00000610521.1





IFNA5
AATTCAAAAATCAACTGCAAGTCAATCTGTTCTCGAGAACAGATTGACTTGCAGTTGA
2500
chr9
ENST00000610521.1





IFNA6
AATTCAAAAAGACAGACATGACTTCAGATTTCTCGAGAAATCTGAAGTCATGTCTGTC
2501
chr9
ENST00000380210.1





IFNA6
AATTCAAAAACTGTCCTCCATGAGGTGATTCCTCGAGGAATCACCTCATGGAGGACAG
2502
chr9
ENST00000380210.1





IFNA6
AATTCAAAAAAGGCTTCTAGACAAACTCTATCTCGAGATAGAGTTTGTCTAGAAGCCT
2503
chr9
ENST00000380210.1





IFNA8
AATTCAAAAATAACTATCTATAGGGCTTAAACTCGAGTTTAAGCCCTATAGATAGTTA
2504
chr9
ENST00000380205.1





IFNA8
AATTCAAAAACCAGGAGGAGTTTGATGATAACTCGAGTTATCATCAAACTCCTCCTGG
2505
chr9
ENST00000380205.1





IFNA8
AATTCAAAAAGACCTGGTACAACACGGAAATCTCGAGATTTCCGTGTTGTACCAGGTC
2506
chr9
ENST00000380205.1





IFNAR1
AATTCAAAAAGCCAAGATTCAGGAAATTATTCTCGAGAATAATTTCCTGAATCTTGGC
2507
chr21
ENST00000270139.7





IFNAR1
AATTCAAAAAGCTCTCCCGTTTGTCATTTATCTCGAGATAAATGACAAACGGGAGAGC
2508
chr21
ENST00000270139.7





IFNAR1
AATTCAAAAAATGAACTGTGTCAAGTATAAGCTCGAGCTTATACTTGACACAGTTCAT
2509
chr21
ENST00000270139.7





IFNAR2
AATTCAAAAAGAGTGGAAATTTCACCTATATCTCGAGATATAGGTGAAATTTCCACTC
2510
chr21
ENST00000342136.8





IFNAR2
AATTCAAAAATGTATATCAGCCTCGTGTTTGCTCGAGCAAACACGAGGCTGATATACA
2511
chr21
ENST00000342136.8





IFNAR2
AATTCAAAAAGCAAATACCACAAGATCATTTCTCGAGAAATGATCTTGTGGTATTTGC
2512
chr21
ENST00000342136.8





IFNB1
AATTCAAAAAATTGAATGGGAGGCTTGAATACTCGAGTATTCAAGCCTCCCATTCAAT
2513
chr9
ENST00000380232.3





IFNB1
AATTCAAAAACCTACAAAGAAGCAGCAATTTCTCGAGAAATTGCTGCTTCTTTGTAGG
2514
chr9
ENST00000380232.3





IFNB1
AATTCAAAAACTAATGTCTATCATCAGATAACTCGAGTTATCTGATGATAGACATTAG
2515
chr9
ENST00000380232.3





IFNE
AATTCAAAAAGGTAGTGATAACCTTAGATTACTCGAGTAATCTAAGGTTATCACTACC
2516
chr9
ENST00000448696.4





IFNE
AATTCAAAAAAGCCTCTTCAGGGCAAATATTCTCGAGAATATTTGCCCTGAAGAGGCT
2517
chr9
ENST00000448696.4





IFNE
AATTCAAAAACATAGAGTGGTAATACAATTTCTCGAGAAATTGTATTACCACTCTATG
2518
chr9
ENST00000448696.4





IFNG
AATTCAAAAAGGTTGTCCTGCCTGCAATATTCTCGAGAATATTGCAGGCAGGACAACC
2519
chr12
ENST00000229135.3





IFNG
AATTCAAAAACATTCAGATGTAGCGGATAATCTCGAGATTATCCGCTACATCTGAATG
2520
chr12
ENST00000229135.3





IFNG
AATTCAAAAATGTTACTGCCAGGACCCATATCTCGAGATATGGGTCCTGGCAGTAACA
2521
chr12
ENST00000229135.3





IFNGR1
AATTCAAAAAACGAGCAGGAAGTCGATTATGCTCGAGCATAATCGACTTCCTGCTCGT
2522
chr6
ENST00000367739.8





IFNGR1
AATTCAAAAACATGAACCCTATCGTATATTGCTCGAGCAATATACGATAGGGTTCATG
2523
chr6
ENST00000367739.8





IFNGR1
AATTCAAAAACGGAAGTGAGATCCAGTATAACTCGAGTTATACTGGATCTCACTTCCG
2524
chr6
ENST00000367739.8





IFNK
AATTCAAAAAGAGATTGTGGCTACGCAAATGCTCGAGCATTTGCGTAGCCACAATCTC
2525
chr9
ENST00000276943.2





IFNK
AATTCAAAAACTGTTCAGATTCAAGATTATTCTCGAGAATAATCTTGAATCTGAACAG
2526
chr9
ENST00000276943.2





IFNK
AATTCAAAAATCAGCCAACACACCTTCAAATCTCGAGATTTGAAGGTGTGTTGGCTGA
2527
chr9
ENST00000276943.2





IFNL1
AATTCAAAAACTCACGCGAGACCTCAAATATCTCGAGATATTTGAGGTCTCGCGTGAG
2528
chr19
ENST00000333625.2





IFNL1
AATTCAAAAAGCCACATTGGCAGGTTCAAATCTCGAGATTTGAACCTGCCAATGTGGC
2529
chr19
ENST00000333625.2





IFNL1
AATTCAAAAAGAGTTGCAGCTCTCCTGTCTTCTCGAGAAGACAGGAGAGCTGCAACTC
2530
chr19
ENST00000333625.2





IFNL3
AATTCAAAAAAGGGCCAAAGATGCCTTAGAACTCGAGTTCTAAGGCATCTTTGGCCCT
2531
chr19
ENST00000413851.2





IFNL3
AATTCAAAAAGCCTTTAAGAGGGCCAAAGATCTCGAGATCTTTGGCCCTCTTAAAGGC
2532
chr19
ENST00000413851.2





IFNL3
AATTCAAAAATGCCACATAGCCCAGTTCAAGCTCGAGCTTGAACTGGGCTATGTGGCA
2533
chr19
ENST00000413851.2





IFNW1
AATTCAAAAAAGACTCTTATTTCGGCTTTAACTCGAGTTAAAGCCGAAATAAGAGTCT
2534
chr9
ENST00000380229.3





IFNW1
AATTCAAAAATCAGTCCCTAAGATGTTATTTCTCGAGAAATAACATCTTAGGGACTGA
2535
chr9
ENST00000380229.3





IFNW1
AATTCAAAAACGGTATATTAAGCCAGTATATCTCGAGATATACTGGCTTAATATACCG
2536
chr9
ENST00000380229.3





IL10
AATTCAAAAAAGCTTCTCTGTGAACGATTTACTCGAGTAAATCGTTCACAGAGAAGCT
2537
chr1
ENST00000423557.1





IL10
AATTCAAAAAGCAGGTGAAGAATGCCTTTAACTCGAGTTAAAGGCATTCTTCACCTGC
2538
chr1
ENST00000423557.1





IL10
AATTCAAAAAGCTGGACAACTTGTTGTTAAACTCGAGTTTAACAACAAGTTGTCCAGC
2539
chr1
ENST00000423557.1





IL10RA
AATTCAAAAATCTGTCGCTTCCCGAAGTAACCTCGAGGTTACTTCGGGAAGCGACAGA
2540
chr11
ENST00000227752.7





IL10RA
AATTCAAAAAGAAACAGGATCCTCTAGAAATCTCGAGATTTCTAGAGGATCCTGTTTC
2541
chr11
ENST00000227752.7





IL10RA
AATTCAAAAAGAACTCTTTCCTGTATCATAACTCGAGTTATGATACAGGAAAGAGTTC
2542
chr11
ENST00000227752.7





IL11
AATTCAAAAAATATCCACTTGAGGGCGATTTCTCGAGAAATCGCCCTCAAGTGGATAT
2543
chr19
ENST00000264563.6





IL11
AATTCAAAAACCTTCCAAAGCCAGATCTTATCTCGAGATAAGATCTGGCTTTGGAAGG
2544
chr19
ENST00000264563.6





IL11
AATTCAAAAATGCACAGCTGAGGGACAAATTCTCGAGAATTTGTCCCTCAGCTGTGCA
2545
chr19
ENST00000264563.6





IL11RA
AATTCAAAAACGGCAGATTCCACCTATAATTCTCGAGAATTATAGGTGGAATCTGCCG
2546
chr9
ENST00000318041.13





IL11RA
AATTCAAAAATCGGCAGATTCCACCTATAATCTCGAGATTATAGGTGGAATCTGCCGA
2547
chr9
ENST00000318041.13





IL11RA
AATTCAAAAATGGAGCCAGTACCGGATTAATCTCGAGATTAATCCGGTACTGGCTCCA
2548
chr9
ENST00000318041.13





IL12A
AATTCAAAAATGATACCTCTGATCAAGTATTCTCGAGAATACTTGATCAGAGGTATCA
2549
chr3
ENST00000305579.6





IL12A
AATTCAAAAACCTGTGCCTTAGTAGTATTTACTCGAGTAAATACTACTAAGGCACAGG
2550
chr3
ENST00000305579.6





IL12A
AATTCAAAAACCTGTTTACCATTGGAATTAACTCGAGTTAATTCCAATGGTAAACAGG
2551
chr3
ENST00000305579.6





IL12B
AATTCAAAAAGAATTTGGTCCACTGATATTTCTCGAGAAATATCAGTGGACCAAATTC
2552
chr5
ENST00000231228.2





IL12B
AATTCAAAAACCATGGGCCTTCATGCTATTTCTCGAGAAATAGCATGAAGGCCCATGG
2553
chr5
ENST00000231228.2





IL12B
AATTCAAAAATTAGATGCTAAATGCTCATTGCTCGAGCAATGAGCATTTAGCATCTAA
2554
chr5
ENST00000231228.2





IL12RB1
AATTCAAAAACAGCTCTACAACTCAGTTAAACTCGAGTTTAACTGAGTTGTAGAGCTG
2555
chr19
ENST00000593993.6





IL12RB1
AATTCAAAAAGTCATCTCCTCGAACCAATTTCTCGAGAAATTGGTTCGAGGAGATGAC
2556
chr19
ENST00000593993.6





IL12RB1
AATTCAAAAACCAACGGGACCACCATGTATTCTCGAGAATACATGGTGGTCCCGTTGG
2557
chr19
ENST00000593993.6





IL13
AATTCAAAAAACTTCGAAAGCATCATTATTTCTCGAGAAATAATGATGCTTTCGAAGT
2558
chr5
ENST00000304506.7





IL13
AATTCAAAAAATTGAAGTTGCAGATTCATTTCTCGAGAAATGAATCTGCAACTTCAAT
2559
chr5
ENST00000304506.7





IL13
AATTCAAAAACCTGCTCTTACATTTAAAGAACTCGAGTTCTTTAAATGTAAGAGCAGG
2560
chr5
ENST00000304506.7





IL15
AATTCAAAAAGAAGATCTTATTCAATCTATGCTCGAGCATAGATTGAATAAGATCTTC
2561
chr4
ENST00000296545.11





IL15
AATTCAAAAATAAGGGTGATAGTCAAATTATCTCGAGATAATTTGACTATCACCCTTA
2562
chr4
ENST00000296545.11





IL15
AATTCAAAAACACTCTGCTGCTTAGACATAACTCGAGTTATGTCTAAGCAGCAGAGTG
2563
chr4
ENST00000296545.11





IL16
AATTCAAAAATGGGACCACGTGAGATCATTCCTCGAGGAATGATCTCACGTGGTCCCA
2564
chr15
ENST00000302987.8





IL16
AATTCAAAAAGTTCTGGATGAAGCAACATTACTCGAGTAATGTTGCTTCATCCAGAAC
2565
chr15
ENST00000302987.8





IL16
AATTCAAAAACCCAAACAGTGACATTTATTTCTCGAGAAATAAATGTCACTGTTTGGG
2566
chr15
ENST00000302987.8





IL17A
AATTCAAAAAATCAGTTCTGCCTAGGTAAATCTCGAGATTTACCTAGGCAGAACTGAT
2567
chr6
ENST00000340057.1





IL17A
AATTCAAAAAGAGCTATTTAAGGATCTATTTCTCGAGAAATAGATCCTTAAATAGCTC
2568
chr6
ENST00000340057.1





IL17A
AATTCAAAAAGGTCAACCTGAACATCCATAACTCGAGTTATGGATGTTCAGGTTGACC
2569
chr6
ENST00000340057.1





IL17B
AATTCAAAAATATGCCCGCATGGAGGAGTATCTCGAGATACTCCTCCATGCGGGCATA
2570
chr5
ENST00000261796.3





IL17B
AATTCAAAAAGTGTCACGGATGAAACCGTATCTCGAGATACGGTTTCATCCGTGACAC
2571
chr5
ENST00000261796.3





IL17B
AATTCAAAAAGCAGCTGTGGATGTCCAACAACTCGAGTTGTTGGACATCCACAGCTGC
2572
chr5
ENST00000261796.3





IL17C
AATTCAAAAAGCACCTCTTCCAGCCCTTAAACTCGAGTTTAAGGGCTGGAAGAGGTGC
2573
chr16
ENST00000244241.4





IL17C
AATTCAAAAACTTTGCCTTCCACACCGAGTTCTCGAGAACTCGGTGTGGAAGGCAAAG
2574
chr16
ENST00000244241.4





IL17C
AATTCAAAAAATCTCCAGCCTCAGTAGTTGGCTCGAGCCAACTACTGAGGCTGGAGAT
2575
chr16
ENST00000244241.4





IL17D
AATTCAAAAAAGAGCTACTCTGTTACATTTCCTCGAGGAAATGTAACAGAGTAGCTCT
2576
chr13
ENST00000304920.3





IL17D
AATTCAAAAACAAAGAGATAGGGACGCATATCTCGAGATATGCGTCCCTATCTCTTTG
2577
chr13
ENST00000304920.3





IL17D
AATTCAAAAAAGACAGCATCAACTCCAGCATCTCGAGATGCTGGAGTTGATGCTGTCT
2578
chr13
ENST00000304920.3





IL17F
AATTCAAAAATCATCCACCATGTGCAGTAAGCTCGAGCTTACTGCACATGGTGGATGA
2579
chr6
ENST00000336123.4





IL17F
AATTCAAAAAGTACTTGCTGCTGTCGATATTCTCGAGAATATCGACAGCAGCAAGTAC
2580
chr6
ENST00000336123.4





IL17F
AATTCAAAAACGTTTCCATGTCACGTAACATCTCGAGATGTTACGTGACATGGAAACG
2581
chr6
ENST00000336123.4





IL18
AATTCAAAAACCCGGACCATATTTATTATAACTCGAGTTATAATAAATATGGTCCGGG
2582
chr11
ENST00000280357.11





IL18
AATTCAAAAATGATTCTGACTGTAGAGATAACTCGAGTTATCTCTACAGTCAGAATCA
2583
chr11
ENST00000280357.11





IL18
AATTCAAAAATGGCAAGCTTGAATCTAAATTCTCGAGAATTTAGATTCAAGCTTGCCA
2584
chr11
ENST00000280357.11





IL18BP
AATTCAAAAAGGTCCCTTCTCTCACCAAATTCTCGAGAATTTGGTGAGAGAAGGGACC
2585
chr11
ENST00000260049.9





IL18BP
AATTCAAAAATCCCATGTCTCTGCTCATTTACTCGAGTAAATGAGCAGAGACATGGGA
2586
chr11
ENST00000260049.9





IL18BP
AATTCAAAAACTGGGCAATGGTTCCTTCATTCTCGAGAATGAAGGAACCATTGCCCAG
2587
chr11
ENST00000260049.9





IL19
AATTCAAAAAGTCCACGCTGCTGCCATTAAACTCGAGTTTAATGGCAGCAGCGTGGAC
2588
chr1
ENST00000270218.10





IL19
AATTCAAAAATCCACAGACATGCACCATATACTCGAGTATATGGTGCATGTCTGTGGA
2589
chr1
ENST00000270218.10





IL19
AATTCAAAAATGATGACAAGGAACCTGTATACTCGAGTATACAGGTTCCTTGTCATCA
2590
chr1
ENST00000270218.10





IL1A
AATTCAAAAAGTGGAACCAACACTAACATATCTCGAGATATGTTAGTGTTGGTTCCAC
2591
chr2
ENST00000263339.3





IL1A
AATTCAAAAAGCCCTCAATCAAAGTATAATTCTCGAGAATTATACTTTGATTGAGGGC
2592
chr2
ENST00000263339.3





IL1A
AATTCAAAAATATTACAGATGGGCAAATTAACTCGAGTTAATTTGCCCATCTGTAATA
2593
chr2
ENST00000263339.3





IL1B
AATTCAAAAAATCAATAACAAGCTGGAATTTCTCGAGAAATTCCAGCTTGTTATTGAT
2594
chr2
ENST00000263341.6





IL1B
AATTCAAAAAAGCAACCGCTTCCCTATTTATCTCGAGATAAATAGGGAAGCGGTTGCT
2595
chr2
ENST00000263341.6





IL1B
AATTCAAAAACTGACTTCACCATGCAATTTGCTCGAGCAAATTGCATGGTGAAGTCAG
2596
chr2
ENST00000263341.6





IL1F10
AATTCAAAAATCCTTGTGGGCTCAGTTTAATCTCGAGATTAAACTGAGCCCACAAGGA
2597
chr2
ENST00000341010.6





IL1F10
AATTCAAAAAGGTCTATGGTAGGCAGAATAACTCGAGTTATTCTGCCTACCATAGACC
2598
chr2
ENST00000341010.6





IL1F10
AATTCAAAAATGCAGACCAGAAGGCTCTATACTCGAGTATAGAGCCTTCTGGTCTGCA
2599
chr2
ENST00000341010.6





IL1R1
AATTCAAAAAGCCAAGAATACACATGGTATACTCGAGTATACCATGTGTATTCTTGGC
2600
chr2
ENST00000410023.5





IL1R1
AATTCAAAAAATAATGCACAAGCCATATTTACTCGAGTAAATATGGCTTGTGCATTAT
2601
chr2
ENST00000410023.5





IL1R1
AATTCAAAAATGGTATAGATGCAGCATATATCTCGAGATATATGCTGCATCTATACCA
2602
chr2
ENST00000410023.5





IL1R2
AATTCAAAAACAATCCCGTGTAAGGTGTTTCCTCGAGGAAACACCTTACACGGGATTG
2603
chr2
ENST00000332549.7





IL1R2
AATTCAAAAAGACCATTCCTGTGATCATTTCCTCGAGGAAATGATCACAGGAATGGTC
2604
chr2
ENST00000332549.7





IL1R2
AATTCAAAAACGTTCATCTCATACCCGCAAACTCGAGTTTGCGGGTATGAGATGAACG
2605
chr2
ENST00000332549.7





IL1RAPL1
AATTCAAAAACAAAGCAAGCGGCTGATTATTCTCGAGAATAATCAGCCGCTTGCTTTG
2606
chrX
ENST00000378993.5





IL1RAPL1
AATTCAAAAAGCCAGCGTTCTCCTTCATAAACTCGAGTTTATGAAGGAGAACGCTGGC
2607
chrX
ENST00000378993.5





IL1RAPL1
AATTCAAAAATCAAGCTCCTGACGGTCATTACTCGAGTAATGACCGTCAGGAGCTTGA
2608
chrX
ENST00000378993.5





IL1RL1
AATTCAAAAATTACACCGTGGATTGGTATTACTCGAGTAATACCAATCCACGGTGTAA
2609
chr2
ENST00000233954.5





IL1RL1
AATTCAAAAAAGTTGCTGATTCTGGTATTTACTCGAGTAAATACCAGAATCAGCAACT
2610
chr2
ENST00000233954.5





IL1RL1
AATTCAAAAACGTGAAGGAAGAGGATTTATTCTCGAGAATAAATCCTCTTCCTTCACG
2611
chr2
ENST00000233954.5





IL1RN
AATTCAAAAAGCAAGGACCAAATGTCAATTTCTCGAGAAATTGACATTTGGTCCTTGC
2612
chr2
ENST00000409930.3





IL1RN
AATTCAAAAACGTCATGGTCACCAAATTCTACTCGAGTAGAATTTGGTGACCATGACG
2613
chr2
ENST00000409930.3





IL1RN
AATTCAAAAACTGCCTCCAGAATGGTCTTTCCTCGAGGAAAGACCATTCTGGAGGCAG
2614
chr2
ENST00000409930.3





IL2
AATTCAAAAAGCTACCTATTGTAACTATTATCTCGAGATAATAGTTACAATAGGTAGC
2615
chr4
ENST00000226730.4





IL2
AATTCAAAAACAGCTACAACTGGAGCATTTACTCGAGTAAATGCTCCAGTTGTAGCTG
2616
chr4
ENST00000226730.4





IL2
AATTCAAAAATGCTGGATTTACAGATGATTTCTCGAGAAATCATCTGTAAATCCAGCA
2617
chr4
ENST00000226730.4





IL20
AATTCAAAAACTGATGCTCTGTGAGATATTTCTCGAGAAATATCTCACAGAGCATCAG
2618
chr1
ENST00000367096.7





IL20
AATTCAAAAATGGTCACAGTGTATCTTATTTCTCGAGAAATAAGATACACTGTGACCA
2619
chr1
ENST00000367096.7





IL20
AATTCAAAAAGGACTGAAGACACTCAATTTGCTCGAGCAAATTGAGTGTCTTCAGTCC
2620
chr1
ENST00000367096.7





IL20RA
AATTCAAAAACCAGTATTATGCCAAAGTTAACTCGAGTTAACTTTGGCATAATACTGG
2621
chr6
ENST00000316649.9





IL20RA
AATTCAAAAAGAGGGTCTTCAAGGAGTTAAACTCGAGTTTAACTCCTTGAAGACCCTC
2622
chr6
ENST00000316649.9





IL20RA
AATTCAAAAAGCTTCGCATTTGATGGAAATTCTCGAGAATTTCCATCAAATGCGAAGC
2623
chr6
ENST00000316649.9





IL20RB
AATTCAAAAACAGTGTACTATTCTGTCGAATCTCGAGATTCGACAGAATAGTACACTG
2624
chr3
ENST00000329582.8





IL20RB
AATTCAAAAACCAGAATAATCCTTGAGAGAACTCGAGTTCTCTCAAGGATTATTCTGG
2625
chr3
ENST00000329582.8





IL20RB
AATTCAAAAACTCTGTACTCTCAACCAACATCTCGAGATGTTGGTTGAGAGTACAGAG
2626
chr3
ENST00000329582.8





IL21
AATTCAAAAAAGGAAACCACCTTCCACAAATCTCGAGATTTGTGGAAGGTGGTTTCCT
2627
chr4
ENST00000264497.7





IL21
AATTCAAAAAATGACTTGGTCCCTGAATTTCCTCGAGGAAATTCAGGGACCAAGTCAT
2628
chr4
ENST00000264497.7





IL21
AATTCAAAAACTTTCAGAAGGCCCAACTAAACTCGAGTTTAGTTGGGCCTTCTGAAAG
2629
chr4
ENST00000264497.7





IL22
AATTCAAAAAAGGCTAAGCACATGTCATATTCTCGAGAATATGACATGTGCTTAGCCT
2630
chr12
ENST00000328087.5





IL22
AATTCAAAAAGTTTCCATAATCAGTACTTTACTCGAGTAAAGTACTGATTATGGAAAC
2631
chr12
ENST00000328087.5





IL22
AATTCAAAAAAGACTTTCTAAGCATAGATATCTCGAGATATCTATGCTTAGAAAGTCT
2632
chr12
ENST00000328087.5





IL22RA1
AATTCAAAAAGGACACTTTCTAGTCCTAAACCTCGAGGTTTAGGACTAGAAAGTGTCC
2633
chr1
ENST00000270800.1





IL22RA1
AATTCAAAAAAGGGACACCACAGTACCTAAACTCGAGTTTAGGTACTGTGGTGTCCCT
2634
chr1
ENST00000270800.1





IL22RA1
AATTCAAAAACTGTCCGAGATCACCTACTTACTCGAGTAAGTAGGTGATCTCGGACAG
2635
chr1
ENST00000270800.1





IL22RA2
AATTCAAAAAAGACATACAGGAACCTTATTACTCGAGTAATAAGGTTCCTGTATGTCT
2636
chr6
ENST00000349184.8





IL22RA2
AATTCAAAAACTCGTGTTTGAAGGATCTTATCTCGAGATAAGATCCTTCAAACACGAG
2637
chr6
ENST00000349184.8





IL22RA2
AATTCAAAAATGCTCCAAATTTACCATATAGCTCGAGCTATATGGTAAATTTGGAGCA
2638
chr6
ENST00000349184.8





IL23A
AATTCAAAAAAGCTGCTAGGATCGGATATTTCTCGAGAAATATCCGATCCTAGCAGCT
2639
chr12
ENST00000228534.5





IL23A
AATTCAAAAACTGTGAGCCAACAGGTTAATTCTCGAGAATTAACCTGTTGGCTCACAG
2640
chr12
ENST00000228534.5





IL23A
AATTCAAAAAGGATCCACCAGGGTCTGATTTCTCGAGAAATCAGACCCTGGTGGATCC
2641
chr12
ENST00000228534.5





IL23R
AATTCAAAAACTTTCTTTGATTGGGATATTTCTCGAGAAATATCCCAATCAAAGAAAG
2642
chr1
ENST00000347310.9





IL23R
AATTCAAAAATATCTCACCTCAAGCTATATTCTCGAGAATATAGCTTGAGGTGAGATA
2643
chr1
ENST00000347310.9





IL23R
AATTCAAAAACGACAATACTACAGTTGTATACTCGAGTATACAACTGTAGTATTGTCG
2644
chr1
ENST00000347310.9





IL24
AATTCAAAAACACAGGCGGTTTCTGCTATTCCTCGAGGAATAGCAGAAACCGCCTGTG
2645
chr1
ENST00000294984.6





IL24
AATTCAAAAAGTCAGGACTCTGAAGTCATTCCTCGAGGAATGACTTCAGAGTCCTGAC
2646
chr1
ENST00000294984.6





IL24
AATTCAAAAATCGGATGCTGAGAGCTGTTACCTCGAGGTAACAGCTCTCAGCATCCGA
2647
chr1
ENST00000294984.6





IL25
AATTCAAAAAACAGGCACTTTCTAGATATTTCTCGAGAAATATCTAGAAAGTGCCTGT
2648
chr14
ENST00000329715.2





IL25
AATTCAAAAACCACAACCAGACTGTCTTCTACTCGAGTAGAAGACAGTCTGGTTGTGG
2649
chr14
ENST00000329715.2





IL25
AATTCAAAAATCCTGTAGGGCCAGTGAAGATCTCGAGATCTTCACTGGCCCTACAGGA
2650
chr14
ENST00000329715.2





IL26
AATTCAAAAAAGCTGTTGACGCTCTCTATATCTCGAGATATAGAGAGCGTCAACAGCT
2651
chr12
ENST00000229134.4





IL26
AATTCAAAAAAGTACATTGTGTCAACTTAATCTCGAGATTAAGTTGACACAATGTACT
2652
chr12
ENST00000229134.4





IL26
AATTCAAAAACGATTCCAGAAGACCGCATAACTCGAGTTATGCGGTCTTCTGGAATCG
2653
chr12
ENST00000229134.4





IL27
AATTCAAAAAACTCCTTGGAGCTCGTCTTATCTCGAGATAAGACGAGCTCCAAGGAGT
2654
chr16
ENST00000356897.1





IL27
AATTCAAAAAACTTTAGGACTGGAGTCTTGGCTCGAGCCAAGACTCCAGTCCTAAAGT
2655
chr16
ENST00000356897.1





IL27
AATTCAAAAACATCATCAGCCTTGGACAAGGCTCGAGCCTTGTCCAAGGCTGATGATG
2656
chr16
ENST00000356897.1





IL2RA
AATTCAAAAACCTCGTCACAACAACAGATTTCTCGAGAAATCTGTTGTTGTGACGAGG
2657
chr10
ENST00000379959.7





IL2RA
AATTCAAAAAACCCTATACAACTGGACATTGCTCGAGCAATGTCCAGTTGTATAGGGT
2658
chr10
ENST00000379959.7





IL2RA
AATTCAAAAAACTCGGAACACAACGAAACAACTCGAGTTGTTTCGTTGTGTTCCGAGT
2659
chr10
ENST00000379959.7





IL2RB
