MODIFIED IMMUNE CELLS HAVING ENHANCED ANTI-NEOPLASIA ACTIVITY AND IMMUNOSUPPRESSION RESISTANCE

1 . A method for producing a modified immune cell with reduced immunogenicity and/or increased anti-neoplasia activity by multiplexed editing, the method comprising: modifying a target nucleobase in at least four genes or regulatory elements thereof in an immune cell, thereby generating the modified immune cell with reduced immunogenicity and/or increased anti-neoplasia activity.

2 . (canceled)

3 . The method of claim 1 , wherein at least one of the four genes is a checkpoint inhibitor gene, an immune response regulation gene, or an immunogenic gene.

4 . (canceled)

5 . The method of claim 1 , wherein expression of at least one of the four genes is reduced by at least 80% as compared to a control cell without the modification.

6 - 8 . (canceled)

9 . The method of claim 1 , wherein the four genes encode polypeptides that form a TCR complex.

10 . The method of claim 1 , wherein one of the four genes encodes a polypeptide selected from the group consisting of TRAC, a check point inhibitor, PDCD1, a T cell marker, CD52, CD7, CD3 epsilon, CD3 gamma, CD3 delta, TRBC1, TRBC2, CD4, CD5, CD7, CD30, CD33, CD52, CD70, B2M, and CIITA.

11 - 28 . (canceled)

29 . The method of claim 1 , wherein the modifying comprises deaminating the single target nucleobase.

30 . The method of claim 29 , wherein the deaminating is performed by a polypeptide comprising a deaminase.

31 . The method of claim 30 , wherein the deaminase is associated with a nucleic acid programmable DNA binding protein (napDNAbp) to form a base editor.

32 . The method of claim 31 , wherein the deaminase is fused to the nucleic acid programmable DNA binding protein (napDNAbp).

33 . (canceled)

34 . The method of claim 32 , wherein the napDNAbp comprises a Cas9 nickase or nuclease dead Cas9.

35 . The method of claim 32 , wherein the deaminase is a cytidine deaminase that converts a cytosine to a thymine or an adenosine deaminase that converts an adenosine (A) to a guanine (G).

36 . (canceled)

37 . The method of claim 35 , wherein the base editor further comprises a uracil glycosylase inhibitor.

38 - 42 . (canceled)

43 . The method of claim 40 , wherein the modifying comprises contacting the immune cell with a base editor and a guide nucleic acid sequence comprising a sequence selected from the group consisting of UUCGUAUCUGUAAAACCAAG, CCUACCUGUCACCAGGACCA, CUCUUACCUGUACCAUAACC, CACCUACCUAAGAACCAUCC, ACUCACGCUGGAUAGCCUCC, ACUCACCCAGCAUCCCCAGC, CACUCACCUUAGCCUGAGCA, and CACGCACCUGGACAGCUGAC.

44 - 55 . (canceled)

56 . The method of claim 1 , wherein the single target nucleobase is in an exon, a splice donor site or a splice acceptor site.

57 . The method of claim 1 , wherein the target nucleobase is in a splice acceptor or splice donor of a TRAC, PDCD1, CD52, CD7, B2M, CD2, CD5, or CIITA gene.

58 - 64 . (canceled)

65 . The method of claim 1 , wherein the immune cell is a human cytotoxic T cell, a regulatory T cell, a T helper cell, a dendritic cell, a B cell, or a NK cell.

66 - 69 . (canceled)

70 . The method of claim 1 , wherein the immune cell is derived from a single human donor.

71 . The method of claim 1 , further comprising contacting the immune cell with a lentivirus comprising a polynucleotide that encodes an exogenous functional chimeric antigen receptor (CAR) or a functional fragment thereof.

72 - 74 . (canceled)

75 . The method of claim 71 , wherein the CAR specifically binds a marker associated with neoplasia.

76 . The method of claim 75 , wherein the neoplasia is a T cell cancer, a B cell cancer, a lymphoma, a leukemia, or a multiple myeloma.

77 . The method of claim 76 , wherein the CAR specifically binds CD7 or BCMA.

78 - 85 . (canceled)

86 . A modified immune cell produced according to the method of claim 1 .

