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1. WO2020198340 - DNMT3A KNOCKOUT CAR T CELLS FOR ADOPTIVE IMMUNOTHERAPY

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CLAIMS

What is claimed:

1. A modified immune cell or precursor cell thereof, comprising:

a CRISPR-mediated modification in an endogenous gene locus encoding DNMT3A, wherein the modification is capable of downregulating gene expression of endogenous DNMT3A; and

an exogenous T cell receptor (TCR) and/or chimeric antigen receptor (CAR) comprising affinity for an antigen on a target cell.

2. The modified immune cell or precursor cell of claim 1, wherein the modification is selected from the group consisting of a substitution, an insertion, a deletion, and an insertion/deletion.

3. The modified immune cell or precursor cell of claim 1 or 2, wherein the modification is located in an exon, a splice donor, or a splice acceptor of the endogenous gene locus encoding DNMT3A.

4. The modified immune cell or precursor cell of any preceding claim, wherein the modification is located in any one of exons 7-15, or 19 of the endogenous gene locus encoding DNMT3A.

5. The modified immune cell or precursor cell of any preceding claim, wherein the modification is mediated by a CRISPR system comprising a CRISPR nuclease and a guide RNA.

6. The modified immune cell or precursor cell of any preceding claim, wherein the modification is mediated by CRISPR/Cas9.

7. The modified immune cell or precursor cell of any preceding claim, wherein the guide RNA comprises a guide sequence that is sufficiently complementary with a target sequence in the endogenous gene locus encoding DNMT3A.

8. The modified immune cell or precursor cell of any preceding claim, wherein the guide RNA comprises a guide sequence that is sufficiently complementary with a target sequence in any one of exons 7-15, or 19 of the endogenous gene locus encoding DNMT3A.

9. The modified immune cell or precursor cell of any preceding claim, wherein the guide RNA comprises a nucleic acid sequence set forth in any one of SEQ ID NOs: 1-59.

10. A modified immune cell or precursor cell thereof, comprising a nucleic acid encoding an exogenous T cell receptor (TCR) and/or chimeric antigen receptor (CAR) inserted into an endogenous gene locus encoding DNMT3A, wherein the nucleic acid encoding an exogenous TCR and/or CAR is inserted as a result of CRISPR-mediated homology directed repair (HDR), and wherein the TCR and/or CAR comprises affinity for an antigen on a target cell.

11. The modified immune cell or precursor cell of claim 10, wherein the nucleic acid encoding an exogenous TCR and/or CAR is inserted into any one of exons 7-15, or 19 of the endogenous gene locus encoding DNMT3A.

12. The modified immune cell or precursor cell of claim 10 or 11, wherein the nucleic acid encoding an exogenous TCR and/or CAR is inserted into exon 7 of the endogenous gene locus encoding DNMT3A.

13. The modified immune cell or precursor cell of claim 10 or 11, wherein the nucleic acid encoding an exogenous TCR and/or CAR is inserted into exon 8 of the endogenous gene locus encoding DNMT3A.

14. The modified immune cell or precursor cell of any preceding claim, wherein the exogenous TCR is selected from the group consisting of a wild-type TCR, a high affinity TCR, and a chimeric TCR.

15. The modified immune cell or precursor cell of any preceding claim, wherein the exogenous CAR comprises an antigen binding domain, a transmembrane domain, and

an intracellular domain.

16. The modified immune cell or precursor cell of any preceding claim, wherein the exogenous CAR comprises an antigen binding domain selected from the group consisting of an antibody, an scFv, and a Fab.

17. The modified immune cell or precursor cell of any preceding claim, wherein the exogenous CAR comprises an antigen binding domain comprising specificity for the antigen.

18. The modified immune cell or precursor cell of any preceding claim, wherein the exogenous CAR further comprises a hinge domain.

19. The modified immune cell or precursor cell of any preceding claim, wherein the exogenous CAR comprises a hinge domain selected from the group consisting of an Fc fragment of an antibody, a hinge region of an antibody, a CH2 region of an antibody, a CH3 region of an antibody, an artificial hinge domain, a hinge comprising an amino acid sequence of CD8, or any combination thereof.

20. The modified immune cell or precursor cell of any preceding claim, wherein the exogenous CAR comprises a transmembrane domain selected from the group consisting of an artificial hydrophobic sequence and transmembrane domain of a type I transmembrane protein, an alpha, beta, or zeta chain of a T cell receptor, CD28, CD3 epsilon, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD 134, CD137, and CD154.