AATTCAAAAAAGTCCCAGACCTGGTGGATTTCTCGAGAAATCCACCAGGTCTGGGACT
2660
chr22
ENST00000216223.9





IL2RB
AATTCAAAAAGACCCACAGATGCAACATAAGCTCGAGCTTATGTTGCATCTGTGGGTC
2661
chr22
ENST00000216223.9





IL2RB
AATTCAAAAACCAGACACCCAGTATGAGTTTCTCGAGAAACTCATACTGGGTGTCTGG
2662
chr22
ENST00000216223.9





IL2RG
AATTCAAAAATCGTGTTCGGAGCCGCTTTAACTCGAGTTAAAGCGGCTCCGAACACGA
2663
chrX
ENST00000374202.6





IL2RG
AATTCAAAAACCAACCTCACTCTGCATTATTCTCGAGAATAATGCAGAGTGAGGTTGG
2664
chrX
ENST00000374202.6





IL2RG
AATTCAAAAATTGGCTCCATGGGATTGATTACTCGAGTAATCAATCCCATGGAGCCAA
2665
chrX
ENST00000374202.6





IL3
AATTCAAAAATTATCCCATTGAGACTATTTACTCGAGTAAATAGTCTCAATGGGATAA
2666
chr5
ENST00000296870.2





IL3
AATTCAAAAACGGCATCAGATGAATTGTTAACTCGAGTTAACAATTCATCTGATGCCG
2667
chr5
ENST00000296870.2





IL3
AATTCAAAAAGCAATTGAGAGCATTCTTAAACTCGAGTTTAAGAATGCTCTCAATTGC
2668
chr5
ENST00000296870.2





IL31
AATTCAAAAAGCATATCTCAAGACAATCAGACTCGAGTCTGATTGTCTTGAGATATGC
2669
chr12
ENST00000377035.1





IL31
AATTCAAAAACCAAGTGATGATGTACAGAAACTCGAGTTTCTGTACATCATCACTTGG
2670
chr12
ENST00000377035.1





IL31
AATTCAAAAACCTGACTATTTCTCAACAGTTCTCGAGAACTGTTGAGAAATAGTCAGG
2671
chr12
ENST00000377035.1





IL31RA
AATTCAAAAACATCAAACGAATGATTCAAATCTCGAGATTTGAATCATTCGTTTGATG
2672
chr5
ENST00000447346.6





IL31RA
AATTCAAAAACGCCTGTTTCATCTGATTTAACTCGAGTTAAATCAGATGAAACAGGCG
2673
chr5
ENST00000447346.6





IL31RA
AATTCAAAAATTTCCTGTGTCTACTACTATACTCGAGTATAGTAGTAGACACAGGAAA
2674
chr5
ENST00000447346.6





IL32
AATTCAAAAATGTCGCCCTGGCATCTTAATACTCGAGTATTAAGATGCCAGGGCGACA
2675
chr16
ENST00000325568.9





IL32
AATTCAAAAAAGAGCTCACTCCTCTACTTGACTCGAGTCAAGTAGAGGAGTGAGCTCT
2676
chr16
ENST00000325568.9





IL32
AATTCAAAAAAGAGCTGGAGGACGACTTCAACTCGAGTTGAAGTCGTCCTCCAGCTCT
2677
chr16
ENST00000325568.9





IL33
AATTCAAAAAGAGTGCTTTGCCTTTGGTATACTCGAGTATACCAAAGGCAAAGCACTC
2678
chr9
ENST00000381434.7





IL33
AATTCAAAAAGCACTCCAACTGTGTTTCATTCTCGAGAATGAAACACAGTTGGAGTGC
2679
chr9
ENST00000381434.7





IL33
AATTCAAAAACCTGTTACTTTAGGAGAGAAACTCGAGTTTCTCTCCTAAAGTAACAGG
2680
chr9
ENST00000381434.7





IL34
AATTCAAAAAGCCGACTTCAGTACATGAAACCTCGAGGTTTCATGTACTGAAGTCGGC
2681
chr16
ENST00000288098.6





IL34
AATTCAAAAACAGAGCCCTCATTGCAGTATGCTCGAGCATACTGCAATGAGGGCTCTG
2682
chr16
ENST00000288098.6





IL34
AATTCAAAAACCGTGTTGTCCCTCTTGAATGCTCGAGCATTCAAGAGGGACAACACGG
2683
chr16
ENST00000288098.6





IL36A
AATTCAAAAACTCCAGTCACTATTGCCTTAACTCGAGTTAAGGCAATAGTGACTGGAG
2684
chr2
ENST00000259211.6





IL36A
AATTCAAAAATTCAGGACCAGACGCTCATAGCTCGAGCTATGAGCGTCTGGTCCTGAA
2685
chr2
ENST00000259211.6





IL36A
AATTCAAAAACTCTGCCTGATGTGTGCTAAACTCGAGTTTAGCACACATCAGGCAGAG
2686
chr2
ENST00000259211.6





IL36B
AATTCAAAAAATCCTATGCTATTCGTGATTCCTCGAGGAATCACGAATAGCATAGGAT
2687
chr2
ENST00000327407.2





IL36B
AATTCAAAAACCTGAGTGGAAATTCTTTAATCTCGAGATTAAAGAATTTCCACTCAGG
2688
chr2
ENST00000327407.2





IL36B
AATTCAAAAATTGGACTACATAACCTGTAAACTCGAGTTTACAGGTTATGTAGTCCAA
2689
chr2
ENST00000327407.2





IL36G
AATTCAAAAATGATATCATCCAGTCTTTATACTCGAGTATAAAGACTGGATGATATCA
2690
chr2
ENST00000259205.4





IL36G
AATTCAAAAACAGGAGAGCTGGGTGGTATAACTCGAGTTATACCACCCAGCTCTCCTG
2691
chr2
ENST00000259205.4





IL36G
AATTCAAAAAGGGAATCCAGAATCCAGAAATCTCGAGATTTCTGGATTCTGGATTCCC
2692
chr2
ENST00000259205.4





IL36RN
AATTCAAAAACTCGGCATTGAAGGTGCTTTACTCGAGTAAAGCACCTTCAATGCCGAG
2693
chr2
ENST00000346807.7





IL36RN
AATTCAAAAATGGTTCCCAGTTTGGATAAATCTCGAGATTTATCCAAACTGGGAACCA
2694
chr2
ENST00000346807.7





IL36RN
AATTCAAAAAGGGAATCATTCCTGCTTAATGCTCGAGCATTAAGCAGGAATGATTCCC
2695
chr2
ENST00000346807.7





IL37
AATTCAAAAACTCTACTGTGACAAGGATAAACTCGAGTTTATCCTTGTCACAGTAGAG
2696
chr2
ENST00000263326.7





IL37
AATTCAAAAATGCACCTCCTGCAATTGTAATCTCGAGATTACAATTGCAGGAGGTGCA
2697
chr2
ENST00000263326.7





IL37
AATTCAAAAAGTTCACACAAAGATCTTCTTTCTCGAGAAAGAAGATCTTTGTGTGAAC
2698
chr2
ENST00000263326.7





IL4
AATTCAAAAAAGCTGATCCGATTCCTGAAACCTCGAGGTTTCAGGAATCGGATCAGCT
2699
chr5
ENST00000231449.6





IL4
AATTCAAAAACCACGGACACAAGTGCGATATCTCGAGATATCGCACTTGTGTCCGTGG
2700
chr5
ENST00000231449.6





IL4
AATTCAAAAATAGCATGTGCCGGCAACTTTGCTCGAGCAAAGTTGCCGGCACATGCTA
2701
chr5
ENST00000231449.6





IL5
AATTCAAAAAGCAAGAGTTTCTTGGTGTAATCTCGAGATTACACCAAGAAACTCTTGC
2702
chr5
ENST00000231454.5





IL5
AATTCAAAAAGGGTACTGTGGAAAGACTATTCTCGAGAATAGTCTTTCCACAGTACCC
2703
chr5
ENST00000231454.5





IL5
AATTCAAAAAGAAAGAGTCAGGCCTTAATTTCTCGAGAAATTAAGGCCTGACTCTTTC
2704
chr5
ENST00000231454.5





IL6
AATTCAAAAAATGAGCGTTAGGACACTATTTCTCGAGAAATAGTGTCCTAACGCTCAT
2705
chr7
ENST00000258743.9





IL6
AATTCAAAAAGAGTACCTCCAGAACAGATTTCTCGAGAAATCTGTTCTGGAGGTACTC
2706
chr7
ENST00000258743.9





IL6
AATTCAAAAAATGTGAAGCTGAGTTAATTTACTCGAGTAAATTAACTCAGCTTCACAT
2707
chr7
ENST00000258743.9





IL6R
AATTCAAAAAGCAGGCACTTACTACTAATAACTCGAGTTATTAGTAGTAAGTGCCTGC
2708
chr1
ENST00000368485.7





IL6R
AATTCAAAAATATCGGGCTGAACGGTCAAAGCTCGAGCTTTGACCGTTCAGCCCGATA
2709
chr1
ENST00000368485.7





IL6R
AATTCAAAAACTGGACCCTGTGGATGATAAACTCGAGTTTATCATCCACAGGGTCCAG
2710
chr1
ENST00000368485.7





IL6ST
AATTCAAAAAACCGTGCATCGCACCTATTTACTCGAGTAAATAGGTGCGATGCACGGT
2711
chr5
ENST00000336909.9





IL6ST
AATTCAAAAAACTTCAGCAGTACCTATAAAGCTCGAGCTTTATAGGTACTGCTGAAGT
2712
chr5
ENST00000336909.9





IL6ST
AATTCAAAAACGGCCAGAAGATCTACAATTACTCGAGTAATTGTAGATCTTCTGGCCG
2713
chr5
ENST00000336909.9





IL7
AATTCAAAAAGCTCGCAAGTTGAGGCAATTTCTCGAGAAATTGCCTCAACTTGCGAGC
2714
chr8
ENST00000263851.8





IL7
AATTCAAAAAGCTCACTATGAATCTATTATACTCGAGTATAATAGATTCATAGTGAGC
2715
chr8
ENST00000263851.8





IL7
AATTCAAAAAGTGTTTCCTAAAGAGACTATTCTCGAGAATAGTCTCTTTAGGAAACAC
2716
chr8
ENST00000263851.8





IL9
AATTCAAAAAACCACCATGCAAACAAGATACCTCGAGGTATCTTGTTTGCATGGTGGT
2717
chr5
ENST00000274520.1





IL9
AATTCAAAAAGAACAACAAGTGTCCATATTTCTCGAGAAATATGGACACTTGTTGTTC
2718
chr5
ENST00000274520.1





IL9
AATTCAAAAACTGAAGAGTCTTCTGGAAATTCTCGAGAATTTCCAGAAGACTCTTCAG
2719
chr5
ENST00000274520.1





INHA
AATTCAAAAACCTCGGATGGAGGTTACTCTTCTCGAGAAGAGTAACCTCCATCCGAGG
2720
chr2
ENST00000243786.2





INHA
AATTCAAAAAGCAGCACTGTGCTTGTATCTACTCGAGTAGATACAAGCACAGTGCTGC
2721
chr2
ENST00000243786.2





INHA
AATTCAAAAATGGAGGTTACTCTTTCAAGTACTCGAGTACTTGAAAGAGTAACCTCCA
2722
chr2
ENST00000243786.2





INHBA
AATTCAAAAAAGGCACTTTCCTACCCAATTACTCGAGTAATTGGGTAGGAAAGTGCCT
2723
chr7
ENST00000242208.4





INHBA
AATTCAAAAAGAGAATGGTGTACCCTTTATTCTCGAGAATAAAGGGTACACCATTCTC
2724
chr7
ENST00000242208.4





INHBA
AATTCAAAAAAGACGCTGCACTTCGAGATTTCTCGAGAAATCTCGAAGTGCAGCGTCT
2725
chr7
ENST00000242208.4





INHBB
AATTCAAAAACAAATGGATGCGGTGACAAATCTCGAGATTTGTCACCGCATCCATTTG
2726
chr2
ENST00000295228.3





INHBB
AATTCAAAAAGCTGGAACGACTGGATCATAGCTCGAGCTATGATCCAGTCGTTCCAGC
2727
chr2
ENST00000295228.3





INHBB
AATTCAAAAATGATGAGTACAACATCGTCAACTCGAGTTGACGATGTTGTACTCATCA
2728
chr2
ENST00000295228.3





INHBC
AATTCAAAAATCAACCAGACTCGTCTTGATTCTCGAGAATCAAGACGAGTCTGGTTGA
2729
chr12
ENST00000309668.2





INHBC
AATTCAAAAACAAGACTGACATACCTGACATCTCGAGATGTCAGGTATGTCAGTCTTG
2730
chr12
ENST00000309668.2





INHBC
AATTCAAAAACAGGCCAGTCTCATGTTCTTTCTCGAGAAAGAACATGAGACTGGCCTG
2731
chr12
ENST00000309668.2





INHBE
AATTCAAAAACTCCTCTACCTGGATCATAATCTCGAGATTATGATCCAGGTAGAGGAG
2732
chr12
ENST00000266646.2





INHBE
AATTCAAAAAACCTGGGCTGGCATACCTTAACTCGAGTTAAGGTATGCCAGCCCAGGT
2733
chr12
ENST00000266646.2





INHBE
AATTCAAAAAGCAGCCCTTCCTAGAGCTTAACTCGAGTTAAGCTCTAGGAAGGGCTGC
2734
chr12
ENST00000266646.2





ITGA4
AATTCAAAAACCAACGCTTCAGTGATCAATCCTCGAGGATTGATCACTGAAGCGTTGG
2735
chr2
ENST00000397033.6





ITGA4
AATTCAAAAATGTAGAACACATCAAGCATTTCTCGAGAAATGCTTGATGTGTTCTACA
2736
chr2
ENST00000397033.6





ITGA4
AATTCAAAAACATGATCTTGTGACATATTATCTCGAGATAATATGTCACAAGATCATG
2737
chr2
ENST00000397033.6





ITGAV
AATTCAAAAATTGAAGTGTACCCTAGCATTTCTCGAGAAATGCTAGGGTACACTTCAA
2738
chr2
ENST00000261023.7





ITGAV
AATTCAAAAACACTCCAAGAACATGACTATTCTCGAGAATAGTCATGTTCTTGGAGTG
2739
chr2
ENST00000261023.7





ITGAV
AATTCAAAAAGTGAGGTCGAAACAGGATAAACTCGAGTTTATCCTGTTTCGACCTCAC
2740
chr2
ENST00000261023.7





ITGB1
AATTCAAAAATTTGTAGGAAGAGGGATAATACTCGAGTATTATCCCTCTTCCTACAAA
2741
chr10
ENST00000302278.7





ITGB1
AATTCAAAAAGCCTTGCATTACTGCTGATATCTCGAGATATCAGCAGTAATGCAAGGC
2742
chr10
ENST00000302278.7





ITGB1
AATTCAAAAAGCCTTGCATTACTGCTGATATCTCGAGATATCAGCAGTAATGCAAGGC
2743
chr10
ENST00000302278.7





ITGB3
AATTCAAAAAGTCGTCAGATTCCAGTACTATCTCGAGATAGTACTGGAATCTGACGAC
2744
chr17
ENST00000559488.5





ITGB3
AATTCAAAAAGTCGTCAGATTCCAGTACTATCTCGAGATAGTACTGGAATCTGACGAC
2745
chr17
ENST00000559488.5





ITGB3
AATTCAAAAACCACGTCTACCTTCACCAATACTCGAGTATTGGTGAAGGTAGACGTGG
2746
chr17
ENST00000559488.5





KIT
AATTCAAAAAACTTCATCTAACGAGATTAAACTCGAGTTTAATCTCGTTAGATGAAGT
2747
chr4
ENST00000288135.5





KIT
AATTCAAAAAGCGACGAGATTAGGCTGTTATCTCGAGATAACAGCCTAATCTCGTCGC
2748
chr4
ENST00000288135.5





KIT
AATTCAAAAAACGAGTTGGCCCTAGACTTAGCTCGAGCTAAGTCTAGGGCCAACTCGT
2749
chr4
ENST00000288135.5





KITLG
AATTCAAAAAGCAGGAATCGTGTGACTAATACTCGAGTATTAGTCACACGATTCCTGC
2750
chr12
ENST00000228280.9





KITLG
AATTCAAAAAGCAGGAATCGTGTGACTAATACTCGAGTATTAGTCACACGATTCCTGC
2751
chr12
ENST00000228280.9





KITLG
AATTCAAAAACCTATTTAATCCTCTCGTCAACTCGAGTTGACGAGAGGATTAAATAGG
2752
chr12
ENST00000228280.9





KLHL20
AATTCAAAAAGCCAATACATGGAGGTTATATCTCGAGATATAACCTCCATGTATTGGC
2753
chr1
ENST00000209884.4





KLHL20
AATTCAAAAAGCCGCAAGAACGACCACTAATCTCGAGATTAGTGGTCGTTCTTGCGGC
2754
chr1
ENST00000209884.4





KLHL20
AATTCAAAAACACATTGTGAATCCCATATTTCTCGAGAAATATGGGATTCACAATGTG
2755
chr1
ENST00000209884.4





LEFTY1
AATTCAAAAAACAAGTTACCTCACCTAATTTCTCGAGAAATTAGGTGAGGTAACTTGT
2756
chr1
ENST00000272134.5





LEFTY1
AATTCAAAAAAGCCCAATGTGTCATTGTTTACTCGAGTAAACAATGACACATTGGGCT
2757
chr1
ENST00000272134.5





LEFTY1
AATTCAAAAATCTCTAGTGAGCCCTGAATTTCTCGAGAAATTCAGGGCTCACTAGAGA
2758
chr1
ENST00000272134.5





LEFTY2
AATTCAAAAAAGTTACTCCATCCCAATTTAGCTCGAGCTAAATTGGGATGGAGTAACT
2759
chr1
ENST00000366820.9





LEFTY2
AATTCAAAAACTAAGCACTTACGTGAGTAAACTCGAGTTTACTCACGTAAGTGCTTAG
2760
chr1
ENST00000366820.9





LEFTY2
AATTCAAAAATAGGCGCCTGGTGTATCCATTCTCGAGAATGGATACACCAGGCGCCTA
2761
chr1
ENST00000366820.9





LIF
AATTCAAAAAGCAGTGCCAATGCCCTCTTTACTCGAGTAAAGAGGGCATTGGCACTGC
2762
chr22
ENST00000249075.3





LIF
AATTCAAAAAACCGCATAGTCGTGTACCTTGCTCGAGCAAGGTACACGACTATGCGGT
2763
chr22
ENST00000249075.3





LIF
AATTCAAAAACAACAACCTGGACAAGCTATGCTCGAGCATAGCTTGTCCAGGTTGTTG
2764
chr22
ENST00000249075.3





LIFR
AATTCAAAAATGACTTGCGACTACGTCATTACTCGAGTAATGACGTAGTCGCAAGTCA
2765
chr5
ENST00000263409.8





LIFR
AATTCAAAAAACTTCTGCAGATTCGATATTACTCGAGTAATATCGAATCTGCAGAAGT
2766
chr5
ENST00000263409.8





LIFR
AATTCAAAAAGTAGGCTCAGACATAACATTTCTCGAGAAATGTTATGTCTGAGCCTAC
2767
chr5
ENST00000263409.8





LTA
AATTCAAAAAGCCCTAGTACTGTCTTCTTTGCTCGAGCAAAGAAGACAGTACTAGGGC
2768
chr6
ENST00000418386.2





LTA
AATTCAAAAAGATCAAGTCACCGGAGCTTTCCTCGAGGAAAGCTCCGGTGACTTGATC
2769
chr6
ENST00000418386.2





LTA
AATTCAAAAAGCTCCCAGAAGATGGTGTATCCTCGAGGATACACCATCTTCTGGGAGC
2770
chr6
ENST00000418386.2





LTB
AATTCAAAAAGCGAGAGGGAAGACCTTCTTTCTCGAGAAAGAAGGTCTTCCCTCTCGC
2771
chr6
ENST00000429299.2





LTB
AATTCAAAAACGAGAGGGTGTACGTCAACATCTCGAGATGTTGACGTACACCCTCTCG
2772
chr6
ENST00000429299.2





LTB
AATTCAAAAAGACGAAGGAACAGGCGTTTCTCTCGAGAGAAACGCCTGTTCCTTCGTC
2773
chr6
ENST00000429299.2





LTBP1
AATTCAAAAAGATGACCTGTGTCGATGTAAACTCGAGTTTACATCGACACAGGTCATC
2774
chr2
ENST00000407925.5





LTBP1
AATTCAAAAAGGTGGAACAGTGCTGTTATTTCTCGAGAAATAACAGCACTGTTCCACC
2775
chr2
ENST00000407925.5





LTBP1
AATTCAAAAACCGTTGAATACCGCCTTGAATCTCGAGATTCAAGGCGGTATTCAACGG
2776
chr2
ENST00000407925.5





LTBP3
AATTCAAAAAGCATCCTCAATGGATGTGAAACTCGAGTTTCACATCCATTGAGGATGC
2777
chr11
ENST00000322147.8





LTBP3
AATTCAAAAATGTTGTTCGGGTCGGAGATTTCTCGAGAAATCTCCGACCCGAACAACA
2778
chr11
ENST00000322147.8





LTBP3
AATTCAAAAAAGCGCTTCAAGGTGGTCTTTGCTCGAGCAAAGACCACCTTGAAGCGCT
2779
chr11
ENST00000322147.8





LTBP4
AATTCAAAAAGCTTCGACATGCCAGACTTTGCTCGAGCAAAGTCTGGCATGTCGAAGC
2780
chr19
ENST00000308370.11





LTBP4
AATTCAAAAATGAAACACTACAGGGTGTATGCTCGAGCATACACCCTGTAGTGTTTCA
2781
chr19
ENST00000308370.11





LTBP4
AATTCAAAAACAACCGGCTTTGAAAGAGTTACTCGAGTAACTCTTTCAAAGCCGGTTG
2782
chr19
ENST00000308370.11





MAF
AATTCAAAAATCAGTGGGATACGCCACATTTCTCGAGAAATGTGGCGTATCCCACTGA
2783
chr16
ENST00000326043.4





MAF
AATTCAAAAATTTATGGTGTGTGCAAGTAAACTCGAGTTTACTTGCACACACCATAAA
2784
chr16
ENST00000326043.4





MAF
AATTCAAAAAGTTAGAGAAGAAGGCTATTAACTCGAGTTAATAGCCTTCTTCTCTAAC
2785
chr16
ENST00000326043.4





MAF
AATTCAAAAATGTTAATGACTTCGATCTGATCTCGAGATCAGATCGAAGTCATTAACA
2786
chr16
ENST00000326043.4





MIF
AATTCAAAAACTACATCAACTATTACGACATCTCGAGATGTCGTAATAGTTGATGTAG
2787
chr22
ENST00000215754.7





MIF
AATTCAAAAACTACATCAACTATTACGACATCTCGAGATGTCGTAATAGTTGATGTAG
2788
chr22
ENST00000215754.7





MIF
AATTCAAAAAGACAGGGTCTACATCAACTATCTCGAGATAGTTGATGTAGACCCTGTC
2789
chr22
ENST00000215754.7





MINOS1-
AATTCAAAAAGCCAAGCTGCACAATTTAATACTCGAGTATTAAATTGTGCAGCTTGGC
2790
chr1
ENST00000602662.1





MINOS1-
AATTCAAAAAGCTGGCACTGTTCCCAGATAACTCGAGTTATCTGGGAACAGTGCCAGC
2791
chr1
ENST00000602662.1





MINOS1-
AATTCAAAAAAGAAAGACCACTGGCAGAAACCTCGAGGTTTCTGCCAGTGGTCTTTCT
2792
chr1
ENST00000602662.1





MSTN
AATTCAAAAAAGGCCCAACTATGGATATATTCTCGAGAATATATCCATAGTTGGGCCT
2793
chr2
ENST00000260950.4





MSTN
AATTCAAAAAGAGCTAGAAGGAGATCAAATTCTCGAGAATTTGATCTCCTTCTAGCTC
2794
chr2
ENST00000260950.4





MSTN
AATTCAAAAAGTATGCTTTAAAGTCTATTTCCTCGAGGAAATAGACTTTAAAGCATAC
2795
chr2
ENST00000260950.4





NAMPT
AATTCAAAAAAGCGATAGCTATGACATTTATCTCGAGATAAATGTCATAGCTATCGCT
2796
chr7
ENST00000222553.7





NAMPT
AATTCAAAAAGTGAAGATCTAAGACATTTAACTCGAGTTAAATGTCTTAGATCTTCAC
2797
chr7
ENST00000222553.7





NAMPT
AATTCAAAAATACAAGGTTACTCACTATAAACTCGAGTTTATAGTGAGTAACCTTGTA
2798
chr7
ENST00000222553.7





NBL1
AATTCAAAAAGCCAAGCTGCACAATTTAATACTCGAGTATTAAATTGTGCAGCTTGGC
2799
chr1
ENST00000375136.7





NBL1
AATTCAAAAAGCTGGCACTGTTCCCAGATAACTCGAGTTATCTGGGAACAGTGCCAGC
2800
chr1
ENST00000375136.7





NBL1
AATTCAAAAAAGAAAGACCACTGGCAGAAACCTCGAGGTTTCTGCCAGTGGTCTTTCT
2801
chr1
ENST00000375136.7





NDP
AATTCAAAAACTGCTAAAGGTTACCGATTTCCTCGAGGAAATCGGTAACCTTTAGCAG
2802
chrX
ENST00000378062.5





NDP
AATTCAAAAACTCTGCATATTCTAGTAATAACTCGAGTTATTACTAGAATATGCAGAG
2803
chrX
ENST00000378062.5





NDP
AATTCAAAAAACGGACAGCTCATTCATAATGCTCGAGCATTATGAATGAGCTGTCCGT
2804
chrX
ENST00000378062.5





NLRP7
AATTCAAAAACCGTTCAAGGAAATTTCTATTCTCGAGAATAGAAATTTCCTTGAACGG
2805
chr19
ENST00000328092.9





NLRP7
AATTCAAAAACGGGTCTCTAAGATGTCTTATCTCGAGATAAGACATCTTAGAGACCCG
2806
chr19
ENST00000328092.9





NLRP7
AATTCAAAAAACCTGCTCAGAAATCATAAATCTCGAGATTTATGATTTCTGAGCAGGT
2807
chr19
ENST00000328092.9





NODAL
AATTCAAAAAGTGCTCCTAGATCACCATAAACTCGAGTTTATGGTGATCTAGGAGCAC
2808
chr10
ENST00000287139.7





NODAL
AATTCAAAAACACCTATAGCTTTCATGTATTCTCGAGAATACATGAAAGCTATAGGTG
2809
chr10
ENST00000287139.7





NODAL
AATTCAAAAAGCATGCTGTATGTGGATAATGCTCGAGCATTATCCACATACAGCATGC
2810
chr10
ENST00000287139.7





NOG
AATTCAAAAAAGGTCAGTATTATACGTTAAACTCGAGTTTAACGTATAATACTGACCT
2811
chr17
ENST00000332822.4





NOG
AATTCAAAAAATTCTGGTTGTTGCTAATAATCTCGAGATTATTAGCAACAACCAGAAT
2812
chr17
ENST00000332822.4





NOG
AATTCAAAAATGCGGAGGAAGTTACAGATGTCTCGAGACATCTGTAACTTCCTCCGCA
2813
chr17
ENST00000332822.4





NRG1
AATTCAAAAAGATATCACAGAGGCCTATAACCTCGAGGTTATAGGCCTCTGTGATATC
2814
chr8
ENST00000287842.7





NRG1
AATTCAAAAAGGCTGATTCTGGAGAGTATATCTCGAGATATACTCTCCAGAATCAGCC
2815
chr8
ENST00000287842.7





NRG1
AATTCAAAAAGACAGTGCCTCTGCCAATATCCTCGAGGATATTGGCAGAGGCACTGTC
2816
chr8
ENST00000287842.7





NRP1
AATTCAAAAATATACTAGAATCACCGCATTTCTCGAGAAATGCGGTGATTCTAGTATA
2817
chr10
ENST00000265371.8





NRP1
AATTCAAAAACAGCCTTGAATGCACTTATATCTCGAGATATAAGTGCATTCAAGGCTG
2818
chr10
ENST00000265371.8





NRP1
AATTCAAAAACAGCCTTGAATGCACTTATATCTCGAGATATAAGTGCATTCAAGGCTG
2819
chr10
ENST00000265371.8





NRP2
AATTCAAAAACGACTGCAAGTATGACTTTATCTCGAGATAAAGTCATACTTGCAGTCG
2820
chr2
ENST00000357785.9





NRP2
AATTCAAAAACCGGATTGCTAATGAACAGATCTCGAGATCTGTTCATTAGCAATCCGG
