CN116783285A - Compositions and methods for genetically modifying CIITA in cells - Google Patents
Compositions and methods for genetically modifying CIITA in cells Download PDFInfo
- Publication number
- CN116783285A CN116783285A CN202180092210.6A CN202180092210A CN116783285A CN 116783285 A CN116783285 A CN 116783285A CN 202180092210 A CN202180092210 A CN 202180092210A CN 116783285 A CN116783285 A CN 116783285A
- Authority
- CN
- China
- Prior art keywords
- chr16
- cell
- hla
- ciita
- genetic modification
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Pending
Links
- 101100382122 Homo sapiens CIITA gene Proteins 0.000 title claims abstract description 439
- 102100026371 MHC class II transactivator Human genes 0.000 title claims abstract description 360
- 108700002010 MHC class II transactivator Proteins 0.000 title claims abstract description 358
- 238000000034 method Methods 0.000 title claims abstract description 186
- 239000000203 mixture Substances 0.000 title claims abstract description 104
- 230000014509 gene expression Effects 0.000 claims abstract description 276
- 102100029966 HLA class II histocompatibility antigen, DP alpha 1 chain Human genes 0.000 claims abstract description 64
- 101000864089 Homo sapiens HLA class II histocompatibility antigen, DP alpha 1 chain Proteins 0.000 claims abstract description 64
- 101000930802 Homo sapiens HLA class II histocompatibility antigen, DQ alpha 1 chain Proteins 0.000 claims abstract description 64
- 101000968032 Homo sapiens HLA class II histocompatibility antigen, DR beta 3 chain Proteins 0.000 claims abstract description 64
- 210000004027 cell Anatomy 0.000 claims description 971
- 238000012239 gene modification Methods 0.000 claims description 370
- 230000005017 genetic modification Effects 0.000 claims description 369
- 235000013617 genetically modified food Nutrition 0.000 claims description 369
- 125000003729 nucleotide group Chemical group 0.000 claims description 315
- 239000002773 nucleotide Substances 0.000 claims description 310
- 108020005004 Guide RNA Proteins 0.000 claims description 275
- 230000002829 reductive effect Effects 0.000 claims description 201
- 108091054438 MHC class II family Proteins 0.000 claims description 181
- 102000043131 MHC class II family Human genes 0.000 claims description 177
- 102000039446 nucleic acids Human genes 0.000 claims description 137
- 108020004707 nucleic acids Proteins 0.000 claims description 137
- 150000007523 nucleic acids Chemical class 0.000 claims description 134
- 239000011230 binding agent Substances 0.000 claims description 118
- 230000004568 DNA-binding Effects 0.000 claims description 116
- 238000006467 substitution reaction Methods 0.000 claims description 92
- 238000000684 flow cytometry Methods 0.000 claims description 91
- 108090000623 proteins and genes Proteins 0.000 claims description 90
- 210000001744 T-lymphocyte Anatomy 0.000 claims description 81
- 101150118346 HLA-A gene Proteins 0.000 claims description 75
- 210000000822 natural killer cell Anatomy 0.000 claims description 74
- 102000015736 beta 2-Microglobulin Human genes 0.000 claims description 68
- 108010081355 beta 2-Microglobulin Proteins 0.000 claims description 68
- 102000005962 receptors Human genes 0.000 claims description 66
- 108020003175 receptors Proteins 0.000 claims description 66
- 108091033409 CRISPR Proteins 0.000 claims description 61
- 102100028971 HLA class I histocompatibility antigen, C alpha chain Human genes 0.000 claims description 59
- 108010052199 HLA-C Antigens Proteins 0.000 claims description 59
- 102000004169 proteins and genes Human genes 0.000 claims description 57
- 108090000765 processed proteins & peptides Proteins 0.000 claims description 56
- 108091008874 T cell receptors Proteins 0.000 claims description 54
- 102000016266 T-Cell Antigen Receptors Human genes 0.000 claims description 54
- 102000004196 processed proteins & peptides Human genes 0.000 claims description 54
- 229920001184 polypeptide Polymers 0.000 claims description 53
- 230000008685 targeting Effects 0.000 claims description 52
- 102000043129 MHC class I family Human genes 0.000 claims description 51
- 108091054437 MHC class I family Proteins 0.000 claims description 51
- 238000010362 genome editing Methods 0.000 claims description 50
- 238000003780 insertion Methods 0.000 claims description 50
- 230000037431 insertion Effects 0.000 claims description 50
- 102100028976 HLA class I histocompatibility antigen, B alpha chain Human genes 0.000 claims description 49
- 239000008194 pharmaceutical composition Substances 0.000 claims description 49
- 239000003112 inhibitor Substances 0.000 claims description 48
- 108010058607 HLA-B Antigens Proteins 0.000 claims description 46
- 238000012217 deletion Methods 0.000 claims description 42
- 230000037430 deletion Effects 0.000 claims description 42
- 230000004048 modification Effects 0.000 claims description 37
- 238000012986 modification Methods 0.000 claims description 37
- 102000004190 Enzymes Human genes 0.000 claims description 29
- 108090000790 Enzymes Proteins 0.000 claims description 29
- 241000193996 Streptococcus pyogenes Species 0.000 claims description 24
- 108020001507 fusion proteins Proteins 0.000 claims description 20
- 102000037865 fusion proteins Human genes 0.000 claims description 20
- -1 lipid nucleic acid Chemical class 0.000 claims description 14
- 102100028970 HLA class I histocompatibility antigen, alpha chain E Human genes 0.000 claims description 11
- 101000986085 Homo sapiens HLA class I histocompatibility antigen, alpha chain E Proteins 0.000 claims description 11
- 239000013598 vector Substances 0.000 claims description 9
- 206010028980 Neoplasm Diseases 0.000 claims description 8
- 201000011510 cancer Diseases 0.000 claims description 8
- 102000004127 Cytokines Human genes 0.000 claims description 6
- 108090000695 Cytokines Proteins 0.000 claims description 6
- 238000002560 therapeutic procedure Methods 0.000 claims description 6
- 101710153660 Nuclear receptor corepressor 2 Proteins 0.000 claims description 5
- 108091008042 inhibitory receptors Proteins 0.000 claims description 5
- RYYWUUFWQRZTIU-UHFFFAOYSA-K thiophosphate Chemical compound [O-]P([O-])([O-])=S RYYWUUFWQRZTIU-UHFFFAOYSA-K 0.000 claims description 5
- 238000012546 transfer Methods 0.000 claims description 5
- 108010088652 Histocompatibility Antigens Class I Proteins 0.000 claims description 4
- 238000004519 manufacturing process Methods 0.000 claims description 3
- 208000023275 Autoimmune disease Diseases 0.000 claims description 2
- 208000035473 Communicable disease Diseases 0.000 claims description 2
- 208000015181 infectious disease Diseases 0.000 claims description 2
- 150000002632 lipids Chemical class 0.000 claims description 2
- 239000002105 nanoparticle Substances 0.000 claims description 2
- 102100028972 HLA class I histocompatibility antigen, A alpha chain Human genes 0.000 claims 5
- 108010075704 HLA-A Antigens Proteins 0.000 claims 5
- 229940122466 DNA-dependent protein kinase inhibitor Drugs 0.000 claims 3
- 239000013603 viral vector Substances 0.000 claims 2
- 102000008394 Immunoglobulin Fragments Human genes 0.000 claims 1
- 108010021625 Immunoglobulin Fragments Proteins 0.000 claims 1