21. The modified immune cell or precursor cell of any preceding claim, wherein the exogenous CAR comprises at least one co-stimulatory domain selected from the group consisting of co-stimulatory domains of proteins in the TNFR superfamily, CD28, 4-1BB (CD137), 0X40 (CD134), PD-1, CD7, LIGHT, CD83L, DAP10,

DAP 12, CD27, CD2, CD5, ICAM-1, LFA-1, Lck, TNFR-I, TNFR-II, Fas, CD30, CD40, ICOS, NKG2C, and B7-H3.

22. The modified immune cell or precursor cell of any preceding claim, wherein the exogenous CAR comprises an intracellular domain comprising an intracellular domain selected from the group consisting of cytoplasmic signaling domains of a human CD3 zeta chain, FcyRIII, FcsRI, a cytoplasmic tail of an Fc receptor, an immunoreceptor tyrosine-based activation motif (ITAM) bearing cytoplasmic receptors, TCR zeta, FcR gamma, FcR beta, CD3 gamma, CD3 delta, CD3 epsilon, CD5, CD22, CD79a, CD79b, and CD66d.

23. The modified immune cell or precursor cell of any preceding claim, wherein the antigen on a target cell is a tumor associated antigen (TAA).

24. The modified immune cell or precursor cell of any preceding claim, wherein the modified cell is resistant to cell exhaustion.

25. The modified immune cell or precursor cell of any preceding claim, wherein the modified cell is an autologous cell.

26. The modified immune cell or precursor cell of any preceding claim, wherein the modified cell is a cell isolated from a human subject.

27. The modified immune cell or precursor cell of any preceding claim, wherein the modified cell is a modified immune cell.

28. The modified immune cell or precursor cell of any preceding claim, wherein the modified cell is a modified T cell.

29. The modified immune cell or precursor cell of any preceding claim, wherein the modified cell is a modified T cell resistant to T cell exhaustion.

30. The modified immune cell or precursor cell of any preceding claim, wherein the cell exhibits a decrease in the expression of one or more exhaustion-related immune checkpoint receptors at least 10 days following in vitro stimulation as compared to an immune cell or precursor cell which lacks a CRISPR-mediated modification in an endogenous gene locus encoding DNMT3A.

31. The modified immune cell or precursor cell of claim 30, wherein the one or more exhaustion-related immune checkpoint receptors are selected from the group consisting of Tim-3, 2B4 and Lag-3.

32. The modified immune cell or precursor cell of any preceding claim, wherein the cell exhibits a central memory T cell (Tcm) phenotype (CCR7+ CD45RO+) at least 10 days following in vitro stimulation.

33. A population of the modified immune cell or percursor cell of any preceding claim, where at least 50% or more (e.g., 50%, 60%, 70%, 80%, 90% or more) of the cells in the population exhibit a decrease in the expression of one or more exhaustion-related immune checkpoint receptors at least 10 days following in vitro stimulation as compared to a corresponding population of immune cells or precursor cell which lacks a CRISPR-mediated modification in an endogenous gene locus encoding DNMT3A.

34. A population of the modified immune cell or percursor cell of any preceding claim, where at least 50% or more (e.g., 50%, 60%, 70%, 80%, 90% or more) of the cells in the population exhibit a central memory T cell (Tcm) phenotype (CCR7+ CD45RO+) at least 10 days following in vitro stimulation.

35. A method for generating a modified immune cell or precursor cell thereof,

comprising:

introducing into an immune or precursor cell a CRISPR system comprising one or more polypeptides and/or nucleic acids capable of downregulating gene expression of endogenous DNMT3A; and

introducing into the immune or precursor cell a nucleic acid encoding an exogenous T cell receptor (TCR) and/or chimeric antigen receptor (CAR), wherein the exogenous TCR and/or CAR comprises affinity for an antigen on a target cell.

36. The method of claim 35, wherein the one or more polypeptides and/or nucleic acids capable of downregulating gene expression of endogenous DNMT3A introduces a CRISPR-mediated modification in an endogenous gene locus encoding DNMT3A.

37. The method of claim 36, wherein the modification is selected from the group

consisting of a substitution, an insertion, a deletion, and an insertion/deletion.

38. The method of any preceding claim, wherein the modification is located in an exon, a splice donor, or a splice acceptor of the endogenous gene locus encoding DNMT3A.

39. The method of any preceding claim, wherein the modification is located in any one of exons 7-15, or 19 of the endogenous gene locus encoding DNMT3A.

40. The method of any preceding claim, wherein the CRISPR system comprises a

CRISPR nuclease and a guide RNA.