2821
chr2
ENST00000357785.9





NRP2
AATTCAAAAACCTCAACTTCAACCCTCACTTCTCGAGAAGTGAGGGTTGAAGTTGAGG
2822
chr2
ENST00000357785.9





OSM
AATTCAAAAAACTTCCTCCTTTCCGTGTTTCCTCGAGGAAACACGGAAAGGAGGAAGT
2823
chr22
ENST00000215781.2





OSM
AATTCAAAAAGGACCGACTTTCCATTGATTCCTCGAGGAATCAATGGAAAGTCGGTCC
2824
chr22
ENST00000215781.2





OSM
AATTCAAAAATGGTCCTTGCACTCCTGTTTCCTCGAGGAAACAGGAGTGCAAGGACCA
2825
chr22
ENST00000215781.2





OSMR
AATTCAAAAATAACCTGACTCATCGAGTTTACTCGAGTAAACTCGATGAGTCAGGTTA
2826
chr5
ENST00000274276.7





OSMR
AATTCAAAAAAGGAGAACCCTCACCTAATAACTCGAGTTATTAGGTGAGGGTTCTCCT
2827
chr5
ENST00000274276.7





OSMR
AATTCAAAAAGCACTCCATAAGGAATAATTTCTCGAGAAATTATTCCTTATGGAGTGC
2828
chr5
ENST00000274276.7





PARK7
AATTCAAAAAGTAGCCGTGATGTGGTCATTTCTCGAGAAATGACCACATCACGGCTAC
2829
chr1
ENST00000338639.9





PARK7
AATTCAAAAAGCAATTGTTGAAGCCCTGAATCTCGAGATTCAGGGCTTCAACAATTGC
2830
chr1
ENST00000338639.9





PARK7
AATTCAAAAAACTCTGAGAATCGTGTGGAAACTCGAGTTTCCACACGATTCTCAGAGT
2831
chr1
ENST00000338639.9





PDPN
AATTCAAAAAGCCTCTGGTATGAGAAATAAACTCGAGTTTATTTCTCATACCAGAGGC
2832
chr1
ENST00000294489.10





PDPN
AATTCAAAAAAGTCCACGCGCAAGAACAAAGCTCGAGCTTTGTTCTTGCGCGTGGACT
2833
chr1
ENST00000294489.10





PDPN
AATTCAAAAATGACCCTGGTTGGAATCATAGCTCGAGCTATGATTCCAACCAGGGTCA
2834
chr1
ENST00000294489.10





PF4
AATTCAAAAAACGCTGAAGAATGGAAGGAAACTCGAGTTTCCTTCCATTCTTCAGCGT
2835
chr4
ENST00000296029.3





PF4
AATTCAAAAACCAACTGATAGCCACGCTGAACTCGAGTTCAGCGTGGCTATCAGTTGG
2836
chr4
ENST00000296029.3





PF4
AATTCAAAAACACGCTGAAGAATGGAAGGAACTCGAGTTCCTTCCATTCTTCAGCGTG
2837
chr4
ENST00000296029.3





PF4V1
AATTCAAAAAGTTGTGGTATAGTCAATCTATCTCGAGATAGATTGACTATACCACAAC
2838
chr4
ENST00000226524.3





PF4V1
AATTCAAAAAAGCTGCCTAAGTGTGCACTTTCTCGAGAAAGTGCACACTTAGGCAGCT
2839
chr4
ENST00000226524.3





PF4V1
AATTCAAAAACTGACACATCACAATTTCATACTCGAGTATGAAATTGTGATGTGTCAG
2840
chr4
ENST00000226524.3





PGLYRP1
AATTCAAAAAGTGCCCACTCAGGTCACTTATCTCGAGATAAGTGACCTGAGTGGGCAC
2841
chr19
ENST00000008938.4





PGLYRP1
AATTCAAAAACTTACGCTATGTGGTGGTATCCTCGAGGATACCACCACATAGCGTAAG
2842
chr19
ENST00000008938.4





PGLYRP1
AATTCAAAAACATCAGCTTCATGGGCAACTACTCGAGTAGTTGCCCATGAAGCTGATG
2843
chr19
ENST00000008938.4





PLP2
AATTCAAAAACCTGTCGGTGATTGAGATGATCTCGAGATCATCTCAATCACCGACAGG
2844
chrX
ENST00000376327.5





PLP2
AATTCAAAAACCTGTCGGTGATTGAGATGATCTCGAGATCATCTCAATCACCGACAGG
2845
chrX
ENST00000376327.5





PLP2
AATTCAAAAACGAAAGGGAATCCTCCTGTTTCTCGAGAAACAGGAGGATTCCCTTTCG
2846
chrX
ENST00000376327.5





PPBP
AATTCAAAAAAGGTGATGAATCTGCTGATTACTCGAGTAATCAGCAGATTCATCACCT
2847
chr4
ENST00000296028.3





PPBP
AATTCAAAAATGTTTCTGCCAAACTTCTTTACTCGAGTAAAGAAGTTTGGCAGAAACA
2848
chr4
ENST00000296028.3





PPBP
AATTCAAAAACTCCCAGGAAGGGTAGAATTTCTCGAGAAATTCTACCCTTCCTGGGAG
2849
chr4
ENST00000296028.3





PXDN
AATTCAAAAAAGATTGCGACTGGACTCAAACCTCGAGGTTTGAGTCCAGTCGCAATCT
2850
chr2
ENST00000252804.8





PXDN
AATTCAAAAAGTTCCTGACGTCAGTCGAAATCTCGAGATTTCGACTGACGTCAGGAAC
2851
chr2
ENST00000252804.8





PXDN
AATTCAAAAAGAAGGATTCTTGACCATCAATCTCGAGATTGATGGTCAAGAATCCTTC
2852
chr2
ENST00000252804.8





RORC
AATTCAAAAACACCTCACAAATTGAAGTGATCTCGAGATCACTTCAATTTGTGAGGTG
2853
chr1
ENST00000318247.6





RORC
AATTCAAAAAGCCCTCATATTCCAACAACTTCTCGAGAAGTTGTTGGAATATGAGGGC
2854
chr1
ENST00000318247.6





RORC
AATTCAAAAAGCTTCTCAAAGCAGGAGCAATCTCGAGATTGCTCCTGCTTTGAGAAGC
2855
chr1
ENST00000318247.6





RORC
AATTCAAAAACGAGGATGAGATTGCCCTCTACTCGAGTAGAGGGCAATCTCATCCTCG
2856
chr1
ENST00000318247.6





SCG2
AATTCAAAAAACGACAAGGATCAAGAATTAGCTCGAGCTAATTCTTGATCCTTGTCGT
2857
chr2
ENST00000305409.2





SCG2
AATTCAAAAACCTGTCTCTTATCCCTTTAATCTCGAGATTAAAGGGATAAGAGACAGG
2858
chr2
ENST00000305409.2





SCG2
AATTCAAAAAGAAAGCAGCCCAGATTATAATCTCGAGATTATAATCTGGGCTGCTTTC
2859
chr2
ENST00000305409.2





SCGB3A1
AATTCAAAAATCATAGAGGGCTCCCAGAAGTCTCGAGACTTCTGGGAGCCCTCTATGA
2860
chr5
ENST00000292641.3





SCGB3A1
AATTCAAAAACCCGTGAACCACCTCATAGAGCTCGAGCTCTATGAGGTGGTTCACGGG
2861
chr5
ENST00000292641.3





SCGB3A1
AATTCAAAAAGACTGGAGCATCTACACCTGACTCGAGTCAGGTGTAGATGCTCCAGTC
2862
chr5
ENST00000292641.3





SECTM1
AATTCAAAAACACCAGAGAAATAACAGACAACTCGAGTTGTCTGTTATTTCTCTGGTG
2863
chr17
ENST00000269389.7





SECTM1
AATTCAAAAACCATGACTCGAATATCTGAAACTCGAGTTTCAGATATTCGAGTCATGG
2864
chr17
ENST00000269389.7





SECTM1
AATTCAAAAACCATGACTCGAATATCTGAAACTCGAGTTTCAGATATTCGAGTCATGG
2865
chr17
ENST00000269389.7





SLURP1
AATTCAAAAAGTGCTTCCTGCAGGACCATTACTCGAGTAATGGTCCTGCAGGAAGCAC
2866
chr8
ENST00000246515.1





SLURP1
AATTCAAAAAAGACCTCTGCAACTCGGAACTCTCGAGAGTTCCGAGTTGCAGAGGTCT
2867
chr8
ENST00000246515.1





SLURP1
AATTCAAAAACTCAAGTGCTACACCTGCAAGCTCGAGCTTGCAGGTGTAGCACTTGAG
2868
chr8
ENST00000246515.1





SOSTDC1
AATTCAAAAAGATGCCACAGAAATCCTTTATCTCGAGATAAAGGATTTCTGTGGCATC
2869
chr7
ENST00000307068.4





SOSTDC1
AATTCAAAAAGGAACTGCGTTCCACCAAATACTCGAGTATTTGGTGGAACGCAGTTCC
2870
chr7
ENST00000307068.4





SOSTDC1
AATTCAAAAATGAATCTTCACAGTAACATTTCTCGAGAAATGTTACTGTGAAGATTCA
2871
chr7
ENST00000307068.4





SP100
AATTCAAAAAGAAGTGAGCCTGTGATCAATACTCGAGTATTGATCACAGGCTCACTTC
2872
chr2
ENST00000340126.8





SP100
AATTCAAAAATATACGCTGCGGTGGATATACCTCGAGGTATATCCACCGCAGCGTATA
2873
chr2
ENST00000340126.8





SP100
AATTCAAAAATCGTGATCTCATCACAAATAACTCGAGTTATTTGTGATGAGATCACGA
2874
chr2
ENST00000340126.8





SPI1
AATTCAAAAAGCCCTATGACACGGATCTATACTCGAGTATAGATCCGTGTCATAGGGC
2875
chr11
ENST00000378538.7





SPI1
AATTCAAAAAAGAGCTTCGCCGAGAACAACTCTCGAGAGTTGTTCTCGGCGAAGCTCT
2876
chr11
ENST00000378538.7





SPI1
AATTCAAAAACGGATCTATACCAACGCCAAACTCGAGTTTGGCGTTGGTATAGATCCG
2877
chr11
ENST00000378538.7





SPI1
AATTCAAAAACCGTATGTAAATCAGATCTCCCTCGAGGGAGATCTGATTTACATACGG
2878
chr11
ENST00000378538.7





SPP1
AATTCAAAAACCACAAGCAGTCCAGATTATACTCGAGTATAATCTGGACTGCTTGTGG
2879
chr4
ENST00000237623.11





SPP1
AATTCAAAAACCGAGGTGATAGTGTGGTTTACTCGAGTAAACCACACTATCACCTCGG
2880
chr4
ENST00000237623.11





SPP1
AATTCAAAAACTTCAGGGTTATGTCTATGTTCTCGAGAACATAGACATAACCCTGAAG
2881
chr4
ENST00000237623.11





TBX21
AATTCAAAAAGCCCTAACTACAGTCGTTTACCTCGAGGTAAACGACTGTAGTTAGGGC
2882
ch17
ENST00000177694.1





TBX21
AATTCAAAAACCTGTTGTGGTCCAAGTTTAACTCGAGTTAAACTTGGACCACAACAGG
2883
ch17
ENST00000177694.1





TBX21
AATTCAAAAACGCTTCCAACACGCATATCTTCTCGAGAAGATATGCGTGTTGGAAGCG
2884
ch17
ENST00000177694.1





TBX21
AATTCAAAAAACAATGTGACCCAGATGATTGCTCGAGCAATCATCTGGGTCACATTGT
2885
ch17
ENST00000177694.1





TCAP
AATTCAAAAACTTTGTAGTTTGCCCAGAGTTCTCGAGAACTCTGGGCAAACTACAAAG
2886
chr17
ENST00000309889.2





TCAP
AATTCAAAAAGGCCATGGCTGCTTTGTAGTTCTCGAGAACTACAAAGCAGCCATGGCC
2887
chr17
ENST00000309889.2





TCAP
AATTCAAAAAGGTGGCTGAGATCACAAAGCACTCGAGTGCTTTGTGATCTCAGCCACC
2888
chr17
ENST00000309889.2





TGFB1
AATTCAAAAAACTGCGGATCTCTGTGTCATTCTCGAGAATGACACAGAGATCCGCAGT
2889
chr19
ENST00000221930.5





TGFB1
AATTCAAAAACCACAACGAAATCTATGACAACTCGAGTTGTCATAGATTTCGTTGTGG
2890
chr19
ENST00000221930.5





TGFB1
AATTCAAAAACCACAACGAAATCTATGACAACTCGAGTTGTCATAGATTTCGTTGTGG
2891
chr19
ENST00000221930.5





TGFB2
AATTCAAAAAGCGGCCTATTGCTTTAGAAATCTCGAGATTTCTAAAGCAATAGGCCGC
2892
chr1
ENST00000366930.8





TGFB2
AATTCAAAAAGCTGGAGCATGCCCGTATTTACTCGAGTAAATACGGGCATGCTCCAGC
2893
chr1
ENST00000366930.8





TGFB2
AATTCAAAAATTGCTGCCTACGTCCACTTTACTCGAGTAAAGTGGACGTAGGCAGCAA
2894
chr1
ENST00000366930.8





TGFB3
AATTCAAAAACATTGCCAAACAGCGCTATATCTCGAGATATAGCGCTGTTTGGCAATG
2895
chr14
ENST00000238682.7





TGFB3
AATTCAAAAAGCTCTAGGGAATCTGGATTATCTCGAGATAATCCAGATTCCCTAGAGC
2896
chr14
ENST00000238682.7





TGFB3
AATTCAAAAACGGAATACTATGCCAAAGAAACTCGAGTTTCTTTGGCATAGTATTCCG
2897
chr14
ENST00000238682.7





TGFBR1
AATTCAAAAAGCCTTGAGAGTAATGGCTAAACTCGAGTTTAGCCATTACTCTCAAGGC
2898
chr9
ENST00000374994.8





TGFBR1
AATTCAAAAACTCATGTTGATGGTCTATATCCTCGAGGATATAGACCATCAACATGAG
2899
chr9
ENST00000374994.8





TGFBR1
AATTCAAAAAGAAGTTGCTGTTAAGATATTCCTCGAGGAATATCTTAACAGCAACTTC
2900
chr9
ENST00000374994.8





TGFBR2
AATTCAAAAACGTTCAGAAGTCGGTTAATAACTCGAGTTATTAACCGACTTCTGAACG
2901
chr3
ENST00000295754.9





TGFBR2
AATTCAAAAACTCTAGGCTTTATCGTGTTTACTCGAGTAAACACGATAAAGCCTAGAG
2902
chr3
ENST00000295754.9





TGFBR2
AATTCAAAAACTCAGGAAATGAGATTGATTTCTCGAGAAATCAATCTCATTTCCTGAG
2903
chr3
ENST00000295754.9





TGFBR3
AATTCAAAAAGGAGTTGGTAAAGGGTTAATACTCGAGTATTAACCCTTTACCAACTCC
2904
chr1
ENST00000212355.8





TGFBR3
AATTCAAAAATAATGGATTTCCGGGAGATATCTCGAGATATCTCCCGGAAATCCATTA
2905
chr1
ENST00000212355.8





TGFBR3
AATTCAAAAACACACCCAGGGCTAGTATAAACTCGAGTTTATACTAGCCCTGGGTGTG
2906
chr1
ENST00000212355.8





THBS1
AATTCAAAAAGTAGGTTATGATGAGTTTAATCTCGAGATTAAACTCATCATAACCTAC
2907
chr15
ENST00000260356.5





THBS1
AATTCAAAAACGTGACTGTAAGATTGTAAATCTCGAGATTTACAATCTTACAGTCACG
2908
chr15
ENST00000260356.5





THBS1
AATTCAAAAAGAGATCCCTAATCATCAAATTCTCGAGAATTTGATGATTAGGGATCTC
2909
chr15
ENST00000260356.5





THNSL2
AATTCAAAAAGAGCCGATCAAGACTGTGTTTCTCGAGAAACACAGTCTTGATCGGCTC
2910
chr2
ENST00000324166.6





THNSL2
AATTCAAAAAGCTGCCATTGAGAGTGTTCAACTCGAGTTGAACACTCTCAATGGCAGC
2911
chr2
ENST00000324166.6





THNSL2
AATTCAAAAAGCTGCCATTGAGAGTGTTCAACTCGAGTTGAACACTCTCAATGGCAGC
2912
chr2
ENST00000324166.6





THPO
AATTCAAAAAGACCTCCGAGTCCTCAGTAAACTCGAGTTTACTGAGGACTCGGAGGTC
2913
chr3
ENST00000204615.11





THPO
AATTCAAAAAAGCTAGCTCTTTGGTCTATTTCTCGAGAAATAGACCAAAGAGCTAGCT
2914
chr3
ENST00000204615.11





THPO
AATTCAAAAACAACCTCCAGCCTGGATATTCCTCGAGGAATATCCAGGCTGGAGGTTG
2915
chr3
ENST00000204615.11





TIMP1
AATTCAAAAAGCACAGTGTTTCCCTGTTTATCTCGAGATAAACAGGGAAACACTGTGC
2916
chrX
ENST00000218388.8





TIMP1
AATTCAAAAAGCACAGTGTTTCCCTGTTTATCTCGAGATAAACAGGGAAACACTGTGC
2917
chrX
ENST00000218388.8





TIMP1
AATTCAAAAAACAGACGGCCTTCTGCAATTCCTCGAGGAATTGCAGAAGGCCGTCTGT
2918
chrX
ENST00000218388.8





TNF
AATTCAAAAAGAACCCAAGCTTAGAACTTTACTCGAGTAAAGTTCTAAGCTTGGGTTC
2919
chr6
ENST00000449264.2





TNF
AATTCAAAAAGGAGCCAGCTCCCTCTATTTACTCGAGTAAATAGAGGGAGCTGGCTCC
2920
chr6
ENST00000449264.2





TNF
AATTCAAAAATGGCGTGGAGCTGAGAGATAACTCGAGTTATCTCTCAGCTCCACGCCA
2921
chr6
ENST00000449264.2





TNFRSF4
AATTCAAAAAGCACGTGGTGTAACCTCAGAACTCGAGTTCTGAGGTTACACCACGTGC
2922
chr1
ENST00000379236.3





TNFRSF4
AATTCAAAAACAGCAATAGCTCGGACGCAATCTCGAGATTGCGTCCGAGCTATTGCTG
2923
chr1
ENST00000379236.3





TNFRSF4
AATTCAAAAAGACAGCTACAAGCCTGGAGTTCTCGAGAACTCCAGGCTTGTAGCTGTC
2924
chr1
ENST00000379236.3





TNFRSF4
AATTCAAAAAGCTTCTACAACGACGTGGTCACTCGAGTGACCACGTCGTTGTAGAAGC
2925
chr1
ENST00000379236.3





TNFRSF11
AATTCAAAAATGTTTACTTGCCCGGTTTAATCTCGAGATTAAACCGGGCAAGTAAACA
2926
chr18
ENST00000586569.2





TNFRSF11
AATTCAAAAATGTACCAGTGAGAAGCATTATCTCGAGATAATGCTTCTCACTGGTACA
2927
chr18
ENST00000586569.2





TNFRSF11
AATTCAAAAATGGGACGGTGCTGTAACAAATCTCGAGATTTGTTACAGCACCGTCCCA
2928
chr18
ENST00000586569.2





TNFRSF11
AATTCAAAAAGAAAGCACTCACAGCTAATTTCTCGAGAAATTAGCTGTGAGTGCTTTC
2929
chr8
ENST00000297350.8





TNFRSF11
AATTCAAAAAGATAAATGCTTGCTGCATAAACTCGAGTTTATGCAGCAAGCATTTATC
2930
chr8
ENST00000297350.8





TNFRSF11
AATTCAAAAACCAGTGTGTGTTCATTGTAAACTCGAGTTTACAATGAACACACACTGG
2931
chr8
ENST00000297350.8





TNFRSF1A
AATTCAAAAAAGAACCAGTACCGGCATTATTCTCGAGAATAATGCCGGTACTGGTTCT
2932
chr12
ENST00000162749.6





TNFRSF1A
AATTCAAAAAGGAGCTGTTGGTGGGAATATACTCGAGTATATTCCCACCAACAGCTCC
2933
chr12
ENST00000162749.6





TNFRSF1A
AATTCAAAAACTTGAAGGAACTACTACTAAGCTCGAGCTTAGTAGTAGTTCCTTCAAG
2934
chr12
ENST00000162749.6





TNFRSF9
AATTCAAAAAGCTCCGTTTCTCTGTTGTTAACTCGAGTTAACAACAGAGAAACGGAGC
2935
chr1
ENST00000377507.7





TNFRSF9
AATTCAAAAACAAGAACACCATCCTACATAACTCGAGTTATGTAGGATGGTGTTCTTG
2936
chr1
ENST00000377507.7





TNFRSF9
AATTCAAAAACAGTCCCTGTCCTCCAAATAGCTCGAGCTATTTGGAGGACAGGGACTG
2937
chr1
ENST00000377507.7





TNFSF9
AATTCAAAAACAAGTTGGACCTTGATATTTACTCGAGTAAATATCAAGGTCCAACTTG
2938
chr19
ENST00000245817.4





TNFSF9
AATTCAAAAACTACTATGTCTTCTTTCAACTCTCGAGAGTTGAAAGAAGACATAGTAG
2939
chr19
ENST00000245817.4





TNFSF9
AATTCAAAAACCCTTCACCGAGGTCGGAATACTCGAGTATTCCGACCTCGGTGAAGGG
2940
chr19
ENST00000245817.4





TNFSF9
AATTCAAAAATGAGCTACAAAGAGGACACGACTCGAGTCGTGTCCTCTTTGTAGCTCA
2941
chr19
ENST00000245817.4





TNFSF10
AATTCAAAAAAGAAATAGTTGTTGGTCTAAACTCGAGTTTAGACCAACAACTATTTCT
2942
chr3
ENST00000241261.6





TNFSF10
AATTCAAAAAAGTTATCCTGACCCTATATTGCTCGAGCAATATAGGGTCAGGATAACT
2943
chr3
ENST00000241261.6





TNFSF10
AATTCAAAAAGACAAACAAATGGTCCAATATCTCGAGATATTGGACCATTTGTTTGTC
2944
chr3
ENST00000241261.6





TNFSF11
AATTCAAAAACTAATGGTGTACGTCACTAAACTCGAGTTTAGTGACGTACACCATTAG
2945
chr13
ENST00000398795.6





TNFSF11
AATTCAAAAACCGGATCAGGATGCAACATACCTCGAGGTATGTTGCATCCTGATCCGG
2946
chr13
ENST00000398795.6





TNFSF11
AATTCAAAAAGCAGTATATTTCTTCGTTCTTCTCGAGAAGAACGAAGAAATATACTGC
2947
chr13
ENST00000398795.6





TNFSF12
AATTCAAAAAGTATTCCCACTCTTATCTTACCTCGAGGTAAGATAAGAGTGGGAATAC
2948
chr17
ENST00000293825.10





TNFSF12
AATTCAAAAAGGGCATTGTGTTCACTGTACTCTCGAGAGTACAGTGAACACAATGCCC
2949
chr17
ENST00000293825.10





TNFSF12
AATTCAAAAACAGATGGAGGTTACACAACTTCTCGAGAAGTTGTGTAACCTCCATCTG
2950
chr17
ENST00000293825.10





TNFSF12-
AATTCAAAAACCTCACCTACTTCGGACTCTTCTCGAGAAGAGTCCGAAGTAGGTGAGG
2951
chr17
ENST00000293826.4





TNFSF12-
AATTCAAAAACTGTACTGTCAGGTGCACTTTCTCGAGAAAGTGCACCTGACAGTACAG
2952
chr17
ENST00000293826.4





TNFSF12-
AATTCAAAAAGCGCAGGCAGATGGAGGTTACCTCGAGGTAACCTCCATCTGCCTGCGC
2953
chr17
ENST00000293826.4





TNFSF13
AATTCAAAAACAGTTGCCCTCTGGTTGAGTTCTCGAGAACTCAACCAGAGGGCAACTG
2954
chr17
ENST00000338784.8





TNFSF13
AATTCAAAAAATGGCTCTGCTGACCCAACAACTCGAGTTGTTGGGTCAGCAGAGCCAT
2955
chr17
ENST00000338784.8





TNFSF13
AATTCAAAAACCCAACAAACAGAGCTGCAGACTCGAGTCTGCAGCTCTGTTTGTTGGG
2956
chr17
ENST00000338784.8





TNFSF13B
AATTCAAAAACCTGAAACACTACCCAATAATCTCGAGATTATTGGGTAGTGTTTCAGG
2957
chr13
ENST00000375887.8





TNFSF13B
AATTCAAAAACTACGCCATGGGACATCTAATCTCGAGATTAGATGTCCCATGGCGTAG
2958
chr13
ENST00000375887.8





TNFSF13B
AATTCAAAAAGTGACTTTGTTTCGATGTATTCTCGAGAATACATCGAAACAAAGTCAC
2959
chr13
ENST00000375887.8





TNFSF14
AATTCAAAAATCCTGGGAGCAGCTGATACAACTCGAGTTGTATCAGCTGCTCCCAGGA
2960
chr19
ENST00000599359.1





TNFSF14
AATTCAAAAAGACAGACCGACATCCCATTCACTCGAGTGAATGGGATGTCGGTCTGTC
2961
chr19
ENST00000599359.1





TNFSF14
AATTCAAAAAATGGGTCTGACACGTGGAGAACTCGAGTTCTCCACGTGTCAGACCCAT
2962
chr19
ENST00000599359.1





TNFSF15
AATTCAAAAAATTAAGACACTGATCACTAAACTCGAGTTTAGTGATCAGTGTCTTAAT
2963
chr9
ENST00000374045.4





TNFSF15
AATTCAAAAAGTCGGGAGACTACTTCATTTACTCGAGTAAATGAAGTAGTCTCCCGAC
2964
chr9
ENST00000374045.4





TNFSF15
AATTCAAAAAACTATAGGAGGAGAGCAAATACTCGAGTATTTGCTCTCCTCCTATAGT
2965
chr9
ENST00000374045.4





TNFSF18
AATTCAAAAACCTTCAGTTGGCTAATCTTTACTCGAGTAAAGATTAGCCAACTGAAGG
2966
chr1
ENST00000404377.3





TNFSF18
AATTCAAAAACTGAACCTCCTTGCGTGAATACTCGAGTATTCACGCAAGGAGGTTCAG
2967
chr1
ENST00000404377.3





TNFSF18
AATTCAAAAAATGATACAAACTCTAACAAACCTCGAGGTTTGTTAGAGTTTGTATCAT
2968
chr1
ENST00000404377.3





TNFSF4
AATTCAAAAAGTGGCCTCTCTGACTTACAAACTCGAGTTTGTAAGTCAGAGAGGCCAC
2969
chr1
ENST00000281834.3





TNFSF4
AATTCAAAAAGCAGAACAACTCAGTCATCATCTCGAGATGATGACTGAGTTGTTCTGC
2970
chr1
ENST00000281834.3





TNFSF4
AATTCAAAAAGCCAAGATTCGAGAGGAACAACTCGAGTTGTTCCTCTCGAATCTTGGC
2971
chr1
ENST00000281834.3





TNFSF8
AATTCAAAAAACTGTATGGTCTTGATCTATTCTCGAGAATAGATCAAGACCATACAGT
2972
chr9
ENST00000223795.2





TNFSF8
AATTCAAAAACAAACTACACAGGGTATTAAACTCGAGTTTAATACCCTGTGTAGTTTG
2973
chr9
ENST00000223795.2





TNFSF8
AATTCAAAAAGTGGATACATTCCAGTACATACTCGAGTATGTACTGGAATGTATCCAC
2974
chr9
ENST00000223795.2





TNFSF9
AATTCAAAAACAAGTTGGACCTTGATATTTACTCGAGTAAATATCAAGGTCCAACTTG
2975
chr19
ENST00000245817.4





TNFSF9
AATTCAAAAACTACTATGTCTTCTTTCAACTCTCGAGAGTTGAAAGAAGACATAGTAG
2976
chr19
ENST00000245817.4





TNFSF9
AATTCAAAAACCCTTCACCGAGGTCGGAATACTCGAGTATTCCGACCTCGGTGAAGGG
2977
chr19
ENST00000245817.4





TRIM16
AATTCAAAAAGCCGTTGTTCAGCGCAAATATCTCGAGATATTTGCGCTGAACAACGGC
2978
chr17
ENST00000336708.11





TRIM16
AATTCAAAAAGCCGTTGTTCAGCGCAAATATCTCGAGATATTTGCGCTGAACAACGGC
2979
chr17
ENST00000336708.11





TRIM16
AATTCAAAAACCGCATCAGGTGAACATCAAACTCGAGTTTGATGTTCACCTGATGCGG
2980
chr17