- 101150069255 KLRC1 gene Proteins 0.000 claims 1
- 101100404845 Macaca mulatta NKG2A gene Proteins 0.000 claims 1
- 102100022682 NKG2-A/NKG2-B type II integral membrane protein Human genes 0.000 claims 1
- 102100029452 T cell receptor alpha chain constant Human genes 0.000 claims 1
- 230000003472 neutralizing effect Effects 0.000 claims 1
- AICOOMRHRUFYCM-ZRRPKQBOSA-N oxazine, 1 Chemical compound C([C@@H]1[C@H](C(C[C@]2(C)[C@@H]([C@H](C)N(C)C)[C@H](O)C[C@]21C)=O)CC1=CC2)C[C@H]1[C@@]1(C)[C@H]2N=C(C(C)C)OC1 AICOOMRHRUFYCM-ZRRPKQBOSA-N 0.000 claims 1
- 238000009172 cell transfer therapy Methods 0.000 abstract description 4
- 235000018102 proteins Nutrition 0.000 description 51
- 108010058597 HLA-DR Antigens Proteins 0.000 description 41
- 102000006354 HLA-DR Antigens Human genes 0.000 description 41
- 108010010378 HLA-DP Antigens Proteins 0.000 description 40
- 102000015789 HLA-DP Antigens Human genes 0.000 description 40
- 108010062347 HLA-DQ Antigens Proteins 0.000 description 40
- 229940088598 enzyme Drugs 0.000 description 28
- 108700028369 Alleles Proteins 0.000 description 23
- 238000001727 in vivo Methods 0.000 description 23
- 108010019670 Chimeric Antigen Receptors Proteins 0.000 description 22
- 241000282414 Homo sapiens Species 0.000 description 22
- 108700018351 Major Histocompatibility Complex Proteins 0.000 description 22
- 230000004044 response Effects 0.000 description 20
- 230000000735 allogeneic effect Effects 0.000 description 19
- 108020004414 DNA Proteins 0.000 description 18
- 230000000694 effects Effects 0.000 description 18
- 108020004999 messenger RNA Proteins 0.000 description 18
- 230000020382 suppression by virus of host antigen processing and presentation of peptide antigen via MHC class I Effects 0.000 description 18
- 108010008532 Deoxyribonuclease I Proteins 0.000 description 16
- 102000007260 Deoxyribonuclease I Human genes 0.000 description 16
- 108010014597 HLA-B44 Antigen Proteins 0.000 description 16
- DRTQHJPVMGBUCF-XVFCMESISA-N Uridine Chemical class O[C@@H]1[C@H](O)[C@@H](CO)O[C@H]1N1C(=O)NC(=O)C=C1 DRTQHJPVMGBUCF-XVFCMESISA-N 0.000 description 14
- 108091032973 (ribonucleotides)n+m Proteins 0.000 description 13
- 239000000427 antigen Substances 0.000 description 13
- 108091007433 antigens Proteins 0.000 description 13
- 102000036639 antigens Human genes 0.000 description 13
- 210000004940 nucleus Anatomy 0.000 description 12
- 239000003795 chemical substances by application Substances 0.000 description 11
- 108091008875 B cell receptors Proteins 0.000 description 10
- 108010004141 HLA-B35 Antigen Proteins 0.000 description 10
- 108090000856 Lyases Proteins 0.000 description 10
- 102000004317 Lyases Human genes 0.000 description 10
- 230000000295 complement effect Effects 0.000 description 10
- 108010021736 HLA-B15 Antigen Proteins 0.000 description 9
- 108010075646 HLA-B18 Antigen Proteins 0.000 description 9
- 101710163270 Nuclease Proteins 0.000 description 9
- 238000000338 in vitro Methods 0.000 description 8
- 108060001084 Luciferase Proteins 0.000 description 7
- 239000005089 Luciferase Substances 0.000 description 7
- 102000003675 cytokine receptors Human genes 0.000 description 7
- 108010057085 cytokine receptors Proteins 0.000 description 7
- 125000005647 linker group Chemical group 0.000 description 7
- 102000009410 Chemokine receptor Human genes 0.000 description 6
- 108050000299 Chemokine receptor Proteins 0.000 description 6
- 108010012236 Chemokines Proteins 0.000 description 6
- 102000019034 Chemokines Human genes 0.000 description 6
- 102100026846 Cytidine deaminase Human genes 0.000 description 6
- 108010031325 Cytidine deaminase Proteins 0.000 description 6
- 230000005867 T cell response Effects 0.000 description 6
- IQFYYKKMVGJFEH-XLPZGREQSA-N Thymidine Chemical compound O=C1NC(=O)C(C)=CN1[C@@H]1O[C@H](CO)[C@@H](O)C1 IQFYYKKMVGJFEH-XLPZGREQSA-N 0.000 description 6
- ISAKRJDGNUQOIC-UHFFFAOYSA-N Uracil Chemical compound O=C1C=CNC(=O)N1 ISAKRJDGNUQOIC-UHFFFAOYSA-N 0.000 description 6
- 235000001014 amino acid Nutrition 0.000 description 6
- 238000003556 assay Methods 0.000 description 6
- OPTASPLRGRRNAP-UHFFFAOYSA-N cytosine Chemical compound NC=1C=CNC(=O)N=1 OPTASPLRGRRNAP-UHFFFAOYSA-N 0.000 description 6
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 6
- 108091028043 Nucleic acid sequence Proteins 0.000 description 5
- 108700020467 WT1 Proteins 0.000 description 5
- 102000040856 WT1 Human genes 0.000 description 5
- 125000003275 alpha amino acid group Chemical group 0.000 description 5
- 238000006243 chemical reaction Methods 0.000 description 5
- 201000010099 disease Diseases 0.000 description 5
- 230000005782 double-strand break Effects 0.000 description 5
- 210000005260 human cell Anatomy 0.000 description 5
- 230000001225 therapeutic effect Effects 0.000 description 5
- KDCGOANMDULRCW-UHFFFAOYSA-N 7H-purine Chemical compound N1=CNC2=NC=NC2=C1 KDCGOANMDULRCW-UHFFFAOYSA-N 0.000 description 4
- 238000010354 CRISPR gene editing Methods 0.000 description 4
- 108010087480 HLA-B40 Antigen Proteins 0.000 description 4
- 108010075326 HLA-B51 Antigen Proteins 0.000 description 4
- 241000282412 Homo Species 0.000 description 4
- 241000699670 Mus sp. Species 0.000 description 4
- 102100030569 Nuclear receptor corepressor 2 Human genes 0.000 description 4
- 108700026244 Open Reading Frames Proteins 0.000 description 4
- 102000004389 Ribonucleoproteins Human genes 0.000 description 4
- 108010081734 Ribonucleoproteins Proteins 0.000 description 4
- 230000004913 activation Effects 0.000 description 4
- OIRDTQYFTABQOQ-KQYNXXCUSA-N adenosine Chemical compound C1=NC=2C(N)=NC=NC=2N1[C@@H]1O[C@H](CO)[C@@H](O)[C@H]1O OIRDTQYFTABQOQ-KQYNXXCUSA-N 0.000 description 4
- 229940024606 amino acid Drugs 0.000 description 4
- 210000003719 b-lymphocyte Anatomy 0.000 description 4
- DRTQHJPVMGBUCF-PSQAKQOGSA-N beta-L-uridine Natural products O[C@H]1[C@@H](O)[C@H](CO)O[C@@H]1N1C(=O)NC(=O)C=C1 DRTQHJPVMGBUCF-PSQAKQOGSA-N 0.000 description 4
- 230000006037 cell lysis Effects 0.000 description 4
- 210000002865 immune cell Anatomy 0.000 description 4
- 230000001965 increasing effect Effects 0.000 description 4
- 229920000642 polymer Polymers 0.000 description 4
- 102000040430 polynucleotide Human genes 0.000 description 4
- 108091033319 polynucleotide Proteins 0.000 description 4
- 239000002157 polynucleotide Substances 0.000 description 4
- 230000028327 secretion Effects 0.000 description 4
- 235000000346 sugar Nutrition 0.000 description 4
- 230000004083 survival effect Effects 0.000 description 4
- RWQNBRDOKXIBIV-UHFFFAOYSA-N thymine Chemical compound CC1=CNC(=O)NC1=O RWQNBRDOKXIBIV-UHFFFAOYSA-N 0.000 description 4
- DRTQHJPVMGBUCF-UHFFFAOYSA-N uracil arabinoside Natural products OC1C(O)C(CO)OC1N1C(=O)NC(=O)C=C1 DRTQHJPVMGBUCF-UHFFFAOYSA-N 0.000 description 4
- 229940045145 uridine Drugs 0.000 description 4
- DWRXFEITVBNRMK-UHFFFAOYSA-N Beta-D-1-Arabinofuranosylthymine Natural products O=C1NC(=O)C(C)=CN1C1C(O)C(O)C(CO)O1 DWRXFEITVBNRMK-UHFFFAOYSA-N 0.000 description 3
- 210000001266 CD8-positive T-lymphocyte Anatomy 0.000 description 3
- 108020004705 Codon Proteins 0.000 description 3