41. The method of claim 40, wherein the CRISPR nuclease is Cas9.

42. The method of claim 40 or 41, wherein the CRISPR nuclease and the guide RNA comprise a ribonucleoprotein (RNP) complex.

43. The method of claim 42, wherein the RNP complex is introduced by electroporation.

44. The method of any preceding claim, wherein the guide RNA comprises a guide

sequence that is sufficiently complementary with a target sequence in the endogenous gene locus encoding DNMT3A.

45. The method of any preceding claim, wherein the guide RNA comprises a guide

sequence that is sufficiently complementary with a target sequence in any one of exons 7-15, or 19 of the endogenous gene locus encoding DNMT3A.

46. The method of any preceding claim, wherein the guide RNA comprises a nucleic acid sequence set forth in any one of SEQ ID NO: 1-59.

47. The method of any preceding claim, wherein the nucleic acid encoding an exogenous TCR and/or CAR is introduced via viral transduction.

48. The method of any preceding claim, wherein the viral transduction comprises

contacting the immune or precursor cell with a viral vector comprising the nucleic acid encoding an exogenous TCR and/or CAR.

49. The method of any preceding claim, wherein the viral vector is selected from the group consisting of a retroviral vector, a lentiviral vector, an adenoviral vector, and an adeno-associated viral vector.

50. The method of any preceding claim, wherein the viral vector is a lentiviral vector.

51. A method for generating a modified immune cell or precursor cell thereof,

comprising:

introducing into an immune or precursor cell one or more polypeptides and/or nucleic acids capable of downregulating gene expression of endogenous DNMT3A; and

introducing into the immune or precursor cell a nucleic acid encoding an exogenous T cell receptor (TCR) and/or chimeric antigen receptor (CAR), wherein the nucleic acid encoding an exogenous TCR and/or CAR is inserted into an endogenous gene locus encoding DNMT3A, and wherein the exogenous TCR and/or CAR comprises affinity for an antigen on a target cell.

52. The method of claim 51, wherein the nucleic acid encoding an exogenous TCR and/or CAR is introduced via viral transduction.

53. The method of claim 52, wherein the viral transduction comprises contacting the immune or precursor cell with a viral vector comprising the nucleic acid encoding an exogenous TCR and/or CAR.

54. The method of any preceding claim, wherein the viral vector is selected from the group consisting of a retroviral vector, a lentiviral vector, an adenoviral vector, and an adeno-associated viral vector

55. The method of any preceding claim, wherein the nucleic acid encoding an exogenous TCR and/or CAR is inserted into an endogenous gene locus encoding DNMT3A, thereby downregulating gene expression of endogenous DNMT3A.

56. The method of any preceding claim, wherein the nucleic acid encoding an exogenous TCR and/or CAR is inserted into an endogenous gene locus encoding DNMT3A as a result of CRISPR-mediated homology directed repair (HDR), thereby downregulating gene expression of endogenous DNMT3A.

57. The method of any preceding claim, wherein the nucleic acid encoding an exogenous TCR and/or CAR is inserted into any one of exons 7-15, or 19 of an endogenous gene locus encoding DNMT3A, thereby downregulating gene expression of endogenous DNMT3A.

58. The method of any preceding claim, wherein the nucleic acid encoding an exogenous TCR and/or CAR is inserted into exon 7 of an endogenous gene locus encoding DNMT3A, thereby downregulating gene expression of endogenous DNMT3A.

59. The method of any preceding claim, wherein the nucleic acid encoding an exogenous TCR and/or CAR is inserted into exon 8 of an endogenous gene locus encoding DNMT3A, thereby downregulating gene expression of endogenous DNMT3A.

60. The method of any preceding claim, wherein the nucleic acid encoding an exogenous TCR and/or CAR is inserted into the endogenous gene locus encoding DNMT3A via homologous recombination.

61. The method of any one of claims 55-60, wherein the nucleic acid encoding an

exogenous TCR and/or CAR is introduced via viral transduction.

62. The method of claim 61, wherein the viral transduction comprises contacting the immune or precursor cell with a viral vector comprising the nucleic acid encoding an exogenous TCR and/or CAR.

63. The method of any one of claim 62, wherein the viral vector is selected from the group consisting of a retroviral vector, a lentiviral vector, an adenoviral vector, and an adeno-associated viral (AAV) vector.