ENST00000336708.11





TSLP
AATTCAAAAAACTCAATGATAGCACCTAAACCTCGAGGTTTAGGTGCTATCATTGAGT
2981
chr5
ENST00000344895.3





TSLP
AATTCAAAAACGTCGCTTCAATCGACCTTTACTCGAGTAAAGGTCGATTGAAGCGACG
2982
chr5
ENST00000344895.3





TSLP
AATTCAAAAACCATCTTTATTATGGTCATATCTCGAGATATGACCATAATAAAGATGG
2983
chr5
ENST00000344895.3





TWSG1
AATTCAAAAAGACTGTGTTGGTATGTGTAATCTCGAGATTACACATACCAACACAGTC
2984
chr18
ENST00000262120.9





TWSG1
AATTCAAAAAGCGCCTTATTCCAGTGACAAACTCGAGTTTGTCACTGGAATAAGGCGC
2985
chr18
ENST00000262120.9





TWSG1
AATTCAAAAAGAATCACTGAGCTGTAACAAACTCGAGTTTGTTACAGCTCAGTGATTC
2986
chr18
ENST00000262120.9





TXLNA
AATTCAAAAAGCACATACTGTGTGGACAATACTCGAGTATTGTCCACACAGTATGTGC
2987
chr1
ENST00000373609.1





TXLNA
AATTCAAAAATGCTGATGCAGACATTGAATACTCGAGTATTCAATGTCTGCATCAGCA
2988
chr1
ENST00000373609.1





TXLNA
AATTCAAAAAAGCGAGGTATTCACCACATTCCTCGAGGAATGTGGTGAATACCTCGCT
2989
chr1
ENST00000373609.1





VASN
AATTCAAAAAAGCTTGACTACGCCGACTTTGCTCGAGCAAAGTCGGCGTAGTCAAGCT
2990
chr16
ENST00000304735.3





VASN
AATTCAAAAAAGCCAACAGGCTGCATGAAATCTCGAGATTTCATGCAGCCTGTTGGCT
2991
chr16
ENST00000304735.3





VASN
AATTCAAAAAGAGATCCTTTCCCATTTATTCCTCGAGGAATAAATGGGAAAGGATCTC
2992
chr16
ENST00000304735.3





VEGFA
AATTCAAAAACAAGATCCGCAGACGTGTAAACTCGAGTTTACACGTCTGCGGATCTTG
2993
chr6
ENST00000425836.6





VEGFA
AATTCAAAAACAAGATCCGCAGACGTGTAAACTCGAGTTTACACGTCTGCGGATCTTG
2994
chr6
ENST00000425836.6





VEGFA
AATTCAAAAAGCGCAAGAAATCCCGGTATAACTCGAGTTATACCGGGATTTCTTGCGC
2995
chr6
ENST00000425836.6





VSTM1
AATTCAAAAAATGAATATGCGGCACTGAAAGCTCGAGCTTTCAGTGCCGCATATTCAT
2996
chr19
ENST00000338372.6





VSTM1
AATTCAAAAACATTCCCAGAATGTGACATTTCTCGAGAAATGTCACATTCTGGGAATG
2997
chr19
ENST00000338372.6





VSTM1
AATTCAAAAAGAGTGACCTATGCTGAGCTAACTCGAGTTAGCTCAGCATAGGTCACTC
2998
chr19
ENST00000338372.6





WFIKKN1
AATTCAAAAATGCGCCCTGATCAGATGTATGCTCGAGCATACATCTGATCAGGGCGCA
2999
chr16
ENST00000319070.2





WFIKKN1
AATTCAAAAAGGACGTGCTCAAGGATGACAACTCGAGTTGTCATCCTTGAGCACGTCC
3000
chr16
ENST00000319070.2





WFIKKN1
AATTCAAAAAAGTGCTGCATCAACGTGTGTGCTCGAGCACACACGTTGATGCAGCACT
3001
chr16
ENST00000319070.2





WFIKKN2
AATTCAAAAATCAAGTTCTTGGGCACCAAGTCTCGAGACTTGGTGCCCAAGAACTTGA
3002
chr17
ENST00000311378.4





WFIKKN2
AATTCAAAAATACAACCGCTGCTATATGGACCTCGAGGTCCATATAGCAGCGGTTGTA
3003
chr17
ENST00000311378.4





WFIKKN2
AATTCAAAAACACCAGCTTGCTCAGATATTCCTCGAGGAATATCTGAGCAAGCTGGTG
3004
chr17
ENST00000311378.4





WNT1
AATTCAAAAACTGTCGAGAAACGGCGTTTATCTCGAGATAAACGCCGTTTCTCGACAG
3005
chr12
ENST00000293549.3





WNT1
AATTCAAAAAGACCTCGTCTACTTCGAGAAACTCGAGTTTCTCGAAGTAGACGAGGTC
3006
chr12
ENST00000293549.3





WNT1
AATTCAAAAAGCCACGAGTTTGGATGTTGTACTCGAGTACAACATCCAAACTCGTGGC
3007
chr12
ENST00000293549.3





WNT2
AATTCAAAAACCACAAATGGTCCCAATTAAGCTCGAGCTTAATTGGGACCATTTGTGG
3008
chr7
ENST00000265441.7





WNT2
AATTCAAAAATCGGGAATCTGCCTTTGTTTACTCGAGTAAACAAAGGCAGATTCCCGA
3009
chr7
ENST00000265441.7





WNT2
AATTCAAAAATTGACTATGGGATCAAATTTGCTCGAGCAAATTTGATCCCATAGTCAA
3010
chr7
ENST00000265441.7





WNT5A
AATTCAAAAACTCCCAGGACCCGCTTATTTACTCGAGTAAATAAGCGGGTCCTGGGAG
3011
chr3
ENST00000264634.8





WNT5A
AATTCAAAAACAAAGAATGCCAGTATCAATTCTCGAGAATTGATACTGGCATTCTTTG
3012
chr3
ENST00000264634.8





WNT5A
AATTCAAAAACCTGTTCAGATGTCAGAAGTACTCGAGTACTTCTGACATCTGAACAGG
3013
chr3
ENST00000264634.8





WNT7A
AATTCAAAAACATAGGAGAAGGCTCACAAATCTCGAGATTTGTGAGCCTTCTCCTATG
3014
chr3
ENST00000285018.4





WNT7A
AATTCAAAAAGGCGCAAGCATCATCTGTAACCTCGAGGTTACAGATGATGCTTGCGCC
3015
chr3
ENST00000285018.4





WNT7A
AATTCAAAAATCTTCGGGAAGGAGCTCAAAGCTCGAGCTTTGAGCTCCTTCCCGAAGA
3016
chr3
ENST00000285018.4





XCL1
AATTCAAAAAGCTGTGACAATGGCAACAATTCTCGAGAATTGTTGCCATTGTCACAGC
3017
chr1
ENST00000367818.3





XCL1
AATTCAAAAATTCAACTTGTCTCTATAATAGCTCGAGCTATTATAGAGACAAGTTGAA
3018
chr1
ENST00000367818.3





XCL1
AATTCAAAAAAGATTCTGGCTAGTGTCTATCCTCGAGGATAGACACTAGCCAGAATCT
3019
chr1
ENST00000367818.3





XCL2
AATTCAAAAAACTGCCAGTTAGCAGAATCAACTCGAGTTGATTCTGCTAACTGGCAGT
3020
chr1
ENST00000367819.2





XCL2
AATTCAAAAAGTAGGGAGTGAAGTCTCACATCTCGAGATGTGAGACTTCACTCCCTAC
3021
chr1
ENST00000367819.2





XCL2
AATTCAAAAAGAAATCCAACACCAGAAATAACTCGAGTTATTTCTGGTGTTGGATTTC
3022
chr1
ENST00000367819.2





ZFP36
AATTCAAAAAGATCCGACCCTGATGAATATGCTCGAGCATATTCATCAGGGTCGGATC
3023
chr19
ENST00000597629.1





ZFP36
AATTCAAAAAATCTGTCTCCTAGAATCTTATCTCGAGATAAGATTCTAGGAGACAGAT
3024
chr19
ENST00000597629.1





ZFP36
AATTCAAAAACCCTTTATTTATGACGACTTTCTCGAGAAAGTCGTCATAAATAAAGGG
3025
chr19
ENST00000597629.1









Example 16—Method of Making and Testing Cells Deficient in Specific Cytokine Expression Using Protein Expression Blockers (PEBL)—CAR19-GM-CSF PEBL

Inject tumor in SCID-Beige mice (3e6 Raji containing Luciferase) if performing in vivo CRS experiment. This should be completed 3 weeks prior to infusion of CAR-T into mice.


The following sERteps may be taken to provide CRS resistance. A selectable marker is required to enrich for this gene modification as cytokines are secreted proteins, thus. This example describes the making of a cell CAR19-GM-CSF PEBL, in which blockade of GM-CSF secretion mitigates risk of CRS. As those of skill in the art will recognize, certain of the steps may be conducted sequentially or out of the order listed below, though perhaps leading to different efficiency.


Step 1: T Cell Activation (Day 0)


Purify T cells from leukapheresis chamber using Miltenyi juman PanT isolation kit. Resuspend in media. Count cells. Determine number of human T cell activation CD3/CD28 beands required to obtain 3:1 bead:cell ratio. Wash beads 2× with T cell media. Dilute cells at 1.256 cells/mL in hXcyte media. Add human T cell activation CD3/CD28 beads. Aliquot 4 ml/well of 1.256 cells/mL solution into 6-well plate. Incubate at 37° C.


Step 2: Transduction of T Cells with PEBL CAR (Day 1)


T cells are transduced with a CAR targeted to (i.e., that recognizes) one or more antigen or protein targets, for example with a Lentivirus containing a CAR construct targeting CD19, combined with the anti-GM-CSF PEBL. Expression from a polycistronic vector is preferable, allowing CD34 expression to mark both CAR and PEBL expression. However, expression can be achieved with the same viral vector expressing both the CAR and PEBL individually, or as independent transductions with separate vectors containing CAR and PEBL. Any other suitable method of transduction may be used, for example, AAV, retrovirus, etc, or through direct insertion of the CAR PEBL complex into a targeted location of the genome using homology directed repair and a donor vector containing the construct.


Step 3: Assessment of Td Efficiency (Day 10)


Take 5×105 cells from each sample and analyze by flow cytometry. Wash samples with RB. Add 3 μL of anti-CD34 PE antibody. Add 5 μL of CD3 APC and 2 μL of anti-FAB BV421 (detects CAR transduction). Wash. Perform flow cytometry. Cells should be CD34-positive indicating expression of CAR and PEBL from a polycistronic vector. Harvest T cells (Day 11).


Purification of CAR-T cells. CAR+(CD34+) and GC-CSF deficient (PEBL+) cells can be enriched using a CD34-positive selection on the Miltenyi Automacs. This enriches the GC-CSF suppressed cells and enriches CAR+ cells.


Step 4: Assessment of CAR-T Activity


Inject 3×107 CAR-T cells per mouse I.P. Assess serum cytokine levels. (Day 12, Day 13, Day 14). Measure serum cytokine levels using Luminex multiplex profiling assay to check for elevations in CRS-related cytokines. Perform a 4-hour chromium release assay against target cells (Raji) to assess in vitro activity (Day 11).


PEBL constructs targeting GM-CSF are provided in FIG. 6 and Tables 17 and 18.









TABLE 17







Anti-GM-CSF PEBL Constructs (SEQ ID NOS 3071-3076, respectively)

















ER-Retention




CD28 Leader
VL
Linker
VH
Domain
2a
trCD34





MALPVTALLLP
DIELTQPPSVS
GGGGS(n)n =
QVQLVESGGG
EQKLISEEDLKD




LALLLHAARP
VAPGQTARISC
1, 2, 3, 4
LVQPGGSLRL
EL (SEQ ID




(SEQ ID
SGDNLPGKYVH
(SEQ ID
SCAASGFTFS
NO: 3028)




NO: 1)
WYQQKPGQAPV
NO: 3069)
SHWMSWVRQA






LVIYYDSNRPS

PGKGLEWVSN






GIPERFSGSNS

IWRGPYIYYA






GNTATLTISGT

DSVKGRFTIS






QAEDEADYYCQ

RDNSKNTLYL






SRTQTTIVFGG

QMNSLRAEDT






GTKLTVLGQ

AVYYCARFQG






(SEQ ID

YGGGFDYWGQ






NO: 3026)

GTLVTVSS








(SEQ ID








NO: 3027)








MALPVTALLLP
DIELTQPPSVS
GGGGS(n)n =
QVQLVESGGG
(GGGGS)4AEK




LALLLHAARP
VAPGQTARISC
1, 2, 3, 4
LVQPGGSLRL
DEL (SEQ ID




(SEQ ID
SGDNLPGKYVH
(SEQ ID
SCAASGFTFS
NO: 3029)




NO: 1)
WYQQKPGQAPV
NO: 3069)
SHWMSWVRQA






LVIYYDSNRPS

PGKGLEWVSN






GIPERFSGSNS

IWRGPYIYYA






GNTATLTISGT

DSVKGRFTIS






QAEDEADYYCQ

RDNSKNTLYL






SRTQTTIVFGG

QMNSLRAEDT






GTKLTVLGQ

AVYYCARFQG






(SEQ ID

YGGGFDYWGQ






NO: 3026)

GTLVTVSS








(SEQ ID








NO: 3027)








MALPVTALLLP
DIELTQPPSVS
GGGGS(n)n =
QVQLVESGGG
TTTPAPRPPTPA




LALLLHAARP
VAPGQTARISC
1, 2, 3, 4
LVQPGGSLRL
PTIASQPLSLRP




(SEQ ID
SGDNLPGKYVH
(SEQ ID
SCAASGFTFS
EACRPAAGGAVH




NO: 1)
WYQQKPGQAPV
NO: 3069)
SHWMSWVRQA
TRGLDFACDIYI





LVIYYDSNRPS

PGKGLEWVSN
WAPLAGTCGVLL





GIPERFSGSNS

IWRGPYIYYA
LSLVITLYCNHR





GNTATLTISGT

DSVKGRFTIS
NLYKYKSRRSFI





QAEDEADYYCQ

RDNSKNTLYL
DEKKMP





SRTQTTIVFGG

QMNSLRAEDT
(SEQ ID





GTKLTVLGQ

AVYYCARFQG
NO: 3030)





(SEQ ID

YGGGFDYWGQ






NO: 3026)

GTLVTVSS








(SEQ ID








NO: 3027)








MALPVTALLLP
DIELTQPPSVS
GGGGS(n)n =
QVQLVESGGG
EQKLISEEDLKD
GSGATNFSLLK
MPRGWTALCLL


LALLLHAARP
VAPGQTARISC
1, 2, 3, 4
LVQPGGSLRL
EL (SEQ ID
QAGDVEENPGP
SLLPSGFMSLD


(SEQ ID
SGDNLPGKYVH
(SEQ ID
SCAASGFTFS
NO: 3028)
(SEQ ID
NNGTATPELPT


NO: 1)
WYQQKPGQAPV
NO: 3069)
SHWMSWVRQA

NO: 3031)
QGTFSNVSTNV



LVIYYDSNRPS

PGKGLEWVSN


SYQETTTPSTL



GIPERFSGSNS

IWRGPYIYYA


GSTSLHPVSQH



GNTATLTISGT

DSVKGRFTIS


GNEATTNITET



QAEDEADYYCQ

RDNSKNTLYL


TVKFTSTSVIT



SRTQTTIVFGG

QMNSLRAEDT


SVYGNTNSSVQ



GTKLTVLGQ

AVYYCARFQG


SQTSVISTVFT



(SEQ ID

YGGGFDYWGQ


TPANVSTPETT



NO: 3026)

GTLVTVSS


LKPSLSPGNVS





(SEQ ID


DLSTTSTSLAT





NO: 3027)


SPTKPYTSSSP








ILSDIKAEIKC








SGIREVKLTQG








ICLEQNKTSSC








AEFKKDRGEGL








ARVLCGEEQAD








ADAGAQVCSLL








LAQSEVRPQCL








LLVLANRTEIS








SKLQLMKKHQS








DLKKLGILDFT








EQDVASHQSYS








QKTLIALVTSG








ALLAVLGITGY








FLMNRRSWSPI








(SEQ ID








NO: 3032)





MALPVTALLLP
DIELTQPPSVS
GGGGS(n)n =
QVQLVESGGG
(GGGGS)4AEK
GSGATNFSLLK
MPRGWTALCLL


LALLLHAARP
VAPGQTARISC
1, 2, 3, 4
LVQPGGSLRL
DEL (SEQ ID
QAGDVEENPGP
SLLPSGFMSLD


(SEQ ID
SGDNLPGKYVH
(SEQ ID
SCAASGFTFS
NO: 3029)
(SEQ ID
NNGTATPELPT


NO: 1)
WYQQKPGQAPV
NO: 3069)
SHWMSWVRQA

NO: 3031)
QGTFSNVSTNV



LVIYYDSNRPS

PGKGLEWVSN


SYQETTTPSTL



GIPERFSGSNS

IWRGPYIYYA


GSTSLHPVSQH



GNTATLTISGT

DSVKGRFTIS


GNEATTNITET



QAEDEADYYCQ

RDNSKNTLYL


TVKFTSTSVIT



SRTQTTIVFGG

QMNSLRAEDT


SVYGNTNSSVQ



GTKLTVLGQ

AVYYCARFQG


SQTSVISTVFT



(SEQ ID

YGGGFDYWGQ


TPANVSTPETT



NO: 3026)

GTLVTVSS


LKPSLSPGNVS





(SEQ ID


DLSTTSTSLAT





NO: 3027)


SPTKPYTSSSP








ILSDIKAEIKC








SGIREVKLTQG








ICLEQNKTSSC








AEFKKDRGEGL








ARVLCGEEQAD








ADAGAQVCSLL








LAQSEVRPQCL








LLVLANRTEIS








SKLQLMKKHQS








DLKKLGILDFT








EQDVASHQSYS








QKTLIALVTSG








ALLAVLGITGY








FLMNRRSWSPI








(SEQ ID








NO: 3032)





MALPVTALLLP
DIELTQPPSVS
GGGGS(n)n =
QVQLVESGGG
TTTPAPRPPTPA
GSGATNFSLLK
MPRGWTALCLL


LALLLHAARP
VAPGQTARISC
1, 2, 3, 4
LVQPGGSLRL
PTIASQPLSLRP
QAGDVEENPGP
SLLPSGFMSLD


(SEQ ID
SGDNLPGKYVH
(SEQ ID
SCAASGFTFS
EACRPAAGGAVH
(SEQ ID
NNGTATPELPT


NO: 1)
WYQQKPGQAPV
NO: 3069)
SHWMSWVRQA
TRGLDFACDIYI
NO: 3031)
QGTFSNVSTNV



LVIYYDSNRPS

PGKGLEWVSN
WAPLAGTCGVLL

SYQETTTPSTL



GIPERFSGSNS

IWRGPYIYYA
LSLVITLYCNHR

GSTSLHPVSQH



GNTATLTISGT

DSVKGRFTIS
NLYKYKSRRSFI

GNEATTNITET



QAEDEADYYCQ

RDNSKNTLYL
DEKKMP

TVKFTSTSVIT



SRTQTTIVFGG

QMNSLRAEDT
(SEQ ID NO: 

SVYGNTNSSVQ



GTKLTVLGQ

AVYYCARFQG
3030)

SQTSVISTVFT



(SEQ ID

YGGGFDYWGQ


TPANVSTPETT



NO: 3026)

GTLVTVSS


LKPSLSPGNVS





(SEQ ID


DLSTTSTSLAT





NO: 3027)


SPTKPYTSSSP








ILSDIKAEIKC








SGIREVKLTQG








ICLEQNKTSSC








AEFKKDRGEGL








ARVLCGEEQAD








ADAGAQVCSLL








LAQSEVRPQCL








LLVLANRTEIS








SKLQLMKKHQS








DLKKLGILDFT








EQDVASHQSYS








QKTLIALVTSG








ALLAVLGITGY








FLMNRRSWSPI








(SEQ ID








NO: 3032)
















TABLE 18







CD19 CAR with GM-CSF PEBL Constructs (SEQ ID NOS 3077-3082, respectively)




































text missing or illegible when filed





CD8a
CD19

CD19
CD8a
CD2
CD28


CD8a




text missing or illegible when filed





Leader
VH
Linker
VL
Hinge
8tm
Co-stim
CD3z
2a
Leader
VL
Linker
VH

text missing or illegible when filed

2a
Tag





MALPVT
DIQMTQTTS
GGGGS4
EVKLQEQGP
TTTPAPRPP
FWVLVVVGG
RSKRSRLLH
RVKFSRSA
GSGATNFS
MALPVT
DIELQTPP
GGGG
QVQLVESG
EQKLISEE




ALLLPL
SLSASLGDR
(SEQ ID
GLVAPSQSL
TPAPTIASQ
VLACYSLLV
SDYMNMTPR
DAPAYKQG
LLKQAGDV
ALLLPL
SVSVAPGQ
S(n)= 1,
GGLVQPGG
DLKDEL




ALLLHA
VTISCRASQ
NO: 9)
SVTCTVSGV
PLSLRPEAC
TVAFIIFWV
RPGPTRKHY
QNQLYNEL
EENPGP
ALLLHA
TARISCSG
2, 3, 4
SLRLSCAA
(SEQ ID




ARP
DISKYLNWY

SLPDYGVSW
RPAAGGAVH
(SEQ ID
QPYAPPRDF
NLGRREEY
(SEQ ID
ARP
DNLPGKYV
(SEQ ID
SGFTFSSH
NO: 3028)




(SEQ ID
QQKPDGTVK

IRQPPRKGL
TRGLDFACD
NO: 3036)
AAYRS
DVLDKRRG
NO: 3031)
(SEQ ID
HWYQQKPG
NO: 3069)
WMSWVRQA





NO: 1)
LLIYHTSRL

EWLGVIWGS
(SEQ ID

(SEQ ID
RDPEMGGK

NO: 1)
QAPVLVIY

PGKGLEWV






HSGVPSRFS

ETTYYNSAL
NO: 3035)

NO: 3037)
PRRKNPQE


YDSNRPSG

SNIWRGPY






GSGSGTDYS

KSRLTIIKD



GLYNELQK


IPERFSGS

IYYADSVK






LTISNLEQE

NSKSQVFLK



DKMAEAYS


NSGNTATL

GRFTISRD






DIATYFCQQ

MNSLQTDDT



EIGMKGER


TISGTQAE

NSKNTLYL






GNTLPYTFG

AIYYCAKHY



RRGKGHDG


DEADYYCQ

QMNSLRAE






GGTKLEITR

YYGGSYAMD



LYQGLSTA


SRTQTTIV

DTAVYYCA






ADAAPTVSI

YWGQGTSVS



TKDTYDAL


FGGGTKLT

RFQGYGGG






FPPSSN

(SEQ ID



HMQALPPR


VLGQ

FDYWGQGT






(SEQ ID

NO: 3034)



(SEQ ID


(SEQ ID

LVTVSS






NO: 3033)





NO: 3038)


NO: 3026)

(SEQ ID

















NO: 3027)








MALPVT
DIQMTQTTS
GGGGS4
EVKLQEQGP
TTTPAPRPP
FWVLVVVGG
RSKRSRLLH
RVKFSRSA
GSGATNFS
MALPVT
DIELQTPP
GGGG
QVQLVESG
(GGGGS)4




ALLLPL
SLSASLGDR
(SEQ ID
GLVAPSQSL
TPAPTIASQ
VLACYSLLV
SDYMNMTPR
DAPAYKQG
LLKQAGDV
ALLLPL
SVSVAPGQ
S(n)= 1,
GGLVQPGG
AEKDEL




ALLLHA
VTISCRASQ
NO: 9)
SVTCTVSGV
PLSLRPEAC
TVAFIIFWV
RPGPTRKHY
QNQLYNEL
EENPGP
ALLLHA
TARISCSG
2, 3, 4
SLRLSCAA
(SEQ ID




ARP
DISKYLNWY

SLPDYGVSW
RPAAGGAVH
(SEQ ID
QPYAPPRDF
NLGRREEY
(SEQ ID
ARP
DNLPGKYV
(SEQ ID
SGFTFSSH
NO: 3029)




(SEQ ID
QQKPDGTVK

IRQPPRKGL
TRGLDFACD
NO: 3036)
AAYRS
DVLDKRRG
NO: 3031)
(SEQ ID
HWYQQKPG
NO: 3069)
WMSWVRQA





NO: 1)
LLIYHTSRL

EWLGVIWGS
(SEQ ID

(SEQ ID
RDPEMGGK

NO: 1)
QAPVLVIY

PGKGLEWV






HSGVPSRFS

ETTYYNSAL
NO: 3035)

NO: 3037)
PRRKNPQE


YDSNRPSG

SNIWRGPY






GSGSGTDYS

KSRLTIIKD



GLYNELQK


IPERFSGS

IYYADSVK






LTISNLEQE

NSKSQVFLK



DKMAEAYS


NSGNTATL

GRFTISRD






DIATYFCQQ

MNSLQTDDT



EIGMKGER


TISGTQAE

NSKNTLYL






GNTLPYTFG

AIYYCAKHY



RRGKGHDG


DEADYYCQ

QMNSLRAE






GGTKLEITR

YYGGSYAMD



LYQGLSTA


SRTQTTIV

DTAVYYCA






ADAAPTVSI

YWGQGTSVS



TKDTYDAL


FGGGTKLT

RFQGYGGG






FPPSSN

(SEQ ID



HMQALPPR


VLGQ

FDYWGQGT






(SEQ ID

NO: 3034)



(SEQ ID


(SEQ ID

LVTVSS






NO: 3033)





NO: 3038)


NO: 3026)

(SEQ ID

















NO: 3027)








MALPVT
DIQMTQTTS
GGGGS4
EVKLQEQGP
TTTPAPRPP
FWVLVVVGG
RSKRSRLLH
RVKFSRSA
GSGATNFS
MALPVT
DIELQTPP
GGGG
QVQLVESG
TTTPAPRP




ALLLPL
SLSASLGDR
(SEQ ID
GLVAPSQSL
TPAPTIASQ
VLACYSLLV
SDYMNMTPR
DAPAYKQG
LLKQAGDV
ALLLPL
SVSVAPGQ
S(n)= 1,
GGLVQPGG
PTPAPTIA




ALLLHA
VTISCRASQ
NO: 9)
SVTCTVSGV
PLSLRPEAC
TVAFIIFWV
RPGPTRKHY
QNQLYNEL
EENPGP
ALLLHA
TARISCSG
2, 3, 4
SLRLSCAA
SQPLSLRP