- NYHBQMYGNKIUIF-UUOKFMHZSA-N Guanosine Chemical compound C1=NC=2C(=O)NC(N)=NC=2N1[C@@H]1O[C@H](CO)[C@@H](O)[C@H]1O NYHBQMYGNKIUIF-UUOKFMHZSA-N 0.000 description 3
- 108010009256 HLA-B13 Antigen Proteins 0.000 description 3
- 108010087017 HLA-B14 Antigen Proteins 0.000 description 3
- 108010070087 HLA-B37 Antigen Proteins 0.000 description 3
- 108010034908 HLA-B52 Antigen Proteins 0.000 description 3
- 108010056113 HLA-B55 antigen Proteins 0.000 description 3
- 108010033369 HLA-B57 antigen Proteins 0.000 description 3
- 102000018713 Histocompatibility Antigens Class II Human genes 0.000 description 3
- 101000983747 Homo sapiens MHC class II transactivator Proteins 0.000 description 3
- 102000000588 Interleukin-2 Human genes 0.000 description 3
- 108010002350 Interleukin-2 Proteins 0.000 description 3
- 108091093037 Peptide nucleic acid Proteins 0.000 description 3
- 101710126859 Single-stranded DNA-binding protein Proteins 0.000 description 3
- 150000001413 amino acids Chemical class 0.000 description 3
- IQFYYKKMVGJFEH-UHFFFAOYSA-N beta-L-thymidine Natural products O=C1NC(=O)C(C)=CN1C1OC(CO)C(O)C1 IQFYYKKMVGJFEH-UHFFFAOYSA-N 0.000 description 3
- 101150090505 cas10 gene Proteins 0.000 description 3
- 238000002659 cell therapy Methods 0.000 description 3
- 229940104302 cytosine Drugs 0.000 description 3
- 230000009615 deamination Effects 0.000 description 3
- 238000006481 deamination reaction Methods 0.000 description 3
- 210000004443 dendritic cell Anatomy 0.000 description 3
- 230000008030 elimination Effects 0.000 description 3
- 238000003379 elimination reaction Methods 0.000 description 3
- 230000004907 flux Effects 0.000 description 3
- 230000002068 genetic effect Effects 0.000 description 3
- 230000001771 impaired effect Effects 0.000 description 3
- 230000001939 inductive effect Effects 0.000 description 3
- 239000007924 injection Substances 0.000 description 3
- 238000002347 injection Methods 0.000 description 3
- 230000000670 limiting effect Effects 0.000 description 3
- 210000002540 macrophage Anatomy 0.000 description 3
- 239000000463 material Substances 0.000 description 3
- 230000035772 mutation Effects 0.000 description 3
- 230000002441 reversible effect Effects 0.000 description 3
- 125000001424 substituent group Chemical group 0.000 description 3
- 235000019527 sweetened beverage Nutrition 0.000 description 3
- 208000024891 symptom Diseases 0.000 description 3
- 229940104230 thymidine Drugs 0.000 description 3
- 210000001519 tissue Anatomy 0.000 description 3
- 229940035893 uracil Drugs 0.000 description 3
- ZXIATBNUWJBBGT-JXOAFFINSA-N 5-methoxyuridine Chemical compound O=C1NC(=O)C(OC)=CN1[C@H]1[C@H](O)[C@H](O)[C@@H](CO)O1 ZXIATBNUWJBBGT-JXOAFFINSA-N 0.000 description 2
- LRSASMSXMSNRBT-UHFFFAOYSA-N 5-methylcytosine Chemical compound CC1=CNC(=O)N=C1N LRSASMSXMSNRBT-UHFFFAOYSA-N 0.000 description 2
- 101150076800 B2M gene Proteins 0.000 description 2
- 239000002126 C01EB10 - Adenosine Substances 0.000 description 2
- 101100329224 Coprinopsis cinerea (strain Okayama-7 / 130 / ATCC MYA-4618 / FGSC 9003) cpf1 gene Proteins 0.000 description 2
- 102000053602 DNA Human genes 0.000 description 2
- 101710150423 DNA nickase Proteins 0.000 description 2
- 238000002965 ELISA Methods 0.000 description 2
- 229940122069 Glycosidase inhibitor Drugs 0.000 description 2
- 102000025850 HLA-A2 Antigen Human genes 0.000 description 2
- 108010074032 HLA-A2 Antigen Proteins 0.000 description 2
- 108010061486 HLA-B27 Antigen Proteins 0.000 description 2
- 102000012153 HLA-B27 Antigen Human genes 0.000 description 2
- 108010086707 HLA-B38 Antigen Proteins 0.000 description 2
- 101000633786 Homo sapiens SLAM family member 6 Proteins 0.000 description 2
- 241000699666 Mus <mouse, genus> Species 0.000 description 2
- 108091005461 Nucleic proteins Chemical group 0.000 description 2
- 229930185560 Pseudouridine Natural products 0.000 description 2
- PTJWIQPHWPFNBW-UHFFFAOYSA-N Pseudouridine C Natural products OC1C(O)C(CO)OC1C1=CNC(=O)NC1=O PTJWIQPHWPFNBW-UHFFFAOYSA-N 0.000 description 2
- CZPWVGJYEJSRLH-UHFFFAOYSA-N Pyrimidine Chemical compound C1=CN=CN=C1 CZPWVGJYEJSRLH-UHFFFAOYSA-N 0.000 description 2
- 238000010357 RNA editing Methods 0.000 description 2
- 230000026279 RNA modification Effects 0.000 description 2
- 108010008281 Recombinant Fusion Proteins Proteins 0.000 description 2
- 102000007056 Recombinant Fusion Proteins Human genes 0.000 description 2
- 102100029197 SLAM family member 6 Human genes 0.000 description 2
- 102100022433 Single-stranded DNA cytosine deaminase Human genes 0.000 description 2
- 230000006052 T cell proliferation Effects 0.000 description 2
- 102000006943 Uracil-DNA Glycosidase Human genes 0.000 description 2
- 108010072685 Uracil-DNA Glycosidase Proteins 0.000 description 2
- 230000003213 activating effect Effects 0.000 description 2
- 229960005305 adenosine Drugs 0.000 description 2
- 238000013459 approach Methods 0.000 description 2
- 230000000712 assembly Effects 0.000 description 2
- 238000000429 assembly Methods 0.000 description 2
- WGDUUQDYDIIBKT-UHFFFAOYSA-N beta-Pseudouridine Natural products OC1OC(CN2C=CC(=O)NC2=O)C(O)C1O WGDUUQDYDIIBKT-UHFFFAOYSA-N 0.000 description 2
- 230000027455 binding Effects 0.000 description 2
- 210000004899 c-terminal region Anatomy 0.000 description 2
- 101150059443 cas12a gene Proteins 0.000 description 2
- 101150038500 cas9 gene Proteins 0.000 description 2
- 210000000349 chromosome Anatomy 0.000 description 2
- 238000003776 cleavage reaction Methods 0.000 description 2
- 238000012258 culturing Methods 0.000 description 2
- 230000016396 cytokine production Effects 0.000 description 2
- 230000009089 cytolysis Effects 0.000 description 2
- 210000001151 cytotoxic T lymphocyte Anatomy 0.000 description 2
- 230000003247 decreasing effect Effects 0.000 description 2
- 231100000673 dose–response relationship Toxicity 0.000 description 2
- 239000003937 drug carrier Substances 0.000 description 2
- 238000001943 fluorescence-activated cell sorting Methods 0.000 description 2
- 238000012268 genome sequencing Methods 0.000 description 2
- 239000003316 glycosidase inhibitor Substances 0.000 description 2
- 210000003714 granulocyte Anatomy 0.000 description 2
- UYTPUPDQBNUYGX-UHFFFAOYSA-N guanine Chemical compound O=C1NC(N)=NC2=C1N=CN2 UYTPUPDQBNUYGX-UHFFFAOYSA-N 0.000 description 2
- 210000003958 hematopoietic stem cell Anatomy 0.000 description 2
- 210000003630 histaminocyte Anatomy 0.000 description 2
- 230000006801 homologous recombination Effects 0.000 description 2
- 238000002744 homologous recombination Methods 0.000 description 2
- 230000028993 immune response Effects 0.000 description 2
- 238000012606 in vitro cell culture Methods 0.000 description 2
- 210000004263 induced pluripotent stem cell Anatomy 0.000 description 2
- 230000002401 inhibitory effect Effects 0.000 description 2
- 230000002147 killing effect Effects 0.000 description 2
- 238000002826 magnetic-activated cell sorting Methods 0.000 description 2
- 125000000956 methoxy group Chemical group [H]C([H])([H])O* 0.000 description 2
- 210000001616 monocyte Anatomy 0.000 description 2
- QJGQUHMNIGDVPM-UHFFFAOYSA-N nitrogen group Chemical group [N] QJGQUHMNIGDVPM-UHFFFAOYSA-N 0.000 description 2
- 229940127073 nucleoside analogue Drugs 0.000 description 2