64. The method of any one of claim 63, wherein the viral vector is an adeno-associated viral (AAV) vector.

65. The method of claim 64, wherein the AAV vector comprises a 5’ ITR and a 3TTR derived from AAV6.

66. The method of claim 64 or 65, wherein the AAV vector comprises a 5’ homology arm and a 3’ homology arm, wherein the 5’ and 3’ homology arms comprise

complementarity to a target sequence in an endogenous gene locus encoding

DNMT3A.

67. The method of any one of claims 64-66, wherein the AAV vector comprises a

Woodchuck Hepatitis Virus post-transcriptional regulatory element (WPRE).

68. The method of any one of claims 64-67, wherein the AAV vector comprises a

polyadenylation (poly A) sequence.

69. The method of 68, wherein the polyA sequence is a bovine growth hormone (BGH) polyA sequence.

70. The method of any preceding claim, wherein the nucleic acid encoding an exogenous TCR and/or CAR is in operable linkage with a promoter.

71. The method of claim 70, wherein the promoter is a phosphoglycerate kinase-1 (PGK) promoter.

72. The method of any preceding claim, wherein the one or more polypeptides and/or nucleic acids capable of downregulating gene expression of endogenous DNMT3A comprise a CRISPR system.

73. The method of any preceding claim, wherein the CRISPR system comprises a

CRISPR nuclease and a guide RNA.

74. The method of claim 73, wherein the CRISPR nuclease is Cas9.

75. The method of any preceding claim, wherein the CRISPR nuclease and the guide RNA comprise a ribonucleoprotein (RNP) complex.

76. The method of claim 75, wherein the RNP complex is introduced by electroporation.

77. A method of treating cancer in a subject in need thereof, comprising administering to the subject the modified immune or precursor cell of any one of claims 1-34, or a modified immune or precursor cell generated by the method of any one of claims 35- 76.

78. A method of treating a disease or disorder in a subject in need thereof, comprising administering to the subject a modified T cell comprising:

a CRISPR-mediated modification in an endogenous gene locus encoding DNMT3A, wherein the modification is capable of downregulating gene expression of endogenous DNMT3A; and

an exogenous T cell receptor (TCR) and/or chimeric antigen receptor (CAR) comprising affinity for an antigen on a target cell.

79. A method of treating a disease or disorder in a subject in need thereof, comprising administering to the subject a modified T cell comprising:

a CRISPR-mediated modification in any one of exons 7-15, or 19 of an endogenous gene locus encoding DNMT3A, wherein the modification is capable of downregulating gene expression of endogenous DNMT3A; and

an exogenous T cell receptor (TCR) and/or chimeric antigen receptor (CAR) comprising affinity for an antigen on a target cell.

80. A method of treating a disease or disorder in a subject in need thereof, comprising administering to the subject a modified T cell comprising a nucleic acid encoding an exogenous T cell receptor (TCR) and/or chimeric antigen receptor (CAR) inserted into an endogenous gene locus encoding DNMT3A, wherein the nucleic acid encoding an exogenous TCR and/or CAR is inserted as a result of CRISPR-mediated homology directed repair (HDR), and wherein the TCR and/or CAR comprises affinity for an antigen on a target cell.

81. A method of treating a disease or disorder in a subject in need thereof, comprising administering to the subject a modified T cell comprising a nucleic acid encoding an exogenous T cell receptor (TCR) and/or chimeric antigen receptor (CAR) inserted into exon 7 or exon 8 of an endogenous gene locus encoding DNMT3A, wherein the nucleic acid encoding an exogenous TCR and/or CAR is inserted as a result of CRISPR-mediated homology directed repair (HDR), and wherein the TCR and/or CAR comprises affinity for an antigen on a target cell.

82. The method of claims 35-81, wherein the antigen on a target cell is a tumor associated antigen (TAA).

83. The method of claims 78-81, wherein the disease or disorder is cancer.

84. The method of claims 78-81, wherein the modified T cell is human.

85. The method of claims 78-81, wherein the modified T cell is autologous.

86. The method of claims 78-81, wherein the subject is human.

87. The method of claims 78-81, wherein the modified T cell exhibits a decrease in the expression of one or more exhaustion-related immune checkpoint receptors at least 10 days following administration to the subject as compared to a T cell which lacks a CRISPR-mediated modification in an endogenous gene locus encoding DNMT3A.

88. The method of claim 87, wherein the one or more exhaustion-related immune

checkpoint receptors are selected from the group consisting of Tim-3, 2B4 and Lag-3.

89. The method of claims 78-81, wherein the modified T cell exhibits a central memory T cell (Tcm) phenotype (CCR7+ CD45RO+) at least 10 days following administration ot the subject.