ARP
DISKYLNWY

SLPDYGVSW
RPAAGGAVH
(SEQ ID
QPYAPPRDF
NLGRREEY
(SEQ ID
ARP
DNLPGKYV
(SEQ ID
SGFTFSSH
EACRPAAG




(SEQ ID
QQKPDGTVK

IRQPPRKGL
TRGLDFACD
NO: 3036)
AAYRS
DVLDKRRG
NO: 3031)
(SEQ ID
HWYQQKPG
NO: 3069)
WMSWVRQA
AAVHTRGL




NO: 1)
LLIYHTSRL

EWLGVIWGS
(SEQ ID

(SEQ ID
RDPEMGGK

NO: 1)
QAPVLVIY

PGKGLEWV
DFACDIYI





HSGVPSRFS

ETTYYNSAL
NO: 3035)

NO: 3037)
PRRKNPQE


YDSNRPSG

SNIWRGPY
WAPLAGTC





GSGSGTDYS

KSRLTIIKD



GLYNELQK


IPERFSGS

IYYADSVK
GVLLLSLV





LTISNLEQE

NSKSQVFLK



DKMAEAYS


NSGNTATL

GRFTISRD
ITLYCNHR





DIATYFCQQ

MNSLQTDDT



EIGMKGER


TISGTQAE

NSKNTLYL
NLYKYKSR





GNTLPYTFG

AIYYCAKHY



RRGKGHDG


DEADYYCQ

QMNSLRAE
RSFIDEKK





GGTKLEITR

YYGGSYAMD



LYQGLSTA


SRTQTTIV

DTAVYYCA
MP





ADAAPTVSI

YWGQGTSVS



TKDTYDAL


FGGGTKLT

RFQGYGGG
(SEQ ID





FPPSSN

(SEQ ID



HMQALPPR


VLGQ

FDYWGQGT
NO: 3030)





(SEQ ID

NO: 3034)



(SEQ ID


(SEQ ID

LVTVSS






NO: 3033)





NO: 3038)


NO: 3026)

(SEQ ID

















NO: 3027)








MALPVT
DIQMTQTTS
GGGGS4
EVKLQEQGP
TTTPAPRPP
FWVLVVVGG
RSKRSRLLH
RVKFSRSA
GSGATNFS
MALPVT
DIELQTPP
GGGG
QVQLVESG
EQKLISEE
GSGATN
MPRGWTA


ALLLPL
SLSASLGDR
(SEQ ID
GLVAPSQSL
TPAPTIASQ
VLACYSLLV
SDYMNMTPR
DAPAYKQG
LLKQAGDV
ALLLPL
SVSVAPGQ
S(n)= 1,
GGLVQPGG
DLKDEL
FSLLKQ
LCLLSLL


ALLLHA
VTISCRASQ
NO: 9)
SVTCTVSGV
PLSLRPEAC
TVAFIIFWV
RPGPTRKHY
QNQLYNEL
EENPGP
ALLLHA
TARISCSG
2, 3, 4
SLRLSCAA
(SEQ ID
AGDVEE
PSGFMSL


ARP
DISKYLNWY

SLPDYGVSW
RPAAGGAVH
(SEQ ID
QPYAPPRDF
NLGRREEY
(SEQ ID
ARP
DNLPGKYV
(SEQ ID
SGFTFSSH
NO: 3028)
NPGP
DNNGTAT


(SEQ ID
QQKPDGTVK

IRQPPRKGL
TRGLDFACD
NO: 3036)
AAYRS
DVLDKRRG
NO: 3031)
(SEQ ID
HWYQQKPG
NO: 3069)
WMSWVRQA

(SEQ
PELPTQG


NO: 1)
LLIYHTSRL

EWLGVIWGS
(SEQ ID

(SEQ ID
RDPEMGGK

NO: 1)
QAPVLVIY

PGKGLEWV

ID NO:
TFSNVST



HSGVPSRFS

ETTYYNSAL
NO: 3035)

NO: 3037)
PRRKNPQE


YDSNRPSG

SNIWRGPY

3031)
NVSYQET



GSGSGTDYS

KSRLTIIKD



GLYNELQK


IPERFSGS

IYYADSVK


TTPSTLG



LTISNLEQE

NSKSQVFLK



DKMAEAYS


NSGNTATL

GRFTISRD


STSLHPV



DIATYFCQQ

MNSLQTDDT



EIGMKGER


TISGTQAE

NSKNTLYL


SQHGNEA



GNTLPYTFG

AIYYCAKHY



RRGKGHDG


DEADYYCQ

QMNSLRAE


TTNITET



GGTKLEITR

YYGGSYAMD



LYQGLSTA


SRTQTTIV

DTAVYYCA


TVKFTST



ADAAPTVSI

YWGQGTSVS



TKDTYDAL


FGGGTKLT

RFQGYGGG


SVITSVY



FPPSSN

(SEQ ID



HMQALPPR


VLGQ

FDYWGQGT


GNTNSSV



(SEQ ID

NO: 3034)



(SEQ ID


(SEQ ID

LVTVSS


QSQTSVI



NO: 3033)





NO: 3038)


NO: 3026)

(SEQ ID


STVFTTP














NO: 3027)


ANVSTPE

















TTLKPSL

















SPGNVSD

















LSTTSTS

















LATSPTK

















PYTSSSP

















ILSDIKA

















EIKCSGI

















REVKLTQ

















GICLEQN

















KTSSCAE

















FKKDRGE

















GLARVLC

















GEEQADA

















DAGAQVC

















SLLLAQS

















EVRPQCL

















LLVLANR

















TEISSKL

















QLMKKHQ

















SDLKKLG

















ILDFTEQ

















DVASHQS

















YSQKTLI

















ALVTSGA

















LLAVLGI

















TGYFLMN

















RRSWSPI

















(SEQ ID

















NO: 10)





MALPVT
DIQMTQTTS
GGGGS4
EVKLQEQGP
TTTPAPRPP
FWVLVVVGG
RSKRSRLLH
RVKFSRSA
GSGATNFS
MALPVT
DIELQTPP
GGGG
QVQLVESG
(GGGGS)4
GSGATN
MPRGWTA


ALLLPL
SLSASLGDR
(SEQ ID
GLVAPSQSL
TPAPTIASQ
VLACYSLLV
SDYMNMTPR
DAPAYKQG
LLKQAGDV
ALLLPL
SVSVAPGQ
S(n)= 1,
GGLVQPGG
AEKDEL
FSLLKQ
LCLLSLL


ALLLHA
VTISCRASQ
NO: 9)
SVTCTVSGV
PLSLRPEAC
TVAFIIFWV
RPGPTRKHY
QNQLYNEL
EENPGP
ALLLHA
TARISCSG
2, 3, 4
SLRLSCAA
(SEQ ID
AGDVEE
PSGFMSL


ARP
DISKYLNWY

SLPDYGVSW
RPAAGGAVH
(SEQ ID
QPYAPPRDF
NLGRREEY
(SEQ ID
ARP
DNLPGKYV
(SEQ ID
SGFTFSSH
NO: 3029)
NPGP
DNNGTAT


(SEQ ID
QQKPDGTVK

IRQPPRKGL
TRGLDFACD
NO: 3036)
AAYRS
DVLDKRRG
NO: 3031)
(SEQ ID
HWYQQKPG
NO: 3069)
WMSWVRQA

(SEQ
PELPTQG


NO: 1)
LLIYHTSRL

EWLGVIWGS
(SEQ ID

(SEQ ID
RDPEMGGK

NO: 1)
QAPVLVIY

PGKGLEWV

ID NO:
TFSNVST



HSGVPSRFS

ETTYYNSAL
NO: 3035)

NO: 3037)
PRRKNPQE


YDSNRPSG

SNIWRGPY

3031)
NVSYQET



GSGSGTDYS

KSRLTIIKD



GLYNELQK


IPERFSGS

IYYADSVK


TTPSTLG



LTISNLEQE

NSKSQVFLK



DKMAEAYS


NSGNTATL

GRFTISRD


STSLHPV



DIATYFCQQ

MNSLQTDDT



EIGMKGER


TISGTQAE

NSKNTLYL


SQHGNEA



GNTLPYTFG

AIYYCAKHY



RRGKGHDG


DEADYYCQ

QMNSLRAE


TTNITET



GGTKLEITR

YYGGSYAMD



LYQGLSTA


SRTQTTIV

DTAVYYCA


TVKFTST



ADAAPTVSI

YWGQGTSVS



TKDTYDAL


FGGGTKLT

RFQGYGGG


SVITSVY



FPPSSN

(SEQ ID



HMQALPPR


VLGQ

FDYWGQGT


GNTNSSV



(SEQ ID

NO: 3034)



(SEQ ID


(SEQ ID

LVTVSS


QSQTSVI



NO: 3033)





NO: 3038)


NO: 3026)

(SEQ ID


STVFTTP














NO: 3027)


ANVSTPE

















TTLKPSL

















SPGNVSD

















LSTTSTS

















LATSPTK

















PYTSSSP

















ILSDIKA

















EIKCSGI

















REVKLTQ

















GICLEQN

















KTSSCAE

















FKKDRGE

















GLARVLC

















GEEQADA

















DAGAQVC

















SLLLAQS

















EVRPQCL

















LLVLANR

















TEISSKL

















QLMKKHQ

















SDLKKLG

















ILDFTEQ

















DVASHQS

















YSQKTLI

















ALVTSGA

















LLAVLGI

















TGYFLMN

















RRSWSPI

















(SEQ ID

















NO: 10)





MALPVT
DIQMTQTTS
GGGGS4
EVKLQEQGP
TTTPAPRPP
FWVLVVVGG
RSKRSRLLH
RVKFSRSA
GSGATNFS
MALPVT
DIELQTPP
GGGG
QVQLVESG
TTTPAPRP
GSGATN
MPRGWTA


ALLLPL
SLSASLGDR
(SEQ ID
GLVAPSQSL
TPAPTIASQ
VLACYSLLV
SDYMNMTPR
DAPAYKQG
LLKQAGDV
ALLLPL
SVSVAPGQ
S(n)= 1,
GGLVQPGG
PTPAPTIA
FSLLKQ
LCLLSLL


ALLLHA
VTISCRASQ
NO: 9)
SVTCTVSGV
PLSLRPEAC
TVAFIIFWV
RPGPTRKHY
QNQLYNEL
EENPGP
ALLLHA
TARISCSG
2, 3, 4
SLRLSCAA
SQPLSLRP
AGDVEE
PSGFMSL


ARP
DISKYLNWY

SLPDYGVSW
RPAAGGAVH
(SEQ ID
QPYAPPRDF
NLGRREEY
(SEQ ID
ARP
DNLPGKYV
(SEQ ID
SGFTFSSH
EACRPAAG
NPGP
DNNGTAT


(SEQ ID
QQKPDGTVK

IRQPPRKGL
TRGLDFACD
NO: 3036)
AAYRS
DVLDKRRG
NO: 3031)
(SEQ ID
HWYQQKPG
NO: 3069)
WMSWVRQA
AAVHTRGL
(SEQ
PELPTQG


NO: 1)
LLIYHTSRL

EWLGVIWGS
(SEQ ID

(SEQ ID
RDPEMGGK

NO: 1)
QAPVLVIY

PGKGLEWV
DFACDIYI
ID NO:
TFSNVST



HSGVPSRFS

ETTYYNSAL
NO: 3035)

NO: 3037)
PRRKNPQE


YDSNRPSG

SNIWRGPY
WAPLAGTC
3031)
NVSYQET



GSGSGTDYS

KSRLTIIKD



GLYNELQK


IPERFSGS

IYYADSVK
GVLLLSLV

TTPSTLG



LTISNLEQE

NSKSQVFLK



DKMAEAYS


NSGNTATL

GRFTISRD
ITLYCNHR

STSLHPV



DIATYFCQQ

MNSLQTDDT



EIGMKGER


TISGTQAE

NSKNTLYL
NLYKYKSR

SQHGNEA



GNTLPYTFG

AIYYCAKHY



RRGKGHDG


DEADYYCQ

QMNSLRAE
RSFIDEKK

TTNITET



GGTKLEITR

YYGGSYAMD



LYQGLSTA


SRTQTTIV

DTAVYYCA
MP

TVKFTST



ADAAPTVSI

YWGQGTSVS



TKDTYDAL


FGGGTKLT

RFQGYGGG
(SEQ ID

SVITSVY



FPPSSN

(SEQ ID



HMQALPPR


VLGQ

FDYWGQGT
NO: 3030)

GNTNSSV



(SEQ ID

NO: 3034)



(SEQ ID


(SEQ ID

LVTVSS


QSQTSVI



NO: 3033)





NO: 3038)


NO: 3026)

(SEQ ID


STVFTTP














NO: 3027)


ANVSTPE

















TTLKPSL

















SPGNVSD

















LSTTSTS

















LATSPTK

















PYTSSSP

















ILSDIKA

















EIKCSGI

















REVKLTQ

















GICLEQN

















KTSSCAE

















FKKDRGE

















GLARVLC

















GEEQADA

















DAGAQVC

















SLLLAQS

















EVRPQCL

















LLVLANR

















TEISSKL

















QLMKKHQ

















SDLKKLG

















ILDFTEQ

















DVASHQS

















YSQKTLI

















ALVTSGA

















LLAVLGI

















TGYFLMN

















RRSWSPI

















(SEQ ID

















NO: 10)






text missing or illegible when filed indicates data missing or illegible when filed







Example 17—In Vitro CRS Assay for Testing Efficacy of Inducing CRS after Deletion of GM-CSF in CART19

Cell lines: CD19 positive B-ALL cell line RAMOS was used to evaluate CRS in an in vitro assay. Prior to assay, RAMOS cells were stably transfected with GFP using lentiviral transduction and cultured in RPMI+10% FBS+ Pen/Strep.


Isolation of normal donor monocytes and T cells: Primary human T cells and monocytes were isolated from normal healthy human donors. T cell cells were isolated from PBMC using CD4+CD8+ selection (Miltenyi Biotec) and monocytes were separated using Miltenyi biotec classical Monocyte Isolation beads according to the manufactures protocol.


T cell culture and CAR-T gene editing: T cells were subsequently resuspended at a concentration of 1×106 cells/mL in Xcyte media supplemented with 50 U/mL IL-2 and 10 ng/ml IL-15 in the presence of anti-CD3/CD28 beads (Bead to cell ratio 3:1). Twenty-four hours after initial stimulation, T cells were transfected with lentiviral vector encoding the CD19 CAR construct in the presence of polybrene (final conc. 6 μg/ml). Stimulatory beads were removed on day 2. CART19 cells were suspended at 4×106 T cells were electroporated in 100 μl buffer P3 with 15 μg spCas9 mRNA (Trilink CA.) and 20 μg of GM-CSF gRNA (Trilink) using a nucleofector 4D, program EO-115. Control CAR19 T cells were electroporated without GM-CSF gRNA. T-cells were subsequently evaluated at day 6 for CD34 (bicistronic marker of CAR-T) and intracellular GM-CSF expression using flowcytometry prior to co-culture assay.


Production of monocyte lineage cells: For iDC generation, monocytes (1×106) were plated in 6 mLs of RPMI 1640 supplemented with supplemented with 0.1 mmol/L MEM Non-Essential Amino Acids, 2 mmol/L L-glutamine, 100 units/mL penicillin, 100 μg/mL streptomycin (Life Technologies) and 10% fetal calf serum (cR10). cR10 was subsequently supplemented with 0.2 μg/mL human IL-4 (Peptotec) and 0.2 μg/mL GM-CSF (Peptotec).


For macrophage and activated macrophage generation cR10 was supplemented with 10% human A/B serum instead of fetal calf serum (hR10). For activated macrophages hR10 medium was supplemented with 30 ng/ml of LPS. On day 4 cytokines and LPS were replenished. Macrophages, activated macrophages and iDC were harvested using 2 mmol/L EDTA (immature dendritic cells) on day 6 post-isolation. Twenty-four hours later, cells were harvested with 2 mmol/L EDTA and stained with CD45, CD80 and CD86 to confirm immature DC and mature DC differentiation.


Co-culture assay: CAR-T cells were combined at the following ratios in 200 ul per 96 well: 12.5K UCART-19 (with or with-out GM-CSF KO), Target, 50K Ramos cells (CD19+) and monocyte derived cells, 1K iDC or 5K macrophages or 5K activated macrophages.


Co-cultures were subsequently incubated for at 37° C. and 100 ul of assay supernatant was collected at 24 hr and 48 hr for cytokine analysis.


Measurement of Cytokine Levels


Cytokine concentration determination from culture supernatants was performed using IL-6 ELISA plate (R&D systems). Prior to analysis supernatant's were centrifuged at 300 g for 10 mins at 4° C. and subsequently diluted 1:10 in assay calibrator diluent. Measurements were performed using standard product protocols.


GM-CSF deficient CART19 induced significantly lower il-6 expression across the different monocyte lineages. FIGS. 8 and 10


Example 18—In Vitro CRS Assay for Testing Efficacy of Inducing CRS after Deletion of GM-CSF in T Cells

Isolation of normal donor monocytes and T cells: Primary human T cells and monocytes were isolated from normal healthy human donors. T cell cells were isolated from PBMC using CD4+CD8+ selection (Miltenyi Biotec) and monocytes were separated using Miltenyi Biotec classical Monocyte Isolation beads according to the manufactures protocol.


T cell culture and gene editing: T cells were subsequently resuspended at a concentration of 106 cells/mL in Xcyte media supplemented with 50 U/mL IL-2 and 10 ng/ml IL-15 in the presence of anti-CD3/CD28 beads (Bead to cell ratio 3:1) (Life Technologies, catalog #111.32D). Twenty-four hours after initial stimulation. Stimulatory beads were removed on day 2.4×106 T cells were electroporated in 100 μl buffer P3 with 15 μg spCas9 mRNA (Trilink CA.) and 20 μg of TRAC gRNA (Trilink) using a nucleofector 4D, program EO-115. Control CAR19 T cells were electroporated without GM-CSF gRNA. T-cells were subsequently evaluated at day 6 for GM-CSF expression prior to co-culture assay using flowcytometry.


Production of monocyte lineage cells: For iDC generation, monocytes (1×106) were plated in 6 mLs of RPMI 1640 supplemented with supplemented with 0.1 mmol/L MEM Non-Essential Amino Acids, 2 mmol/L L-glutamine, 100 units/mL penicillin, 100 μg/mL streptomycin (Life Technologies) and 10% fetal calf serum (cR10). cR10 was subsequently supplemented with 0.2 μg/mL human IL-4 (Peptotec) and 0.2 μg/mL GM-CSF (Peptotec). On day 4 cytokines and LPS were replenished. iDC were harvested using 2 mmol/L EDTA (immature dendritic cells) on day 6 post-isolation.


Co-culture assay: T cells were combined at the following ratios in 200 ul per 96 well: 12.5K T cells (with or with-out GM-CSF KO), 50K anti-CD3/CD28 beads and 1K iDC Co-cultures were subsequently incubated for at 37° C. and 100 ul of assay supernatant was collected at 24 hr and 48 hr for cytokine analysis.


Measurement of cytokine levels: Cytokine concentration determination from culture supernatants was performed using IL-6 ELISA plate (R&D systems). Prior to analysis supernatants were centrifuged at 300×g for 10 mins at 4° C. and subsequently diluted 1:10 in assay calibrator diluent. Measurements were performed using standard product protocols.


Deletion of GM-CSF reduced IL-6 production >3-fold at 24 hrs (FIGS. 7 and 8) and >4-fold at 48 hrs (FIGS. 9 and 10) relative to non-edited T cells.


GM-CSF deficient T cells induced significantly lower IL-6 expression across the different monocyte lineages.


Example 19—Method for Making and Testing a Genome-Edited CAR-T Cell Deficient at Inducing CRS by Insertion of Selectable Marker into Gene Edited Locus

Inject primary B-ALL (2×106) in NSG mice if performing an in vivo CRS experiment. This should be completed 3 weeks prior to infusion of CAR-T into mice.


The following steps may be taken to provide a genome-edited, CRS-resistant, CAR-T cells. This example describes the making of a CD19CARTΔGMCSFΔCD38 mutant IL-7R T cell, in which the deletion of GM-CSF mitigates risk of CRS, deletion of CD3e prevent TCR signaling and GvHD and the mutant IL-7R enhances proliferation of the CAR-T sufficiently to induce CRS in this model. Sequences encoding a mutant, constitutively active IL-7R sequence can be found in Table 19, or found in the art. As those of skill in the art will recognize, certain of the steps may be conducted sequentially or out of the order listed below, though perhaps leading to different efficiency.


Step 1: T Cell Activation (Day 0)


Purify T cells from a leukapheresis chamber using a Miltenyi human PanT isolation kit. Resuspend in media. Count cells. Determine the number of human T cell activation CD3/CD28 beads required to obtain a 3:1 bead:cell ratio. Wash beads 2× with T cell media. Dilute cells at 1.256 cells/mL in hXcyte media. Add human T cell activation CD3/CD28 beads. Aliquot 4 mL/well of 1.256 cell/mL solution into a 6 well plate. Incubate cells at 37° C.


Step 2: T Cell Transduction (Day 1)


CAR-T cells may then be transduced with one or more CARs targeted to (i.e., that recognizes) one or more targets, for example with a lentivirus containing a CAR construct. Any other suitable method of transduction may be used.


Step 3: CRISPR (Day 2)


In a CRS-resistant CAR-T cell, cytokine/chemokine/transcription factor genes or transcription factors may be deleted to prevent secretion of factors that induce secretion of cytokines from myeloid cells. Deletion may be accomplished by electroporating with Cas9 mRNA and gRNA against the target(s). Other techniques, however, could be used to suppress expression of the target. These include other genome editing techniques such as TALENs, RNA interference, and eliciting of internal binding of the antigen to prevent cell surface expression. Examples of gRNAs that may be used include those shown in Tables 8-10, and others known in the art.




















Nuecleofection


Sample ID
gRNA#1
gRNA#2
Cas9
Buffer P3







UCART19
20 μg
20 μg gCD3ε
15 μg Cas9
100 μl



gGM-CSF

mRNA









Cells are harvested, isolated, and purified, for example using magnetic selection with a labelled antibody-coated magnetic beads that bind to a cell-specific protein (available from, e.g., Miltenyi Biotec). For T cells, anti-CD3/CD28 beads could be used. Other purification techniques are known in the art and could be used.









TABLE 19







Sequences or Constitutively Expressing IL-7R






























Trucated

Trucated



CD8a
CD19

CD19
CD8a

CD28


Truncated
IL-7R
IL-7
IL-7R



Leader
VL
Linker
VH
Hinge
CD28tm
Co-stim
CD3z
2a
CD34
ECD
TMD
ICD
Description





MALPVT
DIQMTQTT
GGGGS4
EVKLQESGP
TTTPAPRPP
FWVLVVVG
RSKRSRLLH
RVKFSRSA
GSGATNFS
MPRGWTALC
YREGANDFV
LLTCPTISI
KKRIKPIVW
IL7R with L5 and L6


ALLLPL
SSLSASLG
(SEQ ID
GLVAPSQSL
TPAPTIASQ
GVLACYSL
SDYMNMTPR
DAPAYKQG
LLKQAGDV
LLSLLPSGF
VTFNTSHLQ
LSFFSVALL
PSLPDHKKT
elbows with CPT trans-


ALLLHA
DRVTISCR
NO: 9)
SVTCTVSGV
PLSLRPEAC
LVTVAFII
RPGPTRKHY
QNQLYNEL
EENPGP
MSLDNNGTA
KKYVKVLMH
VILACVLW
LEHLCKKPR
membrane T-ALL


ARP
ASQDISKY

SLPDYGVSW
RPAAGGAVH
FWV
QPYAPPRDF
NLGRREEY
(SEQ ID
TPELPTQGT
DVAYRQEKD
(SEQ ID
KNLNVSFNP
mutation insertion


(SEQ ID
LNWYQQKP

IRQPPRKGL
TRGLDFACD
(SEQ ID
AAYRS
DVLDKRRG
NO: 3031)
FSNVSTNVS
ENKWTHVNL
NO: 3045)
ESFLDCQIH
only


NO: 1)
DGTVKLLI

EWLGVIWGS
(SEQ ID
NO: 3036)
(SEQ ID
RDPEMGGK

YQETTTPST
SSTKLTLLQ

RVDDIQARD




YHTSRLHS

ETTYYNSAL
NO: 3035)

NO: 3037)
PRRKNPQE

LGSTSLHPV
RKLQPAAMY

EVEGFLQDT




GVPSRFSG

KSRLKSRLT



GLYNELQK

SQHGNEATT
EIKVRSIPD

FPQQLEESE




SGSGTDYS

IIKDNSKSQ



DKMAEAYS

NITETTVKF
HYFKGFWSE

KQRLGGDVQ




LTISNLEQ

VFLKMNSLQ



EIGMKGER

TSTSVITSV
WSPSYYFRT

SPNCPSEDV




QGNTLPYT

TDDTAIYYC



RRGKGHDG

YGNTNSSVQ
PEINNSSGE

VITPESFGR




FGGGTKLE

AKHYYYGGS



LYQGLSTA

SQTSVISTV
MDPI

DSSLTCLAG




ITRADAAP

YAMDYWGQG



TKDTYDAL

FTTPANVST
(SEQ ID

NVSACDAPI




TVSIFPPS

TSVTVSS



HMQALPPR

PETTLKPSL
NO: 3040)

LSSSRSLDC




SN

(SEQ ID



(SEQ ID

LPSNVSDLS


RESGKNGPH




(SEQ ID

NO: 3034)



NO: 3038)

TTSTSLATS


VYQDLLLSL




NO: 3033)







PTKPYTSSS


GTTNSTLPP












PILSDIKAE


PFSLQSGIL












IKCSGIREV


TLNPVAQGQ












KLTQGICLE


PILTSLGSN












QNKTSSCAE


QEEAYVTMS












FKKDRGEGL


SFYQNQ












ARVLCGEEQ


(SEQ ID












ADADAGAQV


NO: 3047)












CSLLLAQSE















VRPQCLLLV















LANRTEISS















KLQLMKKHQ















SDLKKLG















(SEQ ID















NO: 3039)









MALPVT
DIQMTQTT
GGGGS4
EVKLQESGP
TTTPAPRPP
FWVLVVVG
RSKRSRLLH
RVKFSRSA
GSGATNFS
MPRGWTALC
YREGANDFV
LLTCPTISI
KKRIKPIVW
IL7R with L5 and L6


ALLLPL
SSLSASLG
(SEQ ID
GLVAPSQSL
TPAPTIASQ
GVLACYSL
SDYMNMTPR
DAPAYKQG
LLKQAGDV
LLSLLPSGF
VTFNTSHLQ
LSFFSVALL
PSLPDHKKT
elbows with CPT trans-


ALLLHA
DRVTISCR
NO: 9)
SVTCTVSGV
PLSLRPEAC
LVTVAFII
RPGPTRKHY
QNQLYNEL
EENPGP
MSLDNNGTA
KKYVKVLMH
VILACVLW
LEHLCKKPR
membrane T-ALL


ARP
ASQDISKY

SLPDYGVSW
RPAAGGAVH
FWV
QPYAPPRDF
NLGRREEY
(SEQ ID
TPELPTQGT
DVAYRQEKD
(SEQ ID
KNLNVSFNP
mutation insertion


(SEQ ID
LNWYQQKP

IRQPPRKGL
TRGLDFACD
(SEQ ID
AAYRS
DVLDKRRG
NO: 3031)
FSNVSTNVS
ENKWTHVNL
NO: 3045)
ESFLDCQIH
and S185C B-ALL


NO: 1)
DGTVKLLI

EWLGVIWGS
(SEQ ID
NO: 3036)
(SEQ ID
RDPEMGGK

YQETTTPST
SCTKLTLLQ

RVDDIQARD
mutation



YHTSRLHS

ETTYYNSAL
NO: 3035)

NO: 3037)
PRRKNPQE

LGSTSLHPV
RKLQPAAMY

EVEGFLQDT




GVPSRFSG

KSRLKSRLT



GLYNELQK

SQHGNEATT
EIKVRSIPD

FPQQLEESE




SGSGTDYS

IIKDNSKSQ



DKMAEAYS

NITETTVKF
HYFKGFWSE

KQRLGGDVQ




LTISNLEQ

VFLKMNSLQ



EIGMKGER

TSTSVITSV
WSPSYYFRT

SPNCPSEDV




QGNTLPYT

TDDTAIYYC



RRGKGHDG

YGNTNSSVQ
PEINNSSGE

VITPESFGR




FGGGTKLE

AKHYYYGGS



LYQGLSTA

SQTSVISTV
MD

DSSLTCLAG




ITRADAAP

YAMDYWGQG



TKDTYDAL

FTTPANVST
(SEQ ID

NVSACDAPI




TVSIFPPS

TSVTVSS



HMQALPPR

PETTLKPSL
NO: 3041)