- 239000000047 product Substances 0.000 description 2
- 230000004952 protein activity Effects 0.000 description 2
- 108020001580 protein domains Proteins 0.000 description 2
- PTJWIQPHWPFNBW-GBNDHIKLSA-N pseudouridine Chemical compound O[C@@H]1[C@H](O)[C@@H](CO)O[C@H]1C1=CNC(=O)NC1=O PTJWIQPHWPFNBW-GBNDHIKLSA-N 0.000 description 2
- 238000000746 purification Methods 0.000 description 2
- 238000003757 reverse transcription PCR Methods 0.000 description 2
- 125000000548 ribosyl group Chemical group C1([C@H](O)[C@H](O)[C@H](O1)CO)* 0.000 description 2
- 238000011808 rodent model Methods 0.000 description 2
- 239000000523 sample Substances 0.000 description 2
- 230000007017 scission Effects 0.000 description 2
- 238000010186 staining Methods 0.000 description 2
- 239000000758 substrate Substances 0.000 description 2
- 238000012360 testing method Methods 0.000 description 2
- 229940113082 thymine Drugs 0.000 description 2
- 230000014616 translation Effects 0.000 description 2
- 238000002054 transplantation Methods 0.000 description 2
- UHDGCWIWMRVCDJ-UHFFFAOYSA-N 1-beta-D-Xylofuranosyl-NH-Cytosine Natural products O=C1N=C(N)C=CN1C1C(O)C(O)C(CO)O1 UHDGCWIWMRVCDJ-UHFFFAOYSA-N 0.000 description 1
- UVBYMVOUBXYSFV-XUTVFYLZSA-N 1-methylpseudouridine Chemical compound O=C1NC(=O)N(C)C=C1[C@H]1[C@H](O)[C@H](O)[C@@H](CO)O1 UVBYMVOUBXYSFV-XUTVFYLZSA-N 0.000 description 1
- MGAXHFMCFLLMNG-UHFFFAOYSA-N 1h-pyrimidine-6-thione Chemical compound SC1=CC=NC=N1 MGAXHFMCFLLMNG-UHFFFAOYSA-N 0.000 description 1
- MXHRCPNRJAMMIM-SHYZEUOFSA-N 2'-deoxyuridine Chemical compound C1[C@H](O)[C@@H](CO)O[C@H]1N1C(=O)NC(=O)C=C1 MXHRCPNRJAMMIM-SHYZEUOFSA-N 0.000 description 1
- ROGPOEHBDKFHIE-WGDKSQQYSA-N 2-amino-9-[(2r,4s,5r)-4-hydroxy-5-(hydroxymethyl)-2-methyloxolan-2-yl]-3h-purin-6-one Chemical compound C1=NC(C(NC(N)=N2)=O)=C2N1[C@@]1(C)C[C@H](O)[C@@H](CO)O1 ROGPOEHBDKFHIE-WGDKSQQYSA-N 0.000 description 1
- ASJSAQIRZKANQN-CRCLSJGQSA-N 2-deoxy-D-ribose Chemical compound OC[C@@H](O)[C@@H](O)CC=O ASJSAQIRZKANQN-CRCLSJGQSA-N 0.000 description 1
- CKTSBUTUHBMZGZ-ULQXZJNLSA-N 4-amino-1-[(2r,4s,5r)-4-hydroxy-5-(hydroxymethyl)oxolan-2-yl]-5-tritiopyrimidin-2-one Chemical compound O=C1N=C(N)C([3H])=CN1[C@@H]1O[C@H](CO)[C@@H](O)C1 CKTSBUTUHBMZGZ-ULQXZJNLSA-N 0.000 description 1
- XZLIYCQRASOFQM-UHFFFAOYSA-N 5h-imidazo[4,5-d]triazine Chemical compound N1=NC=C2NC=NC2=N1 XZLIYCQRASOFQM-UHFFFAOYSA-N 0.000 description 1
- GOZMBJCYMQQACI-UHFFFAOYSA-N 6,7-dimethyl-3-[[methyl-[2-[methyl-[[1-[3-(trifluoromethyl)phenyl]indol-3-yl]methyl]amino]ethyl]amino]methyl]chromen-4-one;dihydrochloride Chemical compound Cl.Cl.C=1OC2=CC(C)=C(C)C=C2C(=O)C=1CN(C)CCN(C)CC(C1=CC=CC=C11)=CN1C1=CC=CC(C(F)(F)F)=C1 GOZMBJCYMQQACI-UHFFFAOYSA-N 0.000 description 1
- BXJHWYVXLGLDMZ-UHFFFAOYSA-N 6-O-methylguanine Chemical compound COC1=NC(N)=NC2=C1NC=N2 BXJHWYVXLGLDMZ-UHFFFAOYSA-N 0.000 description 1
- ZAOGIVYOCDXEAK-UHFFFAOYSA-N 6-n-methyl-7h-purine-2,6-diamine Chemical compound CNC1=NC(N)=NC2=C1NC=N2 ZAOGIVYOCDXEAK-UHFFFAOYSA-N 0.000 description 1
- 108010029988 AICDA (activation-induced cytidine deaminase) Proteins 0.000 description 1
- 208000035657 Abasia Diseases 0.000 description 1
- DCXYFEDJOCDNAF-UHFFFAOYSA-N Asparagine Natural products OC(=O)C(N)CC(N)=O DCXYFEDJOCDNAF-UHFFFAOYSA-N 0.000 description 1
- 125000001433 C-terminal amino-acid group Chemical group 0.000 description 1
- 102100036008 CD48 antigen Human genes 0.000 description 1
- 241000687983 Cerobasis alpha Species 0.000 description 1
- 108020004394 Complementary RNA Proteins 0.000 description 1
- MIKUYHXYGGJMLM-GIMIYPNGSA-N Crotonoside Natural products C1=NC2=C(N)NC(=O)N=C2N1[C@H]1O[C@@H](CO)[C@H](O)[C@@H]1O MIKUYHXYGGJMLM-GIMIYPNGSA-N 0.000 description 1
- UHDGCWIWMRVCDJ-PSQAKQOGSA-N Cytidine Natural products O=C1N=C(N)C=CN1[C@@H]1[C@@H](O)[C@@H](O)[C@H](CO)O1 UHDGCWIWMRVCDJ-PSQAKQOGSA-N 0.000 description 1
- 102220605874 Cytosolic arginine sensor for mTORC1 subunit 2_D10A_mutation Human genes 0.000 description 1
- NYHBQMYGNKIUIF-UHFFFAOYSA-N D-guanosine Natural products C1=2NC(N)=NC(=O)C=2N=CN1C1OC(CO)C(O)C1O NYHBQMYGNKIUIF-UHFFFAOYSA-N 0.000 description 1
- HMFHBZSHGGEWLO-SOOFDHNKSA-N D-ribofuranose Chemical compound OC[C@H]1OC(O)[C@H](O)[C@@H]1O HMFHBZSHGGEWLO-SOOFDHNKSA-N 0.000 description 1
- 102100040263 DNA dC->dU-editing enzyme APOBEC-3A Human genes 0.000 description 1
- 238000010442 DNA editing Methods 0.000 description 1
- 108091027757 Deoxyribozyme Proteins 0.000 description 1
- 101100005249 Escherichia coli (strain K12) ygcB gene Proteins 0.000 description 1
- 108700024394 Exon Proteins 0.000 description 1
- 208000009329 Graft vs Host Disease Diseases 0.000 description 1
- 102100028967 HLA class I histocompatibility antigen, alpha chain G Human genes 0.000 description 1
- 108010024164 HLA-G Antigens Proteins 0.000 description 1
- 101100437218 Homo sapiens B2M gene Proteins 0.000 description 1
- 101000716130 Homo sapiens CD48 antigen Proteins 0.000 description 1
- 101000964378 Homo sapiens DNA dC->dU-editing enzyme APOBEC-3A Proteins 0.000 description 1
- 101100395312 Homo sapiens HLA-C gene Proteins 0.000 description 1
- 101000945331 Homo sapiens Killer cell immunoglobulin-like receptor 2DL4 Proteins 0.000 description 1
- 101000863978 Homo sapiens Protein downstream neighbor of Son Proteins 0.000 description 1
- 101001000998 Homo sapiens Protein phosphatase 1 regulatory subunit 12C Proteins 0.000 description 1
- 101000662909 Homo sapiens T cell receptor beta constant 1 Proteins 0.000 description 1
- 101000662902 Homo sapiens T cell receptor beta constant 2 Proteins 0.000 description 1
- 101000831007 Homo sapiens T-cell immunoreceptor with Ig and ITIM domains Proteins 0.000 description 1
- 229930010555 Inosine Natural products 0.000 description 1
- UGQMRVRMYYASKQ-KQYNXXCUSA-N Inosine Chemical compound O[C@@H]1[C@H](O)[C@@H](CO)O[C@H]1N1C2=NC=NC(O)=C2N=C1 UGQMRVRMYYASKQ-KQYNXXCUSA-N 0.000 description 1
- 108010043610 KIR Receptors Proteins 0.000 description 1
- 102100033633 Killer cell immunoglobulin-like receptor 2DL4 Human genes 0.000 description 1
- DCXYFEDJOCDNAF-REOHCLBHSA-N L-asparagine Chemical compound OC(=O)[C@@H](N)CC(N)=O DCXYFEDJOCDNAF-REOHCLBHSA-N 0.000 description 1
- 241000124008 Mammalia Species 0.000 description 1
- 108091027974 Mature messenger RNA Proteins 0.000 description 1
- 102000018697 Membrane Proteins Human genes 0.000 description 1
- 108010052285 Membrane Proteins Proteins 0.000 description 1
- 101150076359 Mhc gene Proteins 0.000 description 1
- 101000912614 Mus musculus C-type lectin domain family 2 member D Proteins 0.000 description 1
- 125000001429 N-terminal alpha-amino-acid group Chemical group 0.000 description 1
- 230000006051 NK cell activation Effects 0.000 description 1
- 108091008043 NK cell inhibitory receptors Proteins 0.000 description 1
- 241000288906 Primates Species 0.000 description 1
- 108010029485 Protein Isoforms Proteins 0.000 description 1
- 102000001708 Protein Isoforms Human genes 0.000 description 1
- 102100035620 Protein phosphatase 1 regulatory subunit 12C Human genes 0.000 description 1
- 230000004570 RNA-binding Effects 0.000 description 1
- 101000946367 Rattus norvegicus C-type lectin domain family 2 member D11 Proteins 0.000 description 1