87 . (canceled)

88 . A modified immune cell with reduced immunogenicity or increased anti-neoplasia activity, wherein the modified immune cell comprises a single target nucleobase modification in each one of at least four gene sequences or regulatory elements thereof, wherein the gene sequences are selected from the group consisting of CD3, CD5, CD52, CD7, CD2, TRAC, CD3 epsilon, CD3 gamma, CD3 delta, TRBC1, TRBC2, CD4, CD30, CD33, CD70, B2M, and CIITA or a regulatory element of each thereof, and the immune cell is a human immune cell selected from the group consisting of a cytotoxic T cell, a regulatory T cell, a T helper cell, a dendritic cell, a B cell, and a NK cell.

89 - 177 . (canceled)

178 . A composition comprising a base editor comprising a nucleic acid programmable DNA binding protein (napDNAbp), a deaminase, and a uracil glycosylate inhibitor, and a guide nucleic acid sequence, wherein the guide nucleic acid sequence comprises a sequence selected from the group consisting of UUCGUAUCUGUAAAACCAAG, CCUACCUGUCACCAGGACCA, CUCUUACCUGUACCAUAACC, CACCUACCUAAGAACCAUCC, ACUCACGCUGGAUAGCCUCC, ACUCACCCAGCAUCCCCAGC, CACUCACCUUAGCCUGAGCA, and CACGCACCUGGACAGCUGAC.

179 . (canceled)

180 . The composition of claim 178 , wherein the napDNAbp comprises a Cas9 nickase or nuclease dead Cas9 and wherein the deaminase is a cytidine or adenosine deaminase.

181 - 184 . (canceled)

185 . A method for producing a modified immune cell with reduced immunogenicity and/or increased anti-neoplasia activity, the method comprising:

a) modifying a single target nucleobase in a first gene sequence or a regulatory element thereof in an immune cell;

b) modifying a second gene sequence or a regulatory element thereof in the immune cell with a Cas12 polypeptide, wherein the Cas12 polypeptide generates a site-specific cleavage in the second gene sequence; wherein each of the first gene and the second gene is an immunogenic gene, a checkpoint inhibitor gene, or an immune response regulation gene; and

c) contacting the modified immune cell with a lentivirus comprising a polynucleotide encoding an exogenous functional chimeric antigen receptor (CAR) or a functional fragment thereof, thereby generating a modified immune cell with reduced immunogenicity and/or increased anti-neoplasia activity.

186 . (canceled)

187 . The method of claim 185 , wherein the polynucleotide encoding the CAR or the functional fragment thereof is inserted into the site specific cleavage generated by the Cas12 polypeptide.

188 . (canceled)

189 . The method of claim 185 , wherein each of the first gene and the second gene is an immunogenic gene, a checkpoint inhibitor gene, or an immune response regulation gene.

190 - 220 . (canceled)

221 . A modified immune cell with reduced immunogenicity and/or increased anti-neoplasia activity, the modified immune cell comprising:

a) a single target nucleobase modification in a first gene sequence or a regulatory element thereof in an immune cell; and

b) a modification in a second gene sequence or a regulatory element thereof, wherein the modification is an insertion of an exogenous chimeric antigen receptor (CAR) or a functional fragment thereof or an exogenous T cell receptor or a functional fragment thereof;

wherein each of the first gene and the second gene is a immunogenic gene, a checkpoint inhibitor gene, or immune response regulation gene.

222 - 263 . (canceled)

264 . A method for producing a modified immune cell with increased anti-neoplasia activity, the method comprising: modifying a single target nucleobase in a Cbl Proto Oncogene B (CBLB) gene sequence or a regulatory element thereof in an immune cell, wherein the modification reduces an activation threshold of the immune cell compared with an immune cell lacking the modification; thereby generating a modified immune cell with increased anti-neoplasia activity.

265 . A composition comprising the modified immune cell of claim 264 .

266 - 267 . (canceled)

268 . A composition comprising a polynucleotide encoding a base editor polypeptide, wherein the base editor polypeptide comprises a nucleic acid programmable DNA binding protein (napDNAbp) and an adenosine or cytidine deaminase and at least four different guide nucleic acid sequences for base editing.

269 - 277 . (canceled)

278 . An immune cell comprising the composition of claim 268 , wherein the composition is introduced into the immune cell with electroporation, nucleofection, viral transduction, or a combination thereof.

279 - 283 . (canceled)