LSSSRSLDC




SN

(SEQ ID



(SEQ ID

LPSNVSDLS


RESGKNGPH




(SEQ ID

NO: 3034)



NO: 3038)

TTSTSLATS


VYQDLLLSL




NO: 3033)







PTKPYTSSS


GTTNSTLPP












PILSDIKAE


PFSLQSGIL












IKCSGIREV


TLNPVAQGQ












KLTQGICLE


PILTSLGSN












QNKTSSCAE


QEEAYVTMS












FKKDRGEGL


SFYQNQ












ARVLCGEEQ


(SEQ ID












ADADAGAQV


NO: 3047)












CSLLLAQSE















VRPQCLLLV















LANRTEISS















KLQLMKKHQ















SDLKKLG















(SEQ ID















NO: 3039)









MALPVT
DIQMTQTT
GGGGS4
EVKLQESGP
TTTPAPRPP
FWVLVVVG
RSKRSRLLH
RVKFSRSA
GSGATNFS
MPRGWTALC
YREGANDFV
LLTISILS
KKRIKPIVW
IL7R with L5 and L6


ALLLPL
SSLSASLG
(SEQ ID
GLVAPSQSL
TPAPTIASQ
GVLACYSL
SDYMNMTPR
DAPAYKQG
LLKQAGDV
LLSLLPSGF
VTFNTSHLQ
FFSVALLV
PSLPDHKKT
elbows with S185C


ALLLHA
DRVTISCR
NO: 9)
SVTCTVSGV
PLSLRPEAC
LVTVAFII
RPGPTRKHY
QNQLYNEL
EENPGP
MSLDNNGTA
KKYVKVLMH
ILACVLW
LEHLCKKPR
B-ALL mutation only


ARP
ASQDISKY

SLPDYGVSW
RPAAGGAVH
FWV
QPYAPPRDF
NLGRREEY
(SEQ ID
TPELPTQGT
DVAYRQEKD
(SEQ ID
KNLNVSFNP



(SEQ ID
LNWYQQKP

IRQPPRKGL
TRGLDFACD
(SEQ ID
AAYRS
DVLDKRRG
NO: 3031)
FSNVSTNVS
ENKWTHVNL
NO: 3046)
ESFLDCQIH



NO: 1)
DGTVKLLI

EWLGVIWGS
(SEQ ID
NO: 3036)
(SEQ ID
RDPEMGGK

YQETTTPST
SCTKLTLLQ

RVDDIQARD




YHTSRLHS

ETTYYNSAL
NO: 3035)

NO: 3037)
PRRKNPQE

LGSTSLHPV
RKLQPAAMY

EVEGFLQDT




GVPSRFSG

KSRLKSRLT



GLYNELQK

SQHGNEATT
EIKVRSIPD

FPQQLEESE




SGSGTDYS

IIKDNSKSQ



DKMAEAYS

NITETTVKF
HYFKGFWSE

KQRLGGDVQ




LTISNLEQ

VFLKMNSLQ



EIGMKGER

TSTSVITSV
WSPSYYFRT

SPNCPSEDV




QGNTLPYT

TDDTAIYYC



RRGKGHDG

YGNTNSSVQ
PEINNSSGE

VITPESFGR




FGGGTKLE

AKHYYYGGS



LYQGLSTA

SQTSVISTV
MD

DSSLTCLAG




ITRADAAP

YAMDYWGQG



TKDTYDAL

FTTPANVST
(SEQ ID

NVSACDAPI




TVSIFPPS

TSVTVSS



HMQALPPR

PETTLKPSL
NO: 3041)

LSSSRSLDC




SN

(SEQ ID



(SEQ ID

LPSNVSDLS


RESGKNGPH




(SEQ ID

NO: 3034)



NO: 3038)

TTSTSLATS


VYQDLLLSL




NO: 3033)







PTKPYTSSS


GTTNSTLPP












PILSDIKAE


PFSLQSGIL












IKCSGIREV


TLNPVAQGQ












KLTQGICLE


PILTSLGSN












QNKTSSCAE


QEEAYVTMS












FKKDRGEGL


SFYQNQ












ARVLCGEEQ


(SEQ ID












ADADAGAQV


NO: 3047)












CSLLLAQSE















VRPQCLLLV















LANRTEISS















KLQLMKKHQ















SDLKKLG















(SEQ ID















NO: 3039)









MALPVT
DIQMTQTT
GGGGS4
EVKLQESGP
TTTPAPRPP
FWVLVVVG
RSKRSRLLH
RVKFSRSA
GSGATNFS
MPRGWTALC
YREGANDFV
LLTISILSF
KKRIKPIVW
IL7R with L5 and L6


ALLLPL
SSLSASLG
(SEQ ID
GLVAPSQSL
TPAPTIASQ
GVLACYSL
SDYMNMTPR
DAPAYKQG
LLKQAGDV
LLSLLPSGF
VTFNTSHLQ
FSVALLVIL
PSLPDHKKT
elbows WT


ALLLHA
DRVTISCR
NO: 9)
SVTCTVSGV
PLSLRPEAC
LVTVAFII
RPGPTRKHY
QNQLYNEL
EENPGP
MSLDNNGTA
KKYVKVLMH
ACVLW
LEHLCKKPR



ARP
ASQDISKY

SLPDYGVSW
RPAAGGAVH
FWV
QPYAPPRDF
NLGRREEY
(SEQ ID
TPELPTQGT
DVAYRQEKD
(SEQ ID
KNLNVSFNP



(SEQ ID
LNWYQQKP

IRQPPRKGL
TRGLDFACD
(SEQ ID
AAYRS
DVLDKRRG
NO: 3031)
FSNVSTNVS
ENKWTHVNL
NO: 46)
ESFLDCQIH



NO: 1)
DGTVKLLI

EWLGVIWGS
(SEQ ID
NO: 3036)
(SEQ ID
RDPEMGGK

YQETTTPST
SSTKLTLLQ

RVDDIQARD




YHTSRLHS

ETTYYNSAL
NO: 3035)

NO: 3037)
PRRKNPQE

LGSTSLHPV
RKLQPAAMY

EVEGFLQDT




GVPSRFSG

KSRLKSRLT



GLYNELQK

SQHGNEATT
EIKVRSIPD

FPQQLEESE




SGSGTDYS

IIKDNSKSQ



DKMAEAYS

NITETTVKF
HYFKGFWSE

KQRLGGDVQ




LTISNLEQ

VFLKMNSLQ



EIGMKGER

TSTSVITSV
WSPSYYFRT

SPNCPSEDV




QGNTLPYT

TDDTAIYYC



RRGKGHDG

YGNTNSSVQ
PEINNSSGE

VITPESFGR




FGGGTKLE

AKHYYYGGS



LYQGLSTA

SQTSVISTV
MDPI

DSSLTCLAG




ITRADAAP

YAMDYWGQG



TKDTYDAL

FTTPANVST
(SEQ ID

NVSACDAPI




TVSIFPPS

TSVTVSS



HMQALPPR

PETTLKPSL
NO: 3040)

LSSSRSLDC




SN

(SEQ ID



(SEQ ID

LPSNVSDLS


RESGKNGPH




(SEQ ID

NO: 3034)



NO: 3038)

TTSTSLATS


VYQDLLLSL




NO: 3033)







PTKPYTSSS


GTTNSTLPP












PILSDIKAE


PFSLQSGIL












IKCSGIREV


TLNPVAQGQ












KLTQGICLE


PILTSLGSN












QNKTSSCAE


QEEAYVTMS












FKKDRGEGL


SFYQNQ












ARVLCGEEQ


(SEQ ID












ADADAGAQV


NO: 3047)












CSLLLAQSE















VRPQCLLLV















LANRTEISS















KLQLMKKHQ















SDLKKLG















(SEQ ID















NO: 3039)









MALPVT
DIQMTQTT
GGGGS4
EVKLQESGP
TTTPAPRPP
FWVLVVVG
RSKRSRLLH
RVKFSRSA
GSGATNFS
MPRGWTALC
VNLSSTKLT
LLTCPTISI
KKRIKPIVW
IL7R with L6 elbow


ALLLPL
SSLSASLG
(SEQ ID
GLVAPSQSL
TPAPTIASQ
GVLACYSL
SDYMNMTPR
DAPAYKQG
LLKQAGDV
LLSLLPSGF
LLQRKLQPA
LSFFSVALL
PSLPDHKKT
only with CPT trans-


ALLLHA
DRVTISCR
NO: 9)
SVTCTVSGV
PLSLRPEAC
LVTVAFII
RPGPTRKHY
QNQLYNEL
EENPGP
MSLDNNGTA
AMYEIKVRS
VILACVLW
LEHLCKKPR
membrane T-ALL


ARP
ASQDISKY

SLPDYGVSW
RPAAGGAVH
FWV
QPYAPPRDF
NLGRREEY
(SEQ ID
TPELPTQGT
IPDHYFKGF
(SEQ ID
KNLNVSFNP
mutation insertion


(SEQ ID
LNWYQQKP

IRQPPRKGL
TRGLDFACD
(SEQ ID
AAYRS
DVLDKRRG
NO: 3031)
FSNVSTNVS
WSEWSPSYY
NO: 3045)
ESFLDCQIH
only


NO: 1)
DGTVKLLI

EWLGVIWGS
(SEQ ID
NO: 3036)
(SEQ ID
RDPEMGGK

YQETTTPST
FRTPEINNS

RVDDIQARD




YHTSRLHS

ETTYYNSAL
NO: 3035)

NO: 3037)
PRRKNPQE

LGSTSLHPV
SGEMDPI

EVEGFLQDT




GVPSRFSG

KSRLKSRLT



GLYNELQK

SQHGNEATT
(SEQ ID

FPQQLEESE




SGSGTDYS

IIKDNSKSQ



DKMAEAYS

NITETTVKF
NO: 3042)

KQRLGGDVQ




LTISNLEQ

VFLKMNSLQ



EIGMKGER

TSTSVITSV


SPNCPSEDV




QGNTLPYT

TDDTAIYYC



RRGKGHDG

YGNTNSSVQ


VITPESFGR




FGGGTKLE

AKHYYYGGS



LYQGLSTA

SQTSVISTV


DSSLTCLAG




ITRADAAP

YAMDYWGQG



TKDTYDAL

FTTPANVST


NVSACDAPI




TVSIFPPS

TSVTVSS



HMQALPPR

PETTLKPSL


LSSSRSLDC




SN

(SEQ ID



(SEQ ID

LPSNVSDLS


RESGKNGPH




(SEQ ID

NO: 3034)



NO: 3038)

TTSTSLATS


VYQDLLLSL




NO: 3033)







PTKPYTSSS


GTTNSTLPP












PILSDIKAE


PFSLQSGIL












IKCSGIREV


TLNPVAQGQ












KLTQGICLE


PILTSLGSN












QNKTSSCAE


QEEAYVTMS












FKKDRGEGL


SFYQNQ












ARVLCGEEQ


(SEQ ID












ADADAGAQV


NO: 3047)












CSLLLAQSE















VRPQCLLLV















LANRTEISS















KLQLMKKHQ















SDLKKLG















(SEQ ID















NO: 3039)









MALPVT
DIQMTQTT
GGGGS4
EVKLQESGP
TTTPAPRPP
FWVLVVVG
RSKRSRLLH
RVKFSRSA
GSGATNFS
MPRGWTALC
VNLSCTKLT
LLTCPTISI
KKRIKPIVW
IL7R with L6 elbows


ALLLPL
SSLSASLG
(SEQ ID
GLVAPSQSL
TPAPTIASQ
GVLACYSL
SDYMNMTPR
DAPAYKQG
LLKQAGDV
LLSLLPSGF
LLQTKLQPA
LSFFSVALL
PSLPDHKKT
with CPT trans-


ALLLHA
DRVTISCR
NO: 9)
SVTCTVSGV
PLSLRPEAC
LVTVAFII
RPGPTRKHY
QNQLYNEL
EENPGP
MSLDNNGTA
AMYEIKVRS
VILACVLW
LEHLCKKPR
membrane T-ALL


ARP
ASQDISKY

SLPDYGVSW
RPAAGGAVH
FWV
QPYAPPRDF
NLGRREEY
(SEQ ID
TPELPTQGT
IPDHYFKGF
(SEQ ID
KNLNVSFNP
mutation insertion


(SEQ ID
LNWYQQKP

IRQPPRKGL
TRGLDFACD
(SEQ ID
AAYRS
DVLDKRRG
NO: 3031)
FSNVSTNVS
WSEWSPSYY
NO: 3045)
ESFLDCQIH
and S185C B-ALL


NO: 1)
DGTVKLLI

EWLGVIWGS
(SEQ ID
NO: 3036)
(SEQ ID
RDPEMGGK

YQETTTPST
FRTPEINNS

RVDDIQARD
mutation



YHTSRLHS

ETTYYNSAL
NO: 3035)

NO: 3037)
PRRKNPQE

LGSTSLHPV
SGEMDPI

EVEGFLQDT




GVPSRFSG

KSRLKSRLT



GLYNELQK

SQHGNEATT
(SEQ ID

FPQQLEESE




SGSGTDYS

IIKDNSKSQ



DKMAEAYS

NITETTVKF
NO: 3043)

KQRLGGDVQ




LTISNLEQ

VFLKMNSLQ



EIGMKGER

TSTSVITSV


SPNCPSEDV




QGNTLPYT

TDDTAIYYC



RRGKGHDG

YGNTNSSVQ


VITPESFGR




FGGGTKLE

AKHYYYGGS



LYQGLSTA

SQTSVISTV


DSSLTCLAG




ITRADAAP

YAMDYWGQG



TKDTYDAL

FTTPANVST


NVSACDAPI




TVSIFPPS

TSVTVSS



HMQALPPR

PETTLKPSL


LSSSRSLDC




SN

(SEQ ID



(SEQ ID

LPSNVSDLS


RESGKNGPH




(SEQ ID

NO: 3034)



NO: 3038)

TTSTSLATS


VYQDLLLSL




NO: 3033)







PTKPYTSSS


GTTNSTLPP












PILSDIKAE


PFSLQSGIL












IKCSGIREV


TLNPVAQGQ












KLTQGICLE


PILTSLGSN












QNKTSSCAE


QEEAYVTMS












FKKDRGEGL


SFYQNQ












ARVLCGEEQ


(SEQ ID












ADADAGAQV


NO: 3047)












CSLLLAQSE















VRPQCLLLV















LANRTEISS















KLQLMKKHQ















SDLKKLG















(SEQ ID















NO: 3039)









MALPVT
DIQMTQTT
GGGGS4
EVKLQESGP
TTTPAPRPP
FWVLVVVG
RSKRSRLLH
RVKFSRSA
GSGATNFS
MPRGWTALC
VNLSCTKLT
LLTISILSF
KKRIKPIVW
IL7R with L6 elbows


ALLLPL
SSLSASLG
(SEQ ID
GLVAPSQSL
TPAPTIASQ
GVLACYSL
SDYMNMTPR
DAPAYKQG
LLKQAGDV
LLSLLPSGF
LLQTKLQPA
FSVALLVIL
PSLPDHKKT
with S185C B-ALL


ALLLHA
DRVTISCR
NO: 9)
SVTCTVSGV
PLSLRPEAC
LVTVAFII
RPGPTRKHY
QNQLYNEL
EENPGP
MSLDNNGTA
AMYEIKVRS
ACVLW
LEHLCKKPR
mutation only


ARP
ASQDISKY

SLPDYGVSW
RPAAGGAVH
FWV
QPYAPPRDF
NLGRREEY
(SEQ ID
TPELPTQGT
IPDHYFKGF
(SEQ ID
KNLNVSFNP



(SEQ ID
LNWYQQKP

IRQPPRKGL
TRGLDFACD
(SEQ ID
AAYRS
DVLDKRRG
NO: 3031)
FSNVSTNVS
WSEWSPSYY
NO: 3046)
ESFLDCQIH



NO: 1)
DGTVKLLI

EWLGVIWGS
(SEQ ID
NO: 3036)
(SEQ ID
RDPEMGGK

YQETTTPST
FRTPEINNS

RVDDIQARD




YHTSRLHS

ETTYYNSAL
NO: 3035)

NO: 3037)
PRRKNPQE

LGSTSLHPV
SGEMDPI

EVEGFLQDT




GVPSRFSG

KSRLKSRLT



GLYNELQK

SQHGNEATT
(SEQ ID

FPQQLEESE




SGSGTDYS

IIKDNSKSQ



DKMAEAYS

NITETTVKF
NO: 3043)

KQRLGGDVQ




LTISNLEQ

VFLKMNSLQ



EIGMKGER

TSTSVITSV


SPNCPSEDV




QGNTLPYT

TDDTAIYYC



RRGKGHDG

YGNTNSSVQ


VITPESFGR




FGGGTKLE

AKHYYYGGS



LYQGLSTA

SQTSVISTV


DSSLTCLAG




ITRADAAP

YAMDYWGQG



TKDTYDAL

FTTPANVST


NVSACDAPI




TVSIFPPS

TSVTVSS



HMQALPPR

PETTLKPSL


LSSSRSLDC




SN

(SEQ ID



(SEQ ID

LPSNVSDLS


RESGKNGPH




(SEQ ID

NO: 3034)



NO: 3038)

TTSTSLATS


VYQDLLLSL




NO: 3033)







PTKPYTSSS


GTTNSTLPP












PILSDIKAE


PFSLQSGIL












IKCSGIREV


TLNPVAQGQ












KLTQGICLE


PILTSLGSN












QNKTSSCAE


QEEAYVTMS












FKKDRGEGL


SFYQNQ












ARVLCGEEQ


(SEQ ID












ADADAGAQV


NO: 3047)












CSLLLAQSE















VRPQCLLLV















LANRTEISS















KLQLMKKHQ















SDLKKLG















(SEQ ID















NO: 3039)









MALPVT
DIQMTQTT
GGGGS4
EVKLQESGP
TTTPAPRPP
FWVLVVVG
RSKRSRLLH
RVKFSRSA
GSGATNFS
MPRGWTALC
VNLSSTKLT
LLTISILSF
KKRIKPIVW
IL7R with L6 elbows


ALLLPL
SSLSASLG
(SEQ ID
GLVAPSQSL
TPAPTIASQ
GVLACYSL
SDYMNMTPR
DAPAYKQG
LLKQAGDV
LLSLLPSGF
LLQRKLQPA
FSVALLVIL
PSLPDHKKT
WT


ALLLHA
DRVTISCR
NO: 9)
SVTCTVSGV
PLSLRPEAC
LVTVAFII
RPGPTRKHY
QNQLYNEL
EENPGP
MSLDNNGTA
AMYEIKVRS
ACVLW
LEHLCKKPR



ARP
ASQDISKY

SLPDYGVSW
RPAAGGAVH
FWV
QPYAPPRDF
NLGRREEY
(SEQ ID
TPELPTQGT
IPDHYFKGF
(SEQ ID
KNLNVSFNP



(SEQ ID
LNWYQQKP

IRQPPRKGL
TRGLDFACD
(SEQ ID
AAYRS
DVLDKRRG
NO: 3031)
FSNVSTNVS
WSEWSPSYY
NO: 3046)
ESFLDCQIH



NO: 1)
DGTVKLLI

EWLGVIWGS
(SEQ ID
NO: 3036)
(SEQ ID
RDPEMGGK

YQETTTPST
FRTPEINNS

RVDDIQARD




YHTSRLHS

ETTYYNSAL
NO: 3035)

NO: 3037)
PRRKNPQE

LGSTSLHPV
SGEMDPI

EVEGFLQDT




GVPSRFSG

KSRLKSRLT



GLYNELQK

SQHGNEATT
(SEQ ID

FPQQLEESE




SGSGTDYS

IIKDNSKSQ



DKMAEAYS

NITETTVKF
NO: 3042)

KQRLGGDVQ




LTISNLEQ

VFLKMNSLQ



EIGMKGER

TSTSVITSV


SPNCPSEDV




QGNTLPYT

TDDTAIYYC



RRGKGHDG

YGNTNSSVQ


VITPESFGR




FGGGTKLE

AKHYYYGGS



LYQGLSTA

SQTSVISTV


DSSLTCLAG




ITRADAAP

YAMDYWGQG



TKDTYDAL

FTTPANVST


NVSACDAPI




TVSIFPPS

TSVTVSS



HMQALPPR

PETTLKPSL


LSSSRSLDC




SN

(SEQ ID



(SEQ ID

LPSNVSDLS


RESGKNGPH




(SEQ ID

NO: 3034)



NO: 3038)

TTSTSLATS


VYQDLLLSL




NO: 3033)







PTKPYTSSS


GTTNSTLPP












PILSDIKAE


PFSLQSGIL












IKCSGIREV


TLNPVAQGQ












KLTQGICLE


PILTSLGSN












QNKTSSCAE


QEEAYVTMS












FKKDRGEGL


SFYQNQ












ARVLCGEEQ


(SEQ ID












ADADAGAQV


NO: 3047)












CSLLLAQSE















VRPQCLLLV















LANRTEISS















KLQLMKKHQ















SDLKKLG















(SEQ ID















NO: 3039)









MALPVT
DIQMTQTT
GGGGS4
EVKLQESGP
TTTPAPRPP
FWVLVVVG
RSKRSRLLH
RVKFSRSA
GSGATNFS
MPRGWTALC
PEINNSSGE
LLTCPTISI
KKRIKPIVW
IL-7R with CPT T-ALL


ALLLPL
SSLSASLG
(SEQ ID
GLVAPSQSL
TPAPTIASQ
GVLACYSL
SDYMNMTPR
DAPAYKQG
LLKQAGDV
LLSLLPSGF
MDPI
LSFFSVALL
PSLPDHKKT
mutation. Lacking


ALLLHA
DRVTISCR
NO: 9)
SVTCTVSGV
PLSLRPEAC
LVTVAFII
RPGPTRKHY
QNQLYNEL
EENPGP
MSLDNNGTA
(SEQ ID
VILACVLW
LEHLCKKPR
elbows and WSEWS


ARP
ASQDISKY

SLPDYGVSW
RPAAGGAVH
FWV
QPYAPPRDF
NLGRREEY
(SEQ ID
TPELPTQGT
NO: 3044)
(SEQ ID
KNLNVSFNP
(SEQ ID NO: 3070)


(SEQ ID
LNWYQQKP

IRQPPRKGL
TRGLDFACD
(SEQ ID
AAYRS
DVLDKRRG
NO: 3031)
FSNVSTNVS

NO: 3045)
ESFLDCQIH
motif (sugar bridge)


NO: 1)
DGTVKLLI

EWLGVIWGS
(SEQ ID
NO: 3036)
(SEQ ID
RDPEMGGK

YQETTTPST


RVDDIQARD




YHTSRLHS

ETTYYNSAL
NO: 3035)

NO: 3037)
PRRKNPQE

LGSTSLHPV


EVEGFLQDT




GVPSRFSG

KSRLKSRLT



GLYNELQK

SQHGNEATT


FPQQLEESE




SGSGTDYS

IIKDNSKSQ



DKMAEAYS

NITETTVKF


KQRLGGDVQ




LTISNLEQ

VFLKMNSLQ



EIGMKGER

TSTSVITSV


SPNCPSEDV




QGNTLPYT

TDDTAIYYC



RRGKGHDG

YGNTNSSVQ


VITPESFGR




FGGGTKLE

AKHYYYGGS



LYQGLSTA

SQTSVISTV


DSSLTCLAG




ITRADAAP

YAMDYWGQG



TKDTYDAL

FTTPANVST


NVSACDAPI




TVSIFPPS

TSVTVSS



HMQALPPR

PETTLKPSL


LSSSRSLDC




SN

(SEQ ID



(SEQ ID

LPSNVSDLS


RESGKNGPH




(SEQ ID

NO: 3034)



NO: 3038)

TTSTSLATS


VYQDLLLSL




NO: 3033)







PTKPYTSSS


GTTNSTLPP












PILSDIKAE


PFSLQSGIL












IKCSGIREV


TLNPVAQGQ












KLTQGICLE


PILTSLGSN












QNKTSSCAE


QEEAYVTMS












FKKDRGEGL


SFYQNQ












ARVLCGEEQ


(SEQ ID












ADADAGAQV


NO: 3047)












CSLLLAQSE















VRPQCLLLV















LANRTEISS















KLQLMKKHQ















SDLKKLG















(SEQ ID















NO: 3039)









Protocol—Nucleofection Using Nucleofector 4D:

Perform nucleofection protocol as described above (Example 6), using 20 μg of each gRNA (gGM-CSF and gCD38) to each tube of 15 μg Cas9 mRNA.


Step 4: Assessment of CRISPR Activity and Td Efficiency (Day 10)


Take 5×105 cells from each sample and analyze by flow cytometry. Wash samples with RB. Add 5 μl of CD3 APC and 2 μl of anti-FAB BV421 (detects CAR transduction). Wash. Perform Flow cytometry. Cells should be CD38-negative, CD34-positive and FAB+. Harvest T cells (Day 11).


Purification of CAR-T cells. TCR negative cells can be purified using TCRa/b negative selection to remove TCR positive cells.


Step 5: Assessment of CAR-T Activity In Vivo


Inject 5×106 CAR-T per mouse I.V. Assess serum cytokine levels (Day +1, +2, +3 +4, +5, +10, +15). Measure serum cytokine levels using Luminex multiplex cytokine profiling assay to check for elevations in CRS related cytokines. Perform a 4-hr chromium release assay against targets cells (Raji) to assess in vitro activity (Day 11). Monitor efficacy of tumor clearance using flow cytometry of blood to detect hCD45, CD19+ cells.


A modified gRNA protocol for T cell CRISPR and CAR-T transduction of UCART19 is provided below.


Day 0—T Cell Activation:


Purify T cells from a leukapheresis chamber using a Miltenyi human PanT isolation kit. Resuspend in media. Count cells. Determine the number of human T cell activation CD3/CD28 beads required to obtain a 3:1 bead:cell ratio. Wash beads 2× with T cell media. Dilute cells at 1.256 cells/mL in hXcyte media. Add human T cell activation CD3/CD28 beads


T cells may then be transduced with one or more CARs targeted to (i.e., that recognizes) one or more targets, for example with a lentivirus containing a CAR construct. Any other suitable method of transduction may be used.


Day 2: 4×106 cells per reaction are used. EO-115-100 μl transfection volume is programmed, and the entire supplement added to the Nucleofector™ Solution P3. Cell culture plates are prepared by filling appropriate number of wells with desired volume of recommended culture media (2 ml in 6 well plate) and pre-incubating/equilibrating plates in a humidified 37° C./5% CO2 incubator. Beads are magnetically removed (twice to ensure complete removal), then cells counted and cell density determined. The required number of cells are centrifuged at 90×g for 10 minutes at room temperature, the supernatant removed completely. Cells are then resuspended in PBS (1 ml) and transferred to a microcentrifuge tube, and the required number of cells centrifuged at 90×g for 10 minutes at room temperature. The supernatant is removed completely, and the cell pellet resuspended carefully in complete room temperature 4D Nucleofector™ Solution P3, 4×106 per 100 μl). Twenty μg of gRNA (gGM-CSF and gTRAC) are added to each tube of 15 μg Cas9 mRNA. Then 100 μl of cells is added to each tube of Cas9/gRNA, gently mixed and everything transferred into the Nucleocuvette™. The cuvette is gently tapped to remove bubbles. Electroporation is carried out using program (Human T cell stim EO-115). After run completion, the Nucleocuvette™ is carefully removed from the vessel from the retainer using a specialized tool. Cells are resuspended with pre-warmed medium. The media is then taken up from destination well, added to cuvette, and gently pipetted up and down two to three times. This is then transferred to well. This procedure is repeated with media from same well and incubated at 37° C.