- PYMYPHUHKUWMLA-LMVFSUKVSA-N Ribose Natural products OC[C@@H](O)[C@@H](O)[C@@H](O)C=O PYMYPHUHKUWMLA-LMVFSUKVSA-N 0.000 description 1
- 101710143275 Single-stranded DNA cytosine deaminase Proteins 0.000 description 1
- 108091081024 Start codon Proteins 0.000 description 1
- 230000006044 T cell activation Effects 0.000 description 1
- 102100037272 T cell receptor beta constant 1 Human genes 0.000 description 1
- 102100037298 T cell receptor beta constant 2 Human genes 0.000 description 1
- 102100024834 T-cell immunoreceptor with Ig and ITIM domains Human genes 0.000 description 1
- 101100059152 Thermococcus onnurineus (strain NA1) csm1 gene Proteins 0.000 description 1
- 108091028113 Trans-activating crRNA Proteins 0.000 description 1
- 206010052779 Transplant rejections Diseases 0.000 description 1
- 239000002253 acid Substances 0.000 description 1
- 238000011467 adoptive cell therapy Methods 0.000 description 1
- 238000011316 allogeneic transplantation Methods 0.000 description 1
- HMFHBZSHGGEWLO-UHFFFAOYSA-N alpha-D-Furanose-Ribose Natural products OCC1OC(O)C(O)C1O HMFHBZSHGGEWLO-UHFFFAOYSA-N 0.000 description 1
- 238000004458 analytical method Methods 0.000 description 1
- 229960001230 asparagine Drugs 0.000 description 1
- 235000009582 asparagine Nutrition 0.000 description 1
- 230000033590 base-excision repair Effects 0.000 description 1
- 210000001124 body fluid Anatomy 0.000 description 1
- 239000010839 body fluid Substances 0.000 description 1
- 239000000872 buffer Substances 0.000 description 1
- 101150055191 cas3 gene Proteins 0.000 description 1
- 108020001778 catalytic domains Proteins 0.000 description 1
- 230000003197 catalytic effect Effects 0.000 description 1
- 230000030833 cell death Effects 0.000 description 1
- 230000011712 cell development Effects 0.000 description 1
- 239000013592 cell lysate Substances 0.000 description 1
- 230000004663 cell proliferation Effects 0.000 description 1
- 230000015861 cell surface binding Effects 0.000 description 1
- 230000003833 cell viability Effects 0.000 description 1
- 230000001413 cellular effect Effects 0.000 description 1
- 230000036755 cellular response Effects 0.000 description 1
- 125000003636 chemical group Chemical group 0.000 description 1
- 238000003501 co-culture Methods 0.000 description 1
- 239000003184 complementary RNA Substances 0.000 description 1
- 150000001875 compounds Chemical class 0.000 description 1
- 238000010924 continuous production Methods 0.000 description 1
- UHDGCWIWMRVCDJ-ZAKLUEHWSA-N cytidine Chemical compound O=C1N=C(N)C=CN1[C@H]1[C@H](O)[C@@H](O)[C@H](CO)O1 UHDGCWIWMRVCDJ-ZAKLUEHWSA-N 0.000 description 1
- 230000002939 deleterious effect Effects 0.000 description 1
- 230000001419 dependent effect Effects 0.000 description 1
- 239000002274 desiccant Substances 0.000 description 1
- MXHRCPNRJAMMIM-UHFFFAOYSA-N desoxyuridine Natural products C1C(O)C(CO)OC1N1C(=O)NC(=O)C=C1 MXHRCPNRJAMMIM-UHFFFAOYSA-N 0.000 description 1
- 238000011161 development Methods 0.000 description 1
- 230000018109 developmental process Effects 0.000 description 1
- 208000035475 disorder Diseases 0.000 description 1
- 239000003814 drug Substances 0.000 description 1
- 238000004520 electroporation Methods 0.000 description 1
- 238000002474 experimental method Methods 0.000 description 1
- 102000054767 gene variant Human genes 0.000 description 1
- 208000024908 graft versus host disease Diseases 0.000 description 1
- 229940029575 guanosine Drugs 0.000 description 1
- 210000002443 helper t lymphocyte Anatomy 0.000 description 1
- 239000000833 heterodimer Substances 0.000 description 1
- 230000003301 hydrolyzing effect Effects 0.000 description 1
- 210000000987 immune system Anatomy 0.000 description 1
- 230000005847 immunogenicity Effects 0.000 description 1
- 230000001976 improved effect Effects 0.000 description 1
- 238000010348 incorporation Methods 0.000 description 1
- 229960003786 inosine Drugs 0.000 description 1
- 230000003993 interaction Effects 0.000 description 1
- 210000000265 leukocyte Anatomy 0.000 description 1
- 210000004698 lymphocyte Anatomy 0.000 description 1
- 230000002101 lytic effect Effects 0.000 description 1
- 239000003550 marker Substances 0.000 description 1
- 238000005259 measurement Methods 0.000 description 1
- 230000001404 mediated effect Effects 0.000 description 1
- 210000001806 memory b lymphocyte Anatomy 0.000 description 1
- 210000003071 memory t lymphocyte Anatomy 0.000 description 1
- YACKEPLHDIMKIO-UHFFFAOYSA-N methylphosphonic acid Chemical compound CP(O)(O)=O YACKEPLHDIMKIO-UHFFFAOYSA-N 0.000 description 1
- 238000010369 molecular cloning Methods 0.000 description 1
- 239000000178 monomer Substances 0.000 description 1
- 230000006780 non-homologous end joining Effects 0.000 description 1
- 239000002777 nucleoside Substances 0.000 description 1
- 125000003835 nucleoside group Chemical group 0.000 description 1
- 230000008520 organization Effects 0.000 description 1
- 230000036961 partial effect Effects 0.000 description 1
- 210000003819 peripheral blood mononuclear cell Anatomy 0.000 description 1
- 108010048507 poliovirus receptor Proteins 0.000 description 1
- 230000003389 potentiating effect Effects 0.000 description 1
- 230000035755 proliferation Effects 0.000 description 1
- OYRRZWATULMEPF-UHFFFAOYSA-N pyrimidin-4-amine Chemical compound NC1=CC=NC=N1 OYRRZWATULMEPF-UHFFFAOYSA-N 0.000 description 1
- 230000009467 reduction Effects 0.000 description 1
- 230000001105 regulatory effect Effects 0.000 description 1
- 210000003705 ribosome Anatomy 0.000 description 1
- 238000012216 screening Methods 0.000 description 1
- 230000005783 single-strand break Effects 0.000 description 1
- 239000000243 solution Substances 0.000 description 1
- 239000002904 solvent Substances 0.000 description 1
- 125000006850 spacer group Chemical group 0.000 description 1
- 241000894007 species Species 0.000 description 1
- 238000010561 standard procedure Methods 0.000 description 1
- 150000008163 sugars Chemical class 0.000 description 1
- 239000006228 supernatant Substances 0.000 description 1
- 229940124597 therapeutic agent Drugs 0.000 description 1
- 238000013518 transcription Methods 0.000 description 1
- 230000035897 transcription Effects 0.000 description 1
- 108091006106 transcriptional activators Proteins 0.000 description 1
- 238000010361 transduction Methods 0.000 description 1
- 230000026683 transduction Effects 0.000 description 1
- 230000001131 transforming effect Effects 0.000 description 1
- 230000001052 transient effect Effects 0.000 description 1
- 238000013519 translation Methods 0.000 description 1
- 230000001960 triggered effect Effects 0.000 description 1
- 210000004881 tumor cell Anatomy 0.000 description 1
- 230000035899 viability Effects 0.000 description 1
- 238000001262 western blot Methods 0.000 description 1
Landscapes
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
- Medicines Containing Material From Animals Or Micro-Organisms (AREA)
Abstract
The present disclosure provides compositions and methods for reducing MHC class II protein expression in a cell comprising genetically modifying CIITA for use in, for example, adoptive cell transfer therapy.
Description
The disclosures of U.S. provisional application No. 63/130,098, U.S. provisional application No. 63/251,002, U.S. provisional application No. 63/254,971, U.S. provisional application No. 63/288,502, U.S. provisional application No. 63/254,971, and U.S. provisional application No. 63/288,502, U.S. provisional application No. 10, U.S. patent application No. 63/971, and U.S. provisional application No. 63/288,502, U.S. provisional application No. 2021, U.S. 12, 10, each of which are all incorporated herein by reference in their entirety, as required by 35U.S. C.119 (e).
The present application is presented with a sequence listing in electronic format. The sequence listing is provided as a file titled "2021-12-20_01155-0038-00pct_seq_list_st25.Txt", created at 12 months 20 of 2021, of size 410,044 bytes. The information in the sequence listing in electronic format is incorporated herein by reference in its entirety.
Background
The ability to down-regulate MHC class II is critical for many in vivo and ex vivo applications, for example, when allogeneic cells (derived from a donor) are used for transplantation and/or for example, to generate cell populations in vitro that do not activate T cells. In particular, the transfer of allogeneic cells into a subject is of great interest in the field of cell therapy. The use of allogeneic cells is limited by the problem of rejection of immune cells by the recipient subject, which recognize the transplanted cells as foreign and initiate the challenge. To avoid immune rejection problems, cell-based therapies focus on self-methods that use the subject's own cells as the cell source for the therapy, a time-consuming and expensive method.
Typically, immune rejection of allogeneic cells results from a Major Histocompatibility Complex (MHC) molecule mismatch between the donor and recipient. Within the population, MHC molecules exist in a variety of forms, including, for example, many gene variants, i.e., alleles, of any given MHC gene encoding a different form of MHC protein. The main classes of MHC molecules are referred to as MHC class I and MHC class II. MHC class I molecules (e.g., HLA-A, HLA-B, and HLA-C in humans) express and present antigens on all nucleated cells to activate cytotoxic T cells (CD8+ T cells or CTLs). MHC class II molecules (e.g., HLA-DP, HLA-DQ, and HLA-DR in humans) are expressed on only certain cell types (e.g., B cells, dendritic cells, and macrophages) and present antigens to activate helper T cells (CD4+ T cells or Th cells), which in turn provide signals to B cells to produce antibodies.
For example, minor differences in MHC alleles between individuals can cause T cells in the recipient to become activated. During T cell development, an individual's T cell repertoire is tolerant to self MHC molecules, but T cells that recognize another individual's MHC molecules may persist in the circulation and are referred to as alloreactive T cells. Alloreactive T cells can become activated, for example, by the presence of another individual's MHC molecule-expressing cells in the body, causing, for example, graft versus host disease and graft rejection.
Methods and compositions for reducing the susceptibility of allogeneic cells to rejection are of interest, including, for example, reducing MHC protein expression of the cells to avoid recipient T cell responses. Indeed, the ability of genetically modified allogeneic cells for transplantation into a subject is hampered by: multiple gene editing is required to reduce all MHC protein expression while avoiding other deleterious recipient immune responses. For example, while strategies that deplete MHC class I proteins may reduce CTL activation, cells that lack MHC class I on the surface are prone to lysis by Natural Killer (NK) cells of the immune system, as NK cell activation is regulated by MHC class I specific inhibitory receptors. Gene editing strategies that deplete MHC class II molecules have also proven difficult, especially in certain cell types, due to low editing efficiency and low cell viability, impeding practical use as cell therapies.
Thus, there is a need for improved methods and compositions for modifying allogeneic cells to overcome recipient immune rejection problems and technical difficulties associated with the multiple genetic modifications required to produce safer transplanted cells.
Disclosure of Invention
The present disclosure provides engineered cells with reduced or eliminated MHC class II surface expression. The engineered cells comprise a genetic modification in the CIITA gene (class II major histocompatibility complex transactivator), which is applicable to cell therapies. The present disclosure further provides compositions and methods for reducing or eliminating the surface expression of MHC class II proteins in a cell by genetically modifying the CIITA gene. The CIITA protein acts as a transcriptional activator (activating MHC class II promoters) and is critical for MHC class II protein expression.
In some embodiments, the disclosure further provides compositions and methods for reducing or eliminating the surface expression of MHC class I proteins in a cell, for example, by genetically modifying B2M (β -2-microglobulin) or by genetically modifying the HLA-A gene. B2M proteins form heterodimers with MHC class I molecules and are required for MHC class I protein expression on the cell surface. In some embodiments comprising a B2M gene modification, the disclosure further provides for expression of NK cell inhibitor molecules by the cells to reduce or eliminate the lytic activity of NK cells. In some embodiments, the present disclosure further provides compositions and methods for reducing or eliminating the surface expression of HLA-A in cells that are homozygous for HLa-B and homozygous for HLa-C.
In some embodiments, the methods and compositions further provide for insertion of exogenous nucleic acids encoding, for example, a targeted receptor, other polypeptides expressed on the cell surface, or polypeptides secreted from the cell. In some embodiments, the engineered cells are suitable for use as a "cell factory" for secretion of exogenous proteins in a recipient. In some embodiments, the engineered cells are suitable for use as adoptive cell therapies.
The present disclosure provides an engineered cell having reduced or eliminated MHC class II surface expression relative to an unmodified cell comprising a genetic modification in a CIITA gene, wherein the genetic modification comprises at least one nucleotide of an exon within genomic coordinates chr16:10902662-chr16: 10923285.
The present disclosure provides an engineered cell having reduced or eliminated MHC class II surface expression relative to an unmodified cell comprising a genetic modification in a CIITA gene, wherein the genetic modification comprises an insertion/deletion within genomic coordinates selected from the group consisting of: chr16: chr16: the chr16, chr16, 16, and 16 chr16:10907870-10907890, chr16:10907886-10907906, chr16:10907924-10907944, chr16:10907928-10907948, chr16:10907932-10907952, chr16:10907935-10907955, chr16:10907978-10907998, chr16:10907979-10907999, chr16:10908069-10908089, chr16:10908073-10908093, chr16:10908101-10908121, chr16:10909056-10909076, chr16:10909138-10909158, chr16:10910195-10910215, chr16:10910196-10910216, chr16:10915592-10915612, chr16:10915626-10915646, chr16:10916375-10916395, chr16:10916382-10916402, chr16:10916426-10916446, chr16:10916432-10916452, chr16:10918486-10918506, chr16:10918492-10918512, chr16:10918493-10918513, chr16:10922435-10922455, chr16:10922441-10922461, chr16:10922441-10922461, chr16:10922444-10922464, chr16:10922460-10922480, chr16:10923257-10923277 and chr16:10923265-10923285.