Day 5: Assessment of CRISPR activity and Td efficiency. Samples of cells may then be assessed for transduction efficiency by taking a 5×105 cells from each sample and analyzing by flow cytometry. Samples are fix permeabilised and analysed by FACS for CD3, CD34 and intracellular GM-CSF.


Assessment of Genetic Deletion.


To assess genetic deletion of GM-CSF and TRAC, 5×105 cells are harvested from each sample and their DNA extracted. Gene editing efficiency is assessed using target sequencing of the target loci using TIDE-analysis or deep sequencing.


Day 11


Bleed mice and measure tumor burden.


CD3 deplete T cells and inject into mice.


Inject mice with 5×106 CAR+ T cells/mouse.


Mouse groups are provided in Table 20 below:














TABLE 20







Sample
Gene

T cell




ID
editing
Tumor
source
CAR-T
CAR-T





A

2 × 106/







mouse





B
TRAC
2 × 106/
PanT
CAR19 +
5 × 106/




mouse

CD34
mouse


C
TRAC +
2 × 106/
PanT
CAR 19
5 × 106/



GM-CSF
mouse

CD34
mouse


D
TRAC
2 × 106/
PanT
CAR 19 +
5 × 106/




mouse

IL7r + CD34
mouse


E
TRAC +
2 × 106/
PanT
CAR 19 +
5 × 106/



GM-CSF
mouse

IL7r + CD34
mouse










Attune - CAR-T in vivo panel - CRS

















Titer 50 μl







blood or



Bandpass



500K in


Detector
(nm)
Antigen
Colour
Product #
100 μl





BL1
530/30






BL2
695/40
7aad
7AAd

1 ul


RL1
670/14
CD19
APC
BD555415
2 ul


RL2
720/30






RL3
780/60
CD4
APC-H7
BD560158
1 ul


VL1
450/40
mCD45
BV421
BD563890
1 ul


VL2
525/50
CD8
BV510
BD563256
4 ul


VL3
610/20






VL4
660/20
CD3
BV650
BD563852
2 ul


VL5
710/50









Cytokine analysis and assessment of CRS is assed using Millipore luminex multiplex cytokine analysis.


Sequences for preparation of a vector as described herein include, but are not limited to, the following:










Left ITR of vector for insertion of CD34 into GM-CSF locus



SEQ ID NO: 3048



CCTGCAGGCAGCTGCGCGCTCGCTCGCTCACTGAGGCCGCCCGGGCGTCGGGCGACCTTTGGTCGCCCGGCCTCAGTGAGCGAGCGAGC






GCGCAGAGAGGGAGTGGCCAACTCCATCACTAGGGGTTCCT





Left Homology Arm of vector for insertion of CD34 into GM-CSF locus


SEQ ID NO: 3049



cctgagatggatgcagccacagccctggagccagcctgaagctcctggtgtcttctgggggctacatataggagtgtagtccgaacctc






agaggggcaaacctgctctgcagagggaatcaaggttcacataaccagagaggggagtcactcaggaaggtggctccagagccaagagt





cagactctgggtcccgacttgacccagccacaccccctctgaagcttgctgagagtggctgcagtctcgctgctggatgtgcacatggt





ggtcattccctctgctcacaggggcaggggtccccccttactggactgaggttgccccctgctccaggtcctgggtgggagcccatgtg





aactgtcagtggggcaggtctgtgagagctcccctcacactcaagtctctcacagtggccagagaagaggaaggctggagtcagaatga





ggcaccagggcgggcatagcctgcccaaaggcccctgggattacaggcaggatggggagccctatctaagtgtctcccacgccccaccc





cagccattccaggccaggaagtccaaactgtgcccctcagagggagggggcagcctcaggcccattcagactgcccagggagggctgga





gagccctcaggaaggcgggtgggtgggctgtcggttcttggaaaggttcattaatgaaaacccccaagcctgaccacctagggaaaagg





ctcaccgttcccatgtgtggctgataagggccaggagattccacagttcaggtagttcccccgcctccctggcattttgtggtcaccat





taatcatttcctctgtgtatttaagagctcttttgccagtgagcccaGTACACAGAGAGAAAGGCTAAAGTTCTCTGGAGGATGTGGCT





GCAGAGCCTGCTGCTCTTGGGCACTGTGGCCTGCAGCATCTCTGCACCCGCCCGCTCGCCCAGCCCCAGCACGCAGCCCTGGGAGCATG





TGAATGCCATCCAGGAGGCCC





EFS promotor of vector for insertion of CD34 into GM-CSF locus


SEQ ID NO: 3050



GGCTCCGGTGCCCGTCAGTGGGCAGAGCGCACATCGCCCACAGTCCCCGAGAAGTTGGGGGGAGGGGTCGGCAATTGATCCGGTGCCTA






GAGAAGGTGGCGCGGGGTAAACTGGGAAAGTGATGTCGTGTACTGGCTCCGCCTTTTTCCCGAGGGTGGGGGAGAACCGTATATAAGTG





CAGTAGTCGCCGTGAACGTTCTTTTTCGCAACGGGTTTGCCGCCAGAACACAGG





trCD34 of vector for insertion of CD34 into GM-CSF locus


SEQ ID NO: 3051



atgccgcggggctggaccgcgctttgcttgctgagtttgctgccttctgggttcatgagtcttgacaacaacggtactgctaccccaga






gttacctacccagggaacattttcaaatgtttctacaaatgtatcctaccaagaaactacaacacctagtacccttggaagtaccagcc





tgcaccctgtgtctcaacatggcaatgaggccacaacaaacatcacagaaacgacagtcaaattcacatctacctctgtgataacctca





gtttatggaaacacaaactcttctgtccagtcacagacctctgtaatcagcacagtgttcaccaccccagccaacgtttcaactccaga





gacaaccttgaagcctagcctgtcacctggaaatgtttcagacctttcaaccactagcactagccttgcaacatctcccactaaaccct





atacatcatcttctcctatcctaagtgacatcaaggcagaaatcaaatgttcaggcatcagagaagtgaaattgactcagggcatctgc





ctggagcaaaataagacctccagctgtgcggagtttaagaaggacaggggagagggcctggcccgagtgctgtgtggggaggagcaggc





tgatgctgatgctggggcccaggtatgctccctgctccttgcccagtctgaggtgaggcctcagtgtctactgctggtcttggccaaca





gaacagaaatttccagcaaactccaacttatgaaaaagcaccaatctgacctgaaaaagctggggatcctagatttcactgagcaagat





gttgcaagccaccagagctattcccaaaagaccctgattgcactggtcacctcgggagccctgctggctgtcttgggcatcactggcta





tttcctgatgaatcgccgcagctggagccccatttaa





hGMB Poly A of vector for insertion of CD34 into GM-CSF locus


SEQ ID NO: 3052



ACGGGTGGCATCCCTGTGACCCCTCCCCAGTGCCTCTCCTGGCCCTGGAAGTTGCCACTCCAGTGCCCACCAGCCTTGTCCTAATAAAA






TTAAGTTGCATCATTTTGTCTGACTAGGTGTCCTTCTATAATATTATGGGGTGGAGGGGGGTGGTATGGAGCAAGGGGCAAGTTGGGAA





GACAACCTGTAGGGCCTGCGGGGTCTATTGGGAACCAAGCTGGAGTGCAGTGGCACAATCTTGGCTCACTGCAATCTCCGCCTCCTGGG





TTCAAGCGATTCTCCTGCCTCAGCCTCCCGAGTTGTTGGGATTCCAGGCATGCATGACCAGGCTCAGCTAATTTTTGTTTTTTTGGTAG





AGACGGGGTTTCACCATATTGGCCAGGCTGGTCTCCAACTCCTAATCTCAGGTGATCTACCCACCTTGGCCTCCCAAATTGCTGGGATT





ACAGGCGTGAACCACTGCTCCCTTCCCTGTCCTTCTGATTTTGTAGGTAACCACGTGCGGACCGA





RHA of vector for insertion of CD34 into GM-CSF locus


SEQ ID NO: 3053



CGTCTCCTGAACCTGAGTAGAGACACTGCTGCTGAGATGgtaagtgagagaatgtgggcctgtgcctaggccacccagctggcccctga






ctggccacgcctgtcagcttgataacatgacattttccttttctacagAATGAAACAGTAGAAGTCATCTCAGAAATGTTTGACCTCCA





Ggtaagatgcttctctctgacatagctttccagaagcccctgccctggggtggaggtggggactccattttagatggcaccacacaggg





ttgtccactttctctccagtcagctggctgcaggaggagggggtagcaactgggtgctcaagaggctgctggccgtgcccctatggcag





tcacatgagctcctttatcagctgagcggccatgggcagacctagcattcaatggccaggagtcaccaggggacaggtggtaaagtggg





ggtcacttcatgagacaggagctgtgggtttggggcgctcactgtgccccgagaccaagtcctgttgagacagtgctgactacagagag





gcacagaggggtttcaggaacaacccttgcccacccagcaggtccaggtgaggccccacccccctctccctgaatgatggggtgagagt





cacctccttccctaaggctgggctcctctccaggtgccgctgagggtggcctgggcggggcagtgagaagggcaggttcgtgcctgcca





tggacagggcagggtctatgactggacccagcctgtgcccctcccaagccctactcctgggggctgggggcagcagcaaaaaggagtgg





tggagagttcttgtaccactgtgggcacttggccactgctcaccgacgaacgacattttccacagGAGCCGACCTGCCTACAGACCCGC





CTGGAGCTGTACAAGCAGGGCCTGCGGGGCAGCCTCACCAAGCTCAAGGGCCCCTTGACCATGATGGCCAGCCACTACAAGCAGCACTG





CCCTCCAACCCCGgtgagtgc





Right ITR of vector for insertion of CD34 into GM-CSF locus


SEQ ID NO: 51



AGGAACCCCTAGTGATGGAGTTGGCCACTCCCTCTCTGCGCGCTCGCTCGCTCACTGAGGCCGGGCGACCAAAGGTCGCCCGACGCCCG






GGCTTTGCCCGGGCGGCCTCAGTGAGCGAGCGAGCGCGCAGCTGCCTGCAGG





Complete sequence of vector for insertion of CD34 into GM-CSF locus


SEQ ID NO: 3054



CCTGCAGGCAGCTGCGCGCTCGCTCGCTCACTGAGGCCGCCCGGGCGTCGGGCGACCTTTGGTCGCCCGGCCTCAGTGAGCGAGCGAGC






GCGCAGAGAGGGAGTGGCCAACTCCATCACTAGGGGTTCCTcctgagatggatgcagccacagccctggagccagcctgaagctcctgg





tgtcttctgggggctacatataggagtgtagtccgaacctcagaggggcaaacctgctctgcagagggaatcaaggttcacataaccag





agaggggagtcactcaggaaggtggctccagagccaagagtcagactctgggtcccgacttgacccagccacaccccctctgaagcttg





ctgagagtggctgcagtctcgctgctggatgtgcacatggtggtcattccctctgctcacaggggcaggggtccccccttactggactg





aggttgccccctgctccaggtcctgggtgggagcccatgtgaactgtcagtggggcaggtctgtgagagctcccctcacactcaagtct





ctcacagtggccagagaagaggaaggctggagtcagaatgaggcaccagggcgggcatagcctgcccaaaggcccctgggattacaggc





aggatggggagccctatctaagtgtctcccacgccccaccccagccattccaggccaggaagtccaaactgtgcccctcagagggaggg





ggcagcctcaggcccattcagactgcccagggagggctggagagccctcaggaaggcgggtgggtgggctgtcggttcttggaaaggtt





cattaatgaaaacccccaagcctgaccacctagggaaaaggctcaccgttcccatgtgtggctgataagggccaggagattccacagtt





caggtagttcccccgcctccctggcattttgtggtcaccattaatcatttcctctgtgtatttaagagctcttttgccagtgagcccaG





TACACAGAGAGAAAGGCTAAAGTTCTCTGGAGGATGTGGCTGCAGAGCCTGCTGCTCTTGGGCACTGTGGCCTGCAGCATCTCTGCACC





CGCCCGCTCGCCCAGCCCCAGCACGCAGCCCTGGGAGCATGTGAATGCCATCCAGGAGGCCCGGCTCCGGTGCCCGTCAGTGGGCAGAG





CGCACATCGCCCACAGTCCCCGAGAAGTTGGGGGGAGGGGTCGGCAATTGATCCGGTGCCTAGAGAAGGTGGCGCGGGGTAAACTGGGA





AAGTGATGTCGTGTACTGGCTCCGCCTTTTTCCCGAGGGTGGGGGAGAACCGTATATAAGTGCAGTAGTCGCCGTGAACGTTCTTTTTC





GCAACGGGTTTGCCGCCAGAACACAGGatgccgcggggctggaccgcgctttgcttgctgagtttgctgccttctgggttcatgagtct





tgacaacaacggtactgctaccccagagttacctacccagggaacattttcaaatgtttctacaaatgtatcctaccaagaaactacaa





cacctagtacccttggaagtaccagcctgcaccctgtgtctcaacatggcaatgaggccacaacaaacatcacagaaacgacagtcaaa





ttcacatctacctctgtgataacctcagtttatggaaacacaaactcttctgtccagtcacagacctctgtaatcagcacagtgttcac





caccccagccaacgtttcaactccagagacaaccttgaagcctagcctgtcacctggaaatgtttcagacctttcaaccactagcacta





gccttgcaacatctcccactaaaccctatacatcatcttctcctatcctaagtgacatcaaggcagaaatcaaatgttcaggcatcaga





gaagtgaaattgactcagggcatctgcctggagcaaaataagacctccagctgtgcggagtttaagaaggacaggggagagggcctggc





ccgagtgctgtgtggggaggagcaggctgatgctgatgctggggcccaggtatgctccctgctccttgcccagtctgaggtgaggcctc





agtgtctactgctggtcttggccaacagaacagaaatttccagcaaactccaacttatgaaaaagcaccaatctgacctgaaaaagctg





gggatcctagatttcactgagcaagatgttgcaagccaccagagctattcccaaaagaccctgattgcactggtcacctcgggagccct





gctggctgtcttgggcatcactggctatttcctgatgaatcgccgcagctggagccccatttaaACGGGTGGCATCCCTGTGACCCCTC





CCCAGTGCCTCTCCTGGCCCTGGAAGTTGCCACTCCAGTGCCCACCAGCCTTGTCCTAATAAAATTAAGTTGCATCATTTTGTCTGACT





AGGTGTCCTTCTATAATATTATGGGGTGGAGGGGGGTGGTATGGAGCAAGGGGCAAGTTGGGAAGACAACCTGTAGGGCCTGCGGGGTC





TATTGGGAACCAAGCTGGAGTGCAGTGGCACAATCTTGGCTCACTGCAATCTCCGCCTCCTGGGTTCAAGCGATTCTCCTGCCTCAGCC





TCCCGAGTTGTTGGGATTCCAGGCATGCATGACCAGGCTCAGCTAATTTTTGTTTTTTTGGTAGAGACGGGGTTTCACCATATTGGCCA





GGCTGGTCTCCAACTCCTAATCTCAGGTGATCTACCCACCTTGGCCTCCCAAATTGCTGGGATTACAGGCGTGAACCACTGCTCCCTTC





CCTGTCCTTCTGATTTTGTAGGTAACCACGTGCGGACCGACGTCTCCTGAACCTGAGTAGAGACACTGCTGCTGAGATGgtaagtgaga





gaatgtgggcctgtgcctaggccacccagctggcccctgactggccacgcctgtcagcttgataacatgacattttccttttctacagA





ATGAAACAGTAGAAGTCATCTCAGAAATGTTTGACCTCCAGgtaagatgcttctctctgacatagctttccagaagcccctgccctggg





gtggaggtggggactccattttagatggcaccacacagggttgtccactttctctccagtcagctggctgcaggaggagggggtagcaa





ctgggtgctcaagaggctgctggccgtgcccctatggcagtcacatgagctcctttatcagctgagcggccatgggcagacctagcatt





caatggccaggagtcaccaggggacaggtggtaaagtgggggtcacttcatgagacaggagctgtgggtttggggcgctcactgtgccc





cgagaccaagtcctgttgagacagtgctgactacagagaggcacagaggggtttcaggaacaacccttgcccacccagcaggtccaggt





gaggccccacccccctctccctgaatgatggggtgagagtcacctccttccctaaggctgggctcctctccaggtgccgctgagggtgg





cctgggcggggcagtgagaagggcaggttcgtgcctgccatggacagggcagggtctatgactggacccagcctgtgcccctcccaagc





cctactcctgggggctgggggcagcagcaaaaaggagtggtggagagttcttgtaccactgtgggcacttggccactgctcaccgacga





acgacattttccacagGAGCCGACCTGCCTACAGACCCGCCTGGAGCTGTACAAGCAGGGCCTGCGGGGCAGCCTCACCAAGCTCAAGG





GCCCCTTGACCATGATGGCCAGCCACTACAAGCAGCACTGCCCTCCAACCCCGgtgagtgcAGGAACCCCTAGTGATGGAGTTGGCCAC





TCCCTCTCTGCGCGCTCGCTCGCTCACTGAGGCCGGGCGACCAAAGGTCGCCCGACGCCCGGGCTTTGCCCGGGCGGCCTCAGTGAGCG





AGCGAGCGCGCAGCTGCCTGCAGG





left ITR of donor construct for inserting GFP into CD3ϵ locus


SEQ ID NO: 3048



CCTGCAGGCAGCTGCGCGCTCGCTCGCTCACTGAGGCCGCCCGGGCGTCGGGCGACCTTTGGTCGCCCGGCCTCAGTGAGCGAGCGAGC






GCGCAGAGAGGGAGTGGCCAACTCCATCACTAGGGGTTCCT





cloning remnants of donor construct for inserting GFP into CD3ϵ locus


SEQ ID NO: 3055



GCGGCCGCATCGATTGaattc






CD3 L homology arm of donor construct for inserting GFP into CD3ϵ locus


SEQ ID NO: 3056



AGGTAAGTCCACGAATCAGTGATTCAGTGGTGTGGAGAGCTTTATTTCTGAGAAGGCCAGTAGCGCTCCCTTCTGACAAGCAAATCTAA






GACCTGGATGACAGATGACTTCCTGCATTTGGTTGGTTCTTTTGTCATTCATATCTATCTGTAATACAGTTCTGGCTAATTTAAGAGGA





TAAGCTTGAAGACCTCTGGAATTTTTCGGCTTTAGGACTTTAAGGCTTTCTGAGCTTCAGTAGATCTAGATCTAGGAGCTCATGCTGGT





ATATTCTGAATCCGATGTATCTGAGTTACATCTATGAGCTACTTAATAAATATATCTATGAGCTAAATCTCATAGGCTAAGCATGAACC





TCACCTCCAAGACTCGGGGTTCCTAAATGGATGAGACCCTCTTTGGGAAGTCTTGTGGGCAGTGTCTAATTCCACTAGAAAAGTTTTAC





CTACAATTTAAACTTAAACCATGATATTTTCTTACTGCTGTTTCCTTTTTTCATTTTCAGGTGGTATTACACAGACACGTGAGTTTATT





GGTCTTTTATTTATGCCCTGTCTGAGGATGCAGATTGGTGGGTAGATGAGAAGGAACTGATTGAGAGAGATTAACCCCAAGAACTGATA





TCTTCCCAGCATTGCATTCTCAACTCCATTTTAGAAAGGTTCCAAATAGGGACTTCTGTGGGTTTTTCTTTACATCCATCTTACCCTTC





CCAAGTCCCCATGTCCCTGCGTAAACCCTAAAGCCACCTCTCAAAAGGTTCTCTAGTTCCCTTCAAGGTTCTCTAGTTCCCTTCATTCC





ACATATCTCCTCTTCCACACCCTCTAGCCAGTAGAGCTCCCTTCTGACAAGCAAGTCTAAGATCTAGATGACAGATGACTTCCTGCATT





TGGGTGGTTCTTTTGTCACTAATTTGCCTTTTCTAAAATTGTCCTGGTTTCTTCTGCCAATTTCCCTTCTTTCTCCCCAGCATATAAAG





TCTCCATCTCTGGAACCACA





CAR7 of donor construct for inserting GFP into CD3ϵ locus


SEQ ID NO: 3057



atggccttaccagtgaccgccttgctcctgccgctggccttgctgctccacgccgccaggccggatatccagatgacacagactacatc






ctccctgtctgcctctctgggagacagagtcaccatcagttgcagtgcaagtcagggcattagcaattatttaaactggtatcagcaga





aaccagatggaactgttaaactcctgatctattacacatcaagtttacactcaggagtcccatcaaggttcagtggcagtgggtctggg





acagattattctctcaccatcagcaacctggaacctgaagatattgccacttattattgtcagcagtatagcaagcttccgtacacgtt





cggaggggggaccaagctggaaataaaacgtggtggtggtggttctggtggtggtggttctggcggcggcggctccggtggtggtggat





ccgaggtgcaactggtggagtctgggggaggcttagtgaagcctggggggtccctgaaactctcctgtgcagcctctggactcactttc





agtagctatgccatgtcttgggttcgccagactccagagaagaggctggagtgggtcgcatccattagtagtggtggtttcacctacta





tccagacagtgtgaagggccgattcaccatctccagagataatgccaggaacatcctgtatctgcaaatgagcagtctgaggtctgagg





acacggccatgtattactgtgcaagagacgaggtacgggggtacctcgatgtctggggcgcagggaccacggtcaccgtttcccctagg





GCTAGCaccacgacgccagcgccgcgaccaccaacaccggcgcccaccatcgcgtcgcagcccctgtccctgcgcccagaggcgtgccg





gccagcggcggggggcgcagtgcacacgagggggctggacttcgcctgtgatttttgggtgctggtggtggttggtggagtcctggctt





gctatagcttgctagtaacagtggcctttattattttctgggtgaggagtaagaggagcaggctcctgcacagtgactacatgaacatg





actccccgccgccccgggcccacccgcaagcattaccagccctatgccccaccacgcgacttcgcagcctatcgctccagagtgaagtt





cagcaggagcgcagacgcccccgcgtacaagcagggccagaaccagctctataacgagctcaatctaggacgaagagaggagtacgatg





ttttggacaagagacgtggccgggaccctgagatggggggaaagccgagaaggaagaaccctcaggaaggcctgtacaatgaactgcag





aaagataagatggcggaggcctacagtgagattgggatgaaaggcgagcgccggaggggcaaggggcacgatggcctttaccagggtct





cagtacagccaccaaggacacctacgacgcccttcacatgcaggccctgccccctcgcCGGACCGATggaagcggagctactaacttca





gcctgctgaagcaggctggagacgtggaggagaaccctggacctgtttctgaagccatgccgcggggctggaccgcgctttgcttgctg





agtttgctgccttctgggttcatgagtcttgacaacaacggtactgctaccccagagttacctacccagggaacattttcaaatgtttc





tacaaatgtatcctaccaagaaactacaacacctagtacccttggaagtaccagcctgcaccctgtgtctcaacatggcaatgaggcca





caacaaacatcacagaaacgacagtcaaattcacatctacctctgtgataacctcagtttatggaaacacaaactcttctgtccagtca





cagacctctgtaatcagcacagtgttcaccaccccagccaacgtttcaactccagagacaaccttgaagcctagcctgtcacctggaaa





tgtttcagacctttcaaccactagcactagccttgcaacatctcccactaaaccctatacatcatcttctcctatcctaagtgacatca





aggcagaaatcaaatgttcaggcatcagagaagtgaaattgactcagggcatctgcctggagcaaaataagacctccagctgtgcggag





tttaagaaggacaggggagagggcctggcccgagtgctgtgtggggaggagcaggctgatgctgatgctggggcccaggtatgctccct





gctccttgcccagtctgaggtgaggcctcagtgtctactgctggtcttggccaacagaacagaaatttccagcaaactccaacttatga





aaaagcaccaatctgacctgaaaaagctggggatcctagatttcactgagcaagatgttgcaagccaccagagctattcccaaaagacc





ctgattgcactggtcacctcgggagccctgctggctgtcttgggcatcactggctatttcctgatgaatcgccgcagctggagccccat





ttaa





GFP of donor construct for inserting into CD3ϵ locus


SEQ ID NO: 3058



ATGGTGAGCAAGGGCGAGGAGCTGTTCACCGGGGTGGTGCCCATCCTGGTCGAGCTGGACGGCGACGTAAACGGCCACAAGTTCAGCGT






GTCCGGCGAGGGCGAGGGCGATGCCACCTACGGCAAGCTGACCCTGAAGTTCATCTGCACCACCGGCAAGCTGCCCGTGCCCTGGCCCA





CCCTCGTGACCACCCTGACCTACGGCGTGCAGTGCTTCAGCCGCTACCCCGACCACATGAAGCAGCACGACTTCTTCAAGTCCGCCATG





CCCGAAGGCTACGTCCAGGAGCGCACCATCTTCTTCAAGGACGACGGCAACTACAAGACCCGCGCCGAGGTGAAGTTCGAGGGCGACAC





CCTGGTGAACCGCATCGAGCTGAAGGGCATCGACTTCAAGGAGGACGGCAACATCCTGGGGCACAAGCTGGAGTACAACTACAACAGCC





ACAACGTCTATATCATGGCCGACAAGCAGAAGAACGGCATCAAGGTGAACTTCAAGATCCGCCACAACATCGAGGACGGCAGCGTGCAG





CTCGCCGACCACTACCAGCAGAACACCCCCATCGGCGACGGCCCCGTGCTGCTGCCCGACAACCACTACCTGAGCACCCAGTCCGCCCT





GAGCAAAGACCCCAACGAGAAGCGCGATCACATGGTCCTGCTGGAGTTCGTGACCGCCGCCGGGATCACTCTCGGCATGGACGAGCTGT





ACAAGTAA





hGMB poly A of donor construct for inserting GFP into CD3ϵ locus


SEQ ID NO: 3059



ACGGGTGGCATCCCTGTGACCCCTCCCCAGTGCCTCTCCTGGCCCTGGAAGTTGCCACTCCAGTGCCCACCAGCCTTGTCCTAATAAAA






TTAAGTTGCATCATTTTGTCTGACTAGGTGTCCTTCTATAATATTATGGGGTGGAGGGGGGTGGTATGGAGCAAGGGGCAAGTTGGGAA





GACAACCTGTAGGGCCTGCGGGGTCTATTGGGAACCAAGCTGGAGTGCAGTGGCACAATCTTGGCTCACTGCAATCTCCGCCTCCTGGG





TTCAAGCGATTCTCCTGCCTCAGCCTCCCGAGTTGTTGGGATTCCAGGCATGCATGACCAGGCTCAGCTAATTTTTGTTTTTTTGGTAG





AGACGGGGTTTCACCATATTGGCCAGGCTGGTCTCCAACTCCTAATCTCAGGTGATCTACCCACCTTGGCCTCCCAAATTGCTGGGATT





ACAGGCGTGAACCACTGCTCCCTTCCCTGTCCTTCTGATTTTGTAGGTAACCAC





CD3e R homology ARM of donor construct for inserting GFP into CD3ϵ locus


SEQ ID NO: 3060



aGTAtaatattgacatgccctcagtatcctggatctgaaatactatggcaacacaatgataaaaacataggcggtgatgaggatgataa






aaacataggcagtgatgaggatcacctgtcactgaaggaattttcagaattggagcaaagtggttattatgtctgctaccccagaggaa





gcaaaccagaagatgcgaacttttatctctacctgagggcaagagtgtgtgagaactgcatggagatggatgtgatgtcggtggccaca





attgtcatagtggacatctgcatcactgggggcttgctgctgctggtttactactggagcaagaatagaaaggccaaggccaagcctgt





gacacgaggagcgggtgctggcggcaggcaaaggggacaaaacaaggagaggccaccacctgttcccaacccagactatgagcccatcc





ggaaaggccagcgggacctgtattctggcctgaatcagagacgcatctgaccctctggagaacactgcctcccgctggcccaggtctcc





tctccagtccccctgcgactccctgtttcctgggctagtcttggaccccacgagagagaatcgttcctcagcctcatggtgaactcgcg





ccctccagcctgatcccccgctccctcctccctgccttctctgctggtacccagtcctaaaatattgctgcttcctcttcctttgaagc





atcatcagtagtcacaccctcacagctggcctgccctcttgccaggatatttatttgtgctattcactcccttccctttggatgtaact





tctccgttcagttccctccttttcttgcatgtaagttgtcccccatcccaaagtattccatctacttttctatcgccgtccccttttgc