Provided herein is a method of making an engineered cell having reduced or eliminated surface expression of MHC class II proteins relative to an unmodified cell, the method comprising contacting the cell with a composition comprising: (a) CIITA guide RNA comprising (i) a guide sequence selected from the group consisting of SEQ ID NOS: 1-117; (ii) At least 17, 18, 19 or 20 contiguous nucleotides of a sequence selected from the group consisting of SEQ ID NOS: 1-117; (iii) A leader sequence having at least 95%, 90% or 85% identity to a sequence selected from the group consisting of SEQ ID NOS.1-117; (iv) A sequence of 10 contiguous nucleotides ± 10 nucleotides comprising the genomic coordinates listed in table 2; (v) At least 17, 18, 19 or 20 consecutive nucleotides of the sequence from (iv); or (vi) a leader sequence having at least 95%, 90% or 85% identity to a sequence selected from (v); and (b) optionally an RNA-guided DNA binding agent or a nucleic acid encoding an RNA-guided DNA binding agent.
Provided herein is a method of reducing or eliminating surface expression of MHC class II proteins in an engineered cell relative to an unmodified cell comprising contacting the cell with a composition comprising: (a) CIITA guide RNA comprising (i) a guide sequence selected from the group consisting of SEQ ID NOS: 1-117; (ii) At least 17, 18, 19 or 20 contiguous nucleotides of a sequence selected from the group consisting of SEQ ID NOS: 1-117; (iii) A leader sequence having at least 95%, 90% or 85% identity to a sequence selected from the group consisting of SEQ ID NOS.1-117; (iv) A sequence of 10 contiguous nucleotides ± 10 nucleotides comprising the genomic coordinates listed in table 2; (v) At least 17, 18, 19 or 20 consecutive nucleotides of the sequence from (iv); or (vi) a leader sequence having at least 95%, 90% or 85% identity to a sequence selected from (v); and (b) optionally an RNA-guided DNA binding agent or a nucleic acid encoding an RNA-guided DNA binding agent.
Other embodiments are provided and described throughout the claims and drawings.
Drawings
Figures 1A-1B show the results of screening CIITA guides for efficacy of editing T cells with Cas9 in two donors after electroporation with RNP. Fig. 1A shows the percentage of editing after CIITA editing in T cells. FIG. 1B shows the percentage of MHC class II negative cells after CIITA editing in T cells.
Figures 2A-2B show the dose response results of editing T cells with Cas9 and three individual CIITA guides (G013674, G013675, G013676) formulated in LNP compositions. Fig. 2A shows the percent insertion/deletion editing (n=1) in total T cells. Figure 2B shows the percentage of MHC class II negative T cells compared to untreated T cells after CIITA editing.
Fig. 3A-3B show the results of four dose-response screens for CIITA guides (CR 002961, CR009217, CR007982, and CR 007994) editing T cells with Cas 9. Fig. 3A shows the percent editing in T cells. Figure 3B shows the percentage of MHC class II negative T cells after CIITA editing.
Fig. 4A-4B show the results of the efficiency of three CIITA guides (G016086, G016092 and G016067) for editing T cells with BC 22. Fig. 4A shows the percent conversion of C to T. Figure 4B shows the percentage of MHC class II negative T cells.
Fig. 5A to 5B show the results of the insertion of three CIITA primers (G013676, G013675, G015535) of mCherry at the CIITA locus. Fig. 5A shows the percentage of mCherry positive cd4+ and cd8+ T cells. Figure 5B shows the percentage of MHC class II negative T cells with and without mCherry inserted and the percentage compared to untreated T cells.
Fig. 6A-6B show the results of CIITA guide G016086 with Cas9 or BC 22. Fig. 6A shows the percentage of total reads inserted/deleted, conversion of C to a/G, and conversion of C to G with increasing Cas9 mRNA or BC22 mRNA concentrations. Fig. 6B shows the percentage of MHC class II negative T cells with increased Cas9 mRNA or BC22 mRNA concentration.
Fig. 7A to 7F show the results of sequential editing in cd8+ T cells. FIG. 7A shows the percentage of HLA-A positive cells. Figure 7B shows the percentage of MHC class II positive cells. FIG. 7C shows the percentage of WT1TCR positive CD3+, vb8+ cells. Figure 7D shows the percentage of cd3+ cells showing mismatched TCRs. Figure 7E shows the percentage of cd3+, vb 8-cells showing only endogenous TCRs. FIG. 7F shows the percentage of CD3+, vb8+ positive for WT1TCR and negative for HLA-A and MHC class II.
Fig. 8A to 8F show the results of sequential editing in cd4+ T cells. FIG. 8A shows the percentage of HLA-A positive cells. Figure 8B shows the percentage of MHC class II positive cells. FIG. 8C shows the percentage of WT1TCR positive CD3+, vb8+ cells. Figure 8D shows the percentage of cells showing mismatched TCRs. Figure 8E shows the percentage of cd3+, vb 8-cells showing only endogenous TCRs. FIG. 8F shows the percentage of CD3+, vb8+ positive for WT1TCR and negative for HLA-A and MHC class II.
Figures 9A to 9D show the percent insertion/deletion after sequential editing of T cells in T cells for CIITA (figure 9A), HLA-A (figure 9B), TRBC1 (figure 9C) and TRBC2 (figure 9D).
FIG. 10 shows resistance to NK-cell mediated killing HLA-A knockdown (HLA-B/C matched) T cells relative to B2M knockdown T cells (optionally including exogenous HLA-E constructs) expressed as percent T cell lysis. Comparison of HLA-A knockouts, HLA-A, CIITA double knockouts, B2M knockouts, B2M+HLA-E and wild type cells.
FIGS. 11A through 11B show luciferase expression of B2M, CIITA, HLA-A or bis (HLA-A, CIITA) knockout human T cells administered to mice vaccinated with human natural killer cells. FIG. 11A shows the emissivity (photons/s/cm 2/sr) of luciferase-expressing T cells present at various time points after injection. FIG. 11B shows the emissivity (photons/s/cm 2/sr) of luciferase-expressing T cells present in different groups of mice on day 27.
FIGS. 12A through 12B show luciferase expression of B2M and alloWT1 knockout human T cells administered to mice vaccinated with human natural killer cells. FIG. 12A shows total flux (p/s) of luciferase-expressing T cells present at various time points after injection. Fig. 12B shows the total flux (p/s) of luciferase-expressing T cells present in different groups of mice after 31 days.
Figures 13A to 13B show the normalized proliferation percentage of host CD4 (figure 13A) or host CD8 (figure 13B) T cells triggered by HLA class I + HLA class II double knockout or HLA-a and HLA class II double knockout engineered autologous or allogeneic T cells.
Fig. 14A to 14F show a set of percentages cd8+ (fig. 14A), endogenous TCR (fig. 14B), wt1tcr+ (fig. 14C), HLA-A2 knockout (fig. 14D), HLa-DRDPDQ knockout (fig. 14E) and allowt1% (fig. 14F).
Figure 15 shows the total flux (p/s) of luciferase-expressing T cells present at various time points to 18 days post injection.
FIGS. 16A through 16B show IFN-. Gamma.and IL-2 release, respectively, in supernatants of killing assays containing co-cultures of engineered T cells of Allo-WT1, auto-WT1, TCR KO and wild-type (WT) groups with target tumor cells.
FIGS. 17A-17B show CIITA, HLA-A, TRAC and TRBC edits and WT1TCR insertion rates in CD8+ T cells under three conditions. For cd8+ T cells, the percentage of cells expressing the relevant cell surface proteins after sequential T cell engineering is shown in fig. 17A. The percentage of T cells with all expected edits (insertion of WT1-TCR, knock-out combination with HLA-A and CIITA) is shown in figure 17B.
Figure 18 shows the average percent editing at CIITA loci in T cells treated with sgrnas in 100-mer or 91-mer formats.
FIG. 19 shows the average percentage of CD8+ T cells negative for HLA-DR, DP, DQ surface receptors after treatment with sgRNA in 100-mer or 91-mer format targeting CIITA.