agccctctctggggatggactgggtaaatgttgacagaggccctgccccgttcacagatcctggccctgagccagccctgtgctcctcc





ctcccccaacactccctaccaacc





cloning remnants of donor construct for inserting GFP into CD3ϵ locus


SEQ ID NO: 3061



GCGgacCGAGCGGCCGC






right ITR of donor construct for inserting GFP into CD3ϵ locus


SEQ ID NO: 3062



AGGAACCCCTAGTGATGGAGTTGGCCACTCCCTCTCTGCGCGCTCGCTCGCTCACTGAGGCCGGGCGACCAAAGGTCGCCCGACGCCCG






GGCTTTGCCCGGGCGGCCTCAGTGAGCGAGCGAGCGCGCAGCTGCCTGCAGG





cloning remnants of donor construct for inserting GFP into CD3ϵ locus


SEQ ID NO: 3063



CCTGCAGGCAGCTGCGCGCTCGCTCGCTCACTGAGGCCGCCCGGGCGTCGGGCGACCTTTGGTCGCCCGGCCTCAGTGAGCGAGCGAGC






GCGCAGAGAGGGAGTGGCCAACTCCATCACTAGGGGTTCCTGCGGCCGCATCGATTGaattcAGGTAAGTCCACGAATCAGTGATTCAG





TGGTGTGGAGAGCTTTATTTCTGAGAAGGCCAGTAGCGCTCCCTTCTGACAAGCAAATCTAAGACCTGGATGACAGATGACTTCCTGCA





TTTGGTTGGTTCTTTTGTCATTCATATCTATCTGTAATACAGTTCTGGCTAATTTAAGAGGATAAGCTTGAAGACCTCTGGAATTTTTC





GGCTTTAGGACTTTAAGGCTTTCTGAGCTTCAGTAGATCTAGATCTAGGAGCTCATGCTGGTATATTCTGAATCCGATGTATCTGAGTT





ACATCTATGAGCTACTTAATAAATATATCTATGAGCTAAATCTCATAGGCTAAGCATGAACCTCACCTCCAAGACTCGGGGTTCCTAAA





TGGATGAGACCCTCTTTGGGAAGTCTTGTGGGCAGTGTCTAATTCCACTAGAAAAGTTTTACCTACAATTTAAACTTAAACCATGATAT





TTTCTTACTGCTGTTTCCTTTTTTCATTTTCAGGTGGTATTACACAGACACGTGAGTTTATTGGTCTTTTATTTATGCCCTGTCTGAGG





ATGCAGATTGGTGGGTAGATGAGAAGGAACTGATTGAGAGAGATTAACCCCAAGAACTGATATCTTCCCAGCATTGCATTCTCAACTCC





ATTTTAGAAAGGTTCCAAATAGGGACTTCTGTGGGTTTTTCTTTACATCCATCTTACCCTTCCCAAGTCCCCATGTCCCTGCGTAAACC





CTAAAGCCACCTCTCAAAAGGTTCTCTAGTTCCCTTCAAGGTTCTCTAGTTCCCTTCATTCCACATATCTCCTCTTCCACACCCTCTAG





CCAGTAGAGCTCCCTTCTGACAAGCAAGTCTAAGATCTAGATGACAGATGACTTCCTGCATTTGGGTGGTTCTTTTGTCACTAATTTGC





CTTTTCTAAAATTGTCCTGGTTTCTTCTGCCAATTTCCCTTCTTTCTCCCCAGCATATAAAGTCTCCATCTCTGGAACCACAATGGTGA





GCAAGGGCGAGGAGCTGTTCACCGGGGTGGTGCCCATCCTGGTCGAGCTGGACGGCGACGTAAACGGCCACAAGTTCAGCGTGTCCGGC





GAGGGCGAGGGCGATGCCACCTACGGCAAGCTGACCCTGAAGTTCATCTGCACCACCGGCAAGCTGCCCGTGCCCTGGCCCACCCTCGT





GACCACCCTGACCTACGGCGTGCAGTGCTTCAGCCGCTACCCCGACCACATGAAGCAGCACGACTTCTTCAAGTCCGCCATGCCCGAAG





GCTACGTCCAGGAGCGCACCATCTTCTTCAAGGACGACGGCAACTACAAGACCCGCGCCGAGGTGAAGTTCGAGGGCGACACCCTGGTG





AACCGCATCGAGCTGAAGGGCATCGACTTCAAGGAGGACGGCAACATCCTGGGGCACAAGCTGGAGTACAACTACAACAGCCACAACGT





CTATATCATGGCCGACAAGCAGAAGAACGGCATCAAGGTGAACTTCAAGATCCGCCACAACATCGAGGACGGCAGCGTGCAGCTCGCCG





ACCACTACCAGCAGAACACCCCCATCGGCGACGGCCCCGTGCTGCTGCCCGACAACCACTACCTGAGCACCCAGTCCGCCCTGAGCAAA





GACCCCAACGAGAAGCGCGATCACATGGTCCTGCTGGAGTTCGTGACCGCCGCCGGGATCACTCTCGGCATGGACGAGCTGTACAAGTA





AACGGGTGGCATCCCTGTGACCCCTCCCCAGTGCCTCTCCTGGCCCTGGAAGTTGCCACTCCAGTGCCCACCAGCCTTGTCCTAATAAA





ATTAAGTTGCATCATTTTGTCTGACTAGGTGTCCTTCTATAATATTATGGGGTGGAGGGGGGTGGTATGGAGCAAGGGGCAAGTTGGGA





AGACAACCTGTAGGGCCTGCGGGGTCTATTGGGAACCAAGCTGGAGTGCAGTGGCACAATCTTGGCTCACTGCAATCTCCGCCTCCTGG





GTTCAAGCGATTCTCCTGCCTCAGCCTCCCGAGTTGTTGGGATTCCAGGCATGCATGACCAGGCTCAGCTAATTTTTGTTTTTTTGGTA





GAGACGGGGTTTCACCATATTGGCCAGGCTGGTCTCCAACTCCTAATCTCAGGTGATCTACCCACCTTGGCCTCCCAAATTGCTGGGAT





TACAGGCGTGAACCACTGCTCCCTTCCCTGTCCTTCTGATTTTGTAGGTAACCACaGTAtaatattgacatgccctcagtatcctggat





ctgaaatactatggcaacacaatgataaaaacataggcggtgatgaggatgataaaaacataggcagtgatgaggatcacctgtcactg





aaggaattttcagaattggagcaaagtggttattatgtctgctaccccagaggaagcaaaccagaagatgcgaacttttatctctacct





gagggcaagagtgtgtgagaactgcatggagatggatgtgatgtcggtggccacaattgtcatagtggacatctgcatcactgggggct





tgctgctgctggtttactactggagcaagaatagaaaggccaaggccaagcctgtgacacgaggagcgggtgctggcggcaggcaaagg





ggacaaaacaaggagaggccaccacctgttcccaacccagactatgagcccatccggaaaggccagcgggacctgtattctggcctgaa





tcagagacgcatctgaccctctggagaacactgcctcccgctggcccaggtctcctctccagtccccctgcgactccctgtttcctggg





ctagtcttggaccccacgagagagaatcgttcctcagcctcatggtgaactcgcgccctccagcctgatcccccgctccctcctccctg





ccttctctgctggtacccagtcctaaaatattgctgcttcctcttcctttgaagcatcatcagtagtcacaccctcacagctggcctgc





cctcttgccaggatatttatttgtgctattcactcccttccctttggatgtaacttctccgttcagttccctccttttcttgcatgtaa





gttgtcccccatcccaaagtattccatctacttttctatcgccgtccccttttgcagccctctctggggatggactgggtaaatgttga





cagaggccctgccccgttcacagatcctggccctgagccagccctgtgctcctccctcccccaacactccctaccaaccGCGgacCGAG





CGGCCGCAGGAACCCCTAGTGATGGAGTTGGCCACTCCCTCTCTGCGCGCTCGCTCGCTCACTGAGGCCGGGCGACCAAAGGTCGCCCG





ACGCCCGGGCTTTGCCCGGGCGGCCTCAGTGAGCGAGCGAGCGCGCAGCTGCCTGCAGG





complete sequence of vector for insertion of GFP into CD3ϵ locus


SEQ ID NO: 3064



CCTGCAGGCAGCTGCGCGCTCGCTCGCTCACTGAGGCCGCCCGGGCGTCGGGCGACCTTTGGTCGCCCGGCCTCAGTGAGCGAGCGAGC






GCGCAGAGAGGGAGTGGCCAACTCCATCACTAGGGGTTCCTGCGGCCGCATCGATTGaattcAGGTAAGTCCACGAATCAGTGATTCAG





TGGTGTGGAGAGCTTTATTTCTGAGAAGGCCAGTAGCGCTCCCTTCTGACAAGCAAATCTAAGACCTGGATGACAGATGACTTCCTGCA





TTTGGTTGGTTCTTTTGTCATTCATATCTATCTGTAATACAGTTCTGGCTAATTTAAGAGGATAAGCTTGAAGACCTCTGGAATTTTTC





GGCTTTAGGACTTTAAGGCTTTCTGAGCTTCAGTAGATCTAGATCTAGGAGCTCATGCTGGTATATTCTGAATCCGATGTATCTGAGTT





ACATCTATGAGCTACTTAATAAATATATCTATGAGCTAAATCTCATAGGCTAAGCATGAACCTCACCTCCAAGACTCGGGGTTCCTAAA





TGGATGAGACCCTCTTTGGGAAGTCTTGTGGGCAGTGTCTAATTCCACTAGAAAAGTTTTACCTACAATTTAAACTTAAACCATGATAT





TTTCTTACTGCTGTTTCCTTTTTTCATTTTCAGGTGGTATTACACAGACACGTGAGTTTATTGGTCTTTTATTTATGCCCTGTCTGAGG





ATGCAGATTGGTGGGTAGATGAGAAGGAACTGATTGAGAGAGATTAACCCCAAGAACTGATATCTTCCCAGCATTGCATTCTCAACTCC





ATTTTAGAAAGGTTCCAAATAGGGACTTCTGTGGGTTTTTCTTTACATCCATCTTACCCTTCCCAAGTCCCCATGTCCCTGCGTAAACC





CTAAAGCCACCTCTCAAAAGGTTCTCTAGTTCCCTTCAAGGTTCTCTAGTTCCCTTCATTCCACATATCTCCTCTTCCACACCCTCTAG





CCAGTAGAGCTCCCTTCTGACAAGCAAGTCTAAGATCTAGATGACAGATGACTTCCTGCATTTGGGTGGTTCTTTTGTCACTAATTTGC





CTTTTCTAAAATTGTCCTGGTTTCTTCTGCCAATTTCCCTTCTTTCTCCCCAGCATATAAAGTCTCCATCTCTGGAACCACAatggcct





taccagtgaccgccttgctcctgccgctggccttgctgctccacgccgccaggccggatatccagatgacacagactacatcctccctg





tctgcctctctgggagacagagtcaccatcagttgcagtgcaagtcagggcattagcaattatttaaactggtatcagcagaaaccaga





tggaactgttaaactcctgatctattacacatcaagtttacactcaggagtcccatcaaggttcagtggcagtgggtctgggacagatt





attctctcaccatcagcaacctggaacctgaagatattgccacttattattgtcagcagtatagcaagcttccgtacacgttcggaggg





gggaccaagctggaaataaaacgtggtggtggtggttctggtggtggtggttctggcggcggcggctccggtggtggtggatccgaggt





gcaactggtggagtctgggggaggcttagtgaagcctggggggtccctgaaactctcctgtgcagcctctggactcactttcagtagct





atgccatgtcttgggttcgccagactccagagaagaggctggagtgggtcgcatccattagtagtggtggtttcacctactatccagac





agtgtgaagggccgattcaccatctccagagataatgccaggaacatcctgtatctgcaaatgagcagtctgaggtctgaggacacggc





catgtattactgtgcaagagacgaggtacgggggtacctcgatgtctggggcgcagggaccacggtcaccgtttcccctaggGCTAGCa





ccacgacgccagcgccgcgaccaccaacaccggcgcccaccatcgcgtcgcagcccctgtccctgcgcccagaggcgtgccggccagcg





gcggggggcgcagtgcacacgagggggctggacttcgcctgtgatttttgggtgctggtggtggttggtggagtcctggcttgctatag





cttgctagtaacagtggcctttattattttctgggtgaggagtaagaggagcaggctcctgcacagtgactacatgaacatgactcccc





gccgccccgggcccacccgcaagcattaccagccctatgccccaccacgcgacttcgcagcctatcgctccagagtgaagttcagcagg





agcgcagacgcccccgcgtacaagcagggccagaaccagctctataacgagctcaatctaggacgaagagaggagtacgatgttttgga





caagagacgtggccgggaccctgagatggggggaaagccgagaaggaagaaccctcaggaaggcctgtacaatgaactgcagaaagata





agatggcggaggcctacagtgagattgggatgaaaggcgagcgccggaggggcaaggggcacgatggcctttaccagggtctcagtaca





gccaccaaggacacctacgacgcccttcacatgcaggccctgccccctcgcCGGACCGATggaagcggagctactaacttcagcctgct





gaagcaggctggagacgtggaggagaaccctggacctgtttctgaagccatgccgcggggctggaccgcgctttgcttgctgagtttgc





tgccttctgggttcatgagtcttgacaacaacggtactgctaccccagagttacctacccagggaacattttcaaatgtttctacaaat





gtatcctaccaagaaactacaacacctagtacccttggaagtaccagcctgcaccctgtgtctcaacatggcaatgaggccacaacaaa





catcacagaaacgacagtcaaattcacatctacctctgtgataacctcagtttatggaaacacaaactcttctgtccagtcacagacct





ctgtaatcagcacagtgttcaccaccccagccaacgtttcaactccagagacaaccttgaagcctagcctgtcacctggaaatgtttca





gacctttcaaccactagcactagccttgcaacatctcccactaaaccctatacatcatcttctcctatcctaagtgacatcaaggcaga





aatcaaatgttcaggcatcagagaagtgaaattgactcagggcatctgcctggagcaaaataagacctccagctgtgcggagtttaaga





aggacaggggagagggcctggcccgagtgctgtgtggggaggagcaggctgatgctgatgctggggcccaggtatgctccctgctcctt





gcccagtctgaggtgaggcctcagtgtctactgctggtcttggccaacagaacagaaatttccagcaaactccaacttatgaaaaagca





ccaatctgacctgaaaaagctggggatcctagatttcactgagcaagatgttgcaagccaccagagctattcccaaaagaccctgattg





cactggtcacctcgggagccctgctggctgtcttgggcatcactggctatttcctgatgaatcgccgcagctggagccccatttaaACG





GGTGGCATCCCTGTGACCCCTCCCCAGTGCCTCTCCTGGCCCTGGAAGTTGCCACTCCAGTGCCCACCAGCCTTGTCCTAATAAAATTA





AGTTGCATCATTTTGTCTGACTAGGTGTCCTTCTATAATATTATGGGGTGGAGGGGGGTGGTATGGAGCAAGGGGCAAGTTGGGAAGAC





AACCTGTAGGGCCTGCGGGGTCTATTGGGAACCAAGCTGGAGTGCAGTGGCACAATCTTGGCTCACTGCAATCTCCGCCTCCTGGGTTC





AAGCGATTCTCCTGCCTCAGCCTCCCGAGTTGTTGGGATTCCAGGCATGCATGACCAGGCTCAGCTAATTTTTGTTTTTTTGGTAGAGA





CGGGGTTTCACCATATTGGCCAGGCTGGTCTCCAACTCCTAATCTCAGGTGATCTACCCACCTTGGCCTCCCAAATTGCTGGGATTACA





GGCGTGAACCACTGCTCCCTTCCCTGTCCTTCTGATTTTGTAGGTAACCACaGTAtaatattgacatgccctcagtatcctggatctga





aatactatggcaacacaatgataaaaacataggcggtgatgaggatgataaaaacataggcagtgatgaggatcacctgtcactgaagg





aattttcagaattggagcaaagtggttattatgtctgctaccccagaggaagcaaaccagaagatgcgaacttttatctctacctgagg





gcaagagtgtgtgagaactgcatggagatggatgtgatgtcggtggccacaattgtcatagtggacatctgcatcactgggggcttgct





gctgctggtttactactggagcaagaatagaaaggccaaggccaagcctgtgacacgaggagcgggtgctggcggcaggcaaaggggac





aaaacaaggagaggccaccacctgttcccaacccagactatgagcccatccggaaaggccagcgggacctgtattctggcctgaatcag





agacgcatctgaccctctggagaacactgcctcccgctggcccaggtctcctctccagtccccctgcgactccctgtttcctgggctag





tcttggaccccacgagagagaatcgttcctcagcctcatggtgaactcgcgccctccagcctgatcccccgctccctcctccctgcctt





ctctgctggtacccagtcctaaaatattgctgcttcctcttcctttgaagcatcatcagtagtcacaccctcacagctggcctgccctc





ttgccaggatatttatttgtgctattcactcccttccctttggatgtaacttctccgttcagttccctccttttcttgcatgtaagttg





tcccccatcccaaagtattccatctacttttctatcgccgtccccttttgcagccctctctggggatggactgggtaaatgttgacaga





ggccctgccccgttcacagatcctggccctgagccagccctgtgctcctccctcccccaacactccctaccaaccGCGgacCGAGCGGC





CGCAGGAACCCCTAGTGATGGAGTTGGCCACTCCCTCTCTGCGCGCTCGCTCGCTCACTGAGGCCGGGCGACCAAAGGTCGCCCGACGC





CCGGGCTTTGCCCGGGCGGCCTCAGTGAGCGAGCGAGCGCGCAGCTGCCTGCAGG






All references, patents or applications, U.S. or foreign, cited in the application are hereby incorporated by reference as if written herein in their entireties. Where any inconsistencies arise, material literally disclosed herein controls.


From the foregoing description, one skilled in the art can easily ascertain the essential characteristics of this invention, and without departing from the spirit and scope thereof, can make various changes and modification of the invention to adapt it to various usages and conditions.



























E
0.172
0.17
0.073
0.075
0.085
0.084
0.492
0.557
0.42
0.18
0.247
0.19
450 (Test)



0.046
0.045
0.044
0.046
0.045
0.045
0.05
0.053
0.05
0.048
0.048
0.047
540 (Ref)



0.698
0.691
0.161
0.161
0.222
0.216
2.46
2.796
2.054
0.735
1.107
0.794
Pathlength



0.171
0.17
0.073
0.075
0.085
0.084
0.496
0.558
0.42
0.181
0.249
0.19
450



0.046
0.046
0.045
0.047
0.045
0.045
0.05
0.053
0.051
0.048
0.049
0.047
540



0.245
0.246
0.454
0.468
0.383
0.389
0.202
0.2
0.204
0.246
0.225
0.239
Corrected [450]



0.066
0.066
0.277
0.291
0.204
0.209
0.02
0.019
0.025
0.066
0.044
0.059
Corrected [540]


F
0.127
0.124
0.07
0.074
0.075
0.072
0.148
0.143
0.128
0.102
0.115
0.121
450 (Test)



0.044
0.045
0.043
0.047
0.046
0.044
0.055
0.057
0.045
0.045
0.051
0.048
540 (Ref)



0.458
0.436
0.147
0.15
0.162
0.157
0.516
0.479
0.463
0.316
0.353
0.406
Pathlength



0.127
0.124
0.07
0.074
0.075
0.072
0.147
0.144
0.129
0.102
0.116
0.12
450



0.045
0.045
0.044
0.047
0.046
0.044
0.055
0.057
0.045
0.045
0.052
0.048
540



0.277
0.283
0.475
0.493
0.465
0.462
0.286
0.3
0.278
0.323
0.328
0.296
Corrected [450]



0.097
0.104
0.297
0.314
0.285
0.283
0.106
0.119
0.097
0.144
0.147
0.119
Corrected [540]


G
0.098
0.1
0.078
0.073
0.104
0.122
0.143
0.169
0.144
0.125
0.134
0.131
450 (Test)



0.044
0.044
0.046
0.044
0.045
0.05
0.049
0.049
0.05
0.05
0.045
0.046
540 (Ref)



0.301
0.313
0.179
0.161
0.326
0.398
0.523
0.667
0.523
0.415
0.493
0.469
Pathlength



0.099
0.101
0.078
0.073
0.103
0.122
0.144
0.169
0.144
0.125
0.133
0.13
450



0.044
0.044
0.046
0.044
0.045
0.05
0.05
0.049
0.05
0.05
0.045
0.045
540



0.328
0.322
0.437
0.452
0.317
0.306
0.275
0.254
0.275
0.301
0.269
0.277
Corrected [450]



0.148
0.142
0.259
0.272
0.138
0.127
0.095
0.074
0.096
0.12
0.091
0.096
Corrected [540]


H
0.082
0.087
0.113
0.12
0.074
0.081
0.165
0.169
0.15
0.103
0.122
0.119
450 (Test)



0.047
0.051
0.049
0.047
0.047
0.053
0.059
0.059
0.045
0.044
0.046
0.044
540 (Ref)



0.197
0.197
0.356
0.406
0.15
0.157
0.611
0.607
0.579
0.329
0.422
0.412
Pathlength



0.082
0.087
0.113
0.12
0.074
0.081
0.165
0.169
0.15
0.103
0.122
0.118
450



0.047
0.052
0.049
0.047
0.047
0.053
0.055
0.06
0.046
0.044
0.046
0.045
540



0.415
0.443
0.316
0.296
0.493
0.516
0.27
0.279
0.259
0.314
0.289
0.288
Corrected [450]



0.236
0.263
0.137
0.117
0.311
0.338
0.09
0.098
0.079
0.134
0.11
0.109
Corrected [540]








Claims
  • 1. A chimeric antigen receptor (CAR)-bearing immune effector cell that is deficient in a cytokine or in a chemokine or in a transcription factor that is involved in cytokine release syndrome.
  • 2. The cell as recited in claim 1, wherein the cytokine or chemokine or transcription factor deficiency is effected by deletion or suppression of a gene encoding the cytokine or chemokine or transcription factor, or by suppression of a gene transcript of the cytokine or chemokine or transcription factor gene.
  • 3. The cell as recited in claim 2, wherein the deletion or suppression of the gene encoding the cytokine or chemokine or transcription factor is effected by inserting the CAR into a locus of the cytokine or chemokine or transcription factor gene, or wherein the suppression of the gene transcript of the cytokine or chemokine or transcription factor is effected by transfection of one or more types of small interfering RNAs (siRNA) or by transduction of one or more types of short hairpin RNAs (shRNA).
  • 4. The cell as recited in claim 3, wherein the CAR is part of a construct that also includes a selectable marker chosen from a green fluorescence (GFP) gene, a YFP gene, a tCD34 gene, or a tEGFR gene.
  • 5.-6. (canceled)
  • 7. The cell as recited in claim 1, wherein deletion or suppression is effected using CRISPR, Cas9-CRISPR, Transcription Activator-like Effector Nucleases (TALENs), Zinc Finger Nucleases (ZFNs), or Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR).
  • 8. (canceled)
  • 9. The cell as recited in claim 7, wherein the Cas9 is delivered into the cell as mRNA or protein, and/or wherein a guide RNA (gRNA) targeting the gene to be deleted or suppressed is delivered contemporaneously with the Cas9.
  • 10.-15. (canceled)
  • 16. The cell of claim 2, wherein: the cell expresses at least one CAR that is inserted into a locus of the gene encoding the cytokine or chemokine or transcription factor;the gene encoding the cytokine or chemokine or transcription factor is deleted or suppressed by a method chosen from Transcription Activator-like Effector Nucleases (TALENs), Zinc Finger Nucleases (ZFNs), and Clustered Regularly Interspaces Short Palindromic Repeats (CRISPR) editing;the cytokine or chemokine or transcription factor is suppressed by expression of an scFv with an endoplasmic reticulum (ER) binding tether to bind the cytokine or chemokine in the ER and prevent secretion;the cytokine or chemokine or transcription factor gene transcript is suppressed by transfection of small interfering RNAs (siRNAs);the cytokine or chemokine or transcription factor gene transcript is suppressed by transduction of short hairpin RNAs (shRNAs);the gene that is deleted or suppressed is a T cell surface receptor that is integrated into CAR-T cell signaling.
  • 17. The cell as recited in claim 1, wherein the cell is chosen from a chimeric antigen receptor T cell (CAR-T), a CAR-bearing iNKT cell (iNKT-CAR), and a CAR-bearing natural killer (NK) cell (NK-CAR), or a CAR-bearing macrophage.
  • 18. (canceled)
  • 19. The cell as recited in claim 17, wherein the cell is a dual or tandem CAR-T or iNKT-CAR or CAR-bearing macrophage.
  • 20.-23. (canceled)
  • 24. The cell as recited in claim 1, wherein the cytokine or chemokine or transcription factor contributes to the development of cytokine release syndrome and is selected from among those recited in Table 10.
  • 25. (canceled)
  • 26. The cell as recited in claim 24, wherein the cytokine or chemokine or transcription factor is produced by T cells that activate or localize myeloid cells, or is a T cell surface receptor gene that activates myeloid or CAR-T cells.
  • 27.-28. (canceled)
  • 29. The cell as recited in claim 1, wherein the cytokine or chemokine or transcription factor drives T cell/CAR-T cell differentiation, or wherein the cytokine or chemokine is a transcription factor that drives T cell/CAT-T cell differentiation.
  • 30. (canceled)
  • 31. The cell as recited in claim 1, wherein the cytokine or chemokine or transcription factor is chosen from MCP1 (CCL2), MCP-2, GM-CSF, G-CSF, M-CSF, Il-4, and IFNγ.
  • 32. (canceled)
  • 33. The cell as recited in claim 31, wherein the cell is a GM-CSF deficient CAR-T cell or a GM-CSF deficient iNKT-CAR cell.
  • 34.-36. (canceled)
  • 37. The cell as recited in claim 1, wherein the chimeric antigen receptor(s) specifically binds at least one antigen expressed on a malignant cell, a malignant T cell, a malignant B cell, a malignant mesothelial cell, or a malignant plasma cell.
  • 38. The cell as recited in claim 37, wherein the at least one antigen expressed on a malignant cell is chosen from BCMA, CS1, CD38, CD138, CD19, CD33, CD123, CD371, CD117, CD135, Tim-3, CD5, CD7, CD2, CD4, CD3, CD79A, CD79B, APRIL, CD56, and CD1a; or wherein the at least one antigen expressed on a malignant T cell is chosen from CD2, CD3ε, CD4, CD5, CD7, TCRA, and TCRβ; orwherein the at least one antigen expressed on a malignant B cell is chosen from CD19, CD20, CD21, CD22, CD23, CD24, CD25, CD27, CD38, and CD45; orwherein the at least one antigen expressed on a malignant mesothelial cell is mesothelin; orwherein the at least one antigen expressed on a malignant plasma cell is chosen from BCMA, CS1, CD38, and CD19.
  • 39.-47. (canceled)
  • 48. The cell as recited in claim 37, wherein the chimeric antigen receptor expresses the extracellular portion of the APRIL protein, the ligand for BCMA and TACI, effectively co-targeting both BCMA and TACI.
  • 49. (canceled)
  • 50. The cell as recited in claim 1, wherein endogenous T cell receptor mediated signaling is negligible in the cell.
  • 51. The cell as recited in claim 50, wherein the cell does not induce alloreactivity, graft-versus-host disease, or fratricide.
  • 52. (canceled)
  • 53. A method of treatment of cancer in a patient, which has a reduced incidence of cytokine release syndrome and/or CAR-T associated neuropathy, comprising the administration of cells as recited in claim 1.
  • 54. The method as recited in claim 53, wherein the cancer is a hematologic malignancy chosen from a T-cell malignancy, multiple myeloma, or acute myeloid leukemia (AML), or a solid tumor chosen from cervical cancer, pancreatic cancer, ovarian cancer, mesothelioma, or lung cancer.
  • 55.-94. (canceled)
Parent Case Info

This application claims the benefit of U.S. Provisional Application No. 62/679,597, filed Jun. 1, 2018, the disclosure of which is hereby incorporated by reference in its entirety.

Provisional Applications (1)
Number Date Country
62679597 Jun 2018 US
Continuations (1)
Number Date Country
Parent 16428362 May 2019 US
Child 17746645 US