Detailed Description
The present disclosure provides engineered cells, as well as methods and compositions for genetically modifying cells to produce engineered cells and engineered cell populations suitable for use in, for example, adoptive Cell Transfer (ACT) therapies. The present disclosure provided herein overcomes certain obstacles of previous approaches by providing methods and compositions for genetically modifying CIITA to reduce expression of MHC class II proteins on the cell surface. In some embodiments, the disclosure provides engineered cells having reduced or eliminated MHC class II surface expression due to genetic modification in the CIITA gene. In some embodiments, the present disclosure provides compositions and methods for reducing or eliminating the expression of MHC class II proteins and compositions and methods for further reducing the susceptibility of cells to immune rejection. For example, in some embodiments, the methods and compositions comprise reducing or eliminating surface expression of MHC class II proteins by genetically modifying CIITA, and reducing or eliminating surface expression of MHC class I proteins and/or inserting into a cell an exogenous nucleic acid encoding an NK cell inhibitor molecule or a targeted receptor or other polypeptide (expressed or secreted on the cell surface) by genetic modification. The engineered cell compositions produced by the methods disclosed herein have desirable properties including, for example, reduced MHC molecule expression, reduced in vitro and in vivo immunogenicity, increased survival, and increased genetic compatibility with more transplanted subjects.
The term "about" or "approximately" means an acceptable error for a particular value as determined by one of ordinary skill in the art, which depends in part on how the value is measured or determined, or the degree of variation does not substantially affect the characteristics of the subject matter, or within tolerances accepted in the art, such as within 10%, 5%, 2%, or 1%. Accordingly, unless indicated to the contrary, the numerical parameters set forth in the following specification and attached claims are approximations that may vary depending upon the desired properties sought to be obtained. At the very least, and not as an attempt to limit the application of the doctrine of equivalents to the scope of the claims, each numerical parameter should at least be construed in light of the number of reported significant digits and by applying ordinary rounding techniques.
I. Definition of the definition
Unless otherwise indicated, the following terms and phrases as used herein are intended to have the following meanings:
as used herein, the term "or a combination thereof" refers to all permutations and combinations of the items listed before the term. For example, "A, B, C or a combination thereof" is intended to include at least one of the following: A. b, C, AB, AC, BC or ABC, and BA, CA, CB, ACB, CBA, BCA, BAC or CAB if order is important in a particular case. Continuing with this example, explicitly includes repeated combinations including one or more items or terms, such as BB, AAA, AAB, BBC, CBBA, CABA and the like. It will be appreciated by those skilled in the art that unless otherwise apparent from the context, there is typically no limit to the number of items or terms in any combination.
As used herein, the term "kit" refers to an encapsulated set of related components, such as one or more polynucleotides or compositions and one or more related materials, such as a delivery device (e.g., syringe), solvent, solution, buffer, instructions, or desiccant.
As used herein, "allogeneic" cells refer to cells derived from a donor subject of the same species as the recipient subject, wherein the donor subject and the recipient subject have genetic dissimilarities, e.g., genetic inconsistencies at one or more loci. Thus, for example, the cells are allogeneic with respect to the subject to whom the cells are to be administered. As used herein, cells removed or isolated from a donor that are no longer introduced into the original donor are considered allogeneic cells.
As used herein, "autologous" cells refer to cells derived from the same subject to which material is later reintroduced. Thus, for example, a cell is considered autologous if it is removed from the subject and subsequently reintroduced into the same subject.
As used herein, "β2m" or "B2M" refers to the nucleic acid sequence or protein sequence of "β -2 microglobulin"; the human gene has accession NC-000015 (range 44711492.. 44718877), referred to GRCh38.p13. The B2M protein associates with MHC class I molecules as heterodimers on the surface of nucleated cells and are required for MHC class I protein expression.
As used herein, "CIITA" or "C2TA" refers to a nucleic acid sequence or protein sequence of a "class II major histocompatibility complex transactivator"; the human gene has accession NC-000016.10 (range 10866208.. 10941562), referred to GRCh38.p13. CIITA proteins in the nucleus act as upregulators of MHC class II gene transcription and are required for MHC class II protein expression.
As used herein, "MHC" or "MHC molecule" or "MHC protein" or "MHC complex" refers to a major histocompatibility complex molecule(s) and includes, for example, MHC class I and MHC class II molecules. In humans, MHC molecules are referred to as "human leukocyte antigen" complexes or "HLA molecules" or "HLA proteins. The use of the terms "MHC" and "HLA" is not intended to be limiting; as used herein, the term "MHC" may be used to refer to a human MHC molecule, i.e., an HLA molecule. Thus, the terms "MHC" and "HLA" are used interchangeably herein.
The term "HLA-A" as used herein in the context of HLA-A proteins refers to MHC class I protein molecules, which are heterodimers composed of a heavy chain (encoded by the HLA-A gene) and a light chain (i.e. beta-2 microglobulin). The term "HLA-A" or "HLA-A gene" as used herein in the context of nucleic acids refers to a gene encoding the heavy chain of an HLA-A protein molecule. HLA-A gene is also known as "HLa class I histocompatibility, aα chain"; the human gene has accession number nc_000006.12 (29942532.. 29945870). HLA-A genes are known to span a population with thousands of different genotypes of HLA-A genes (and individuals can accept two different alleles of HLA-A genes). The public database of HLA-A alleles (including sequence information) has access to IPD-IMGT/HLa: https:// www.ebi.ac.uk/ipd/imgt/hla/. All alleles of HLA-A are encompassed by the terms "HLA-A" and "HLA-A gene".
The term "HLA-B" as used herein in the context of nucleic acids refers to a gene encoding the heavy chain of an HLA-B protein molecule. HLA-B genes are also known as "HLA class I histocompatibility, Bα chain"; the human gene has accession number nc_000006.12 (31353875.. 31357179).
The term "HLA-C" as used herein in the context of nucleic acids refers to a gene encoding the heavy chain of an HLA-C protein molecule. HLA-C genes are also known as "HLA class I histocompatibility, C.alpha.chain"; the human gene has accession number nc_000006.12 (31268749.. 31272092).
As used herein, the term "within genomic coordinates" includes boundaries of a given genomic coordinate range. For example, if chr6:29942854-chr6:29942913 are given, the coordinates chr6:29942854-chr6:29942913 are covered. Throughout the present disclosure, the genomic coordinate system of reference is based on genome annotations in GRCh38 (also known as hg 38) assembly of the human genome from the genome reference association (Geno me Reference Consortium), available on the national center for biotechnology information website. Tools and methods for transforming genomic coordinates between one assembly and another are known in the art and can be used to transform genomic coordinates provided herein to corresponding coordinates in another assembly of a human genome, including to early assemblies generated by the same organization or using the same algorithm (e.g., from GRCh38 to GRCh 37), and to assemblies generated by different organizations or algorithms (e.g., from GRCh38 to NCBI33, generated by the international union of human genome sequencing (International Human Genome Sequencing Consortium). Available methods and tools known in the art include, but are not limited to NCBI Genome Remapping Servic e available at the national center for biotechnology information (National Center for Bi otechnology Information website) website, UCSC Liftover available at the UCSC Genome Brower website, and Assembly Converter available at the Ensembl. Org website.
"exon" as used herein refers to a nucleic acid within a gene encoding a mature RNA transcript. In the case of the CIITA gene, the genomic coordinates of the start and end of each exon within the gene are known and are provided in table 1.
As used herein, the term "subject" is intended to include living organisms that can elicit an immune response, including, for example, mammals, primates, humans.
"Polynucleotide" and "nucleic acid" are used herein to refer to polymeric compounds comprising nucleosides or nucleoside analogues linked together along a backbone of a nitrogen-containing heterocyclic base or base analogue, including conventional RNA, DNA, mixed RNA-DNA, and polymers that are analogues thereof. The nucleic acid "backbone" may be comprised of a plurality of linkages including one or more of sugar-phosphodiester linkages, peptide-nucleic acid linkages ("peptide nucleic acid" or PNA; PCT No. WO 95/32305), phosphorothioate linkages, methylphosphonate linkages, or combinations thereof. NuclearThe sugar moiety of the acid may be ribose, deoxyribose, or similar compounds having a substituent (e.g., a 2 'methoxy or 2' halo substituent). The nitrogenous base can be a conventional base (A, G, C, T, U), an analog thereof (e.g., a modified uridine such as 5-methoxyuridine, pseudouridine, or N1-methylpseuduridines or other modified uridine), a inosine, a derivative of a purine or pyrimidine (e.g., N 4 Methyl deoxyguanosine, deazapurine or azapurine, deazapyrimidine or azapyrimidine, pyrimidine bases having a substituent at the 5-or 6-position (e.g.5-methylcytosine), purine bases having a substituent at the 2-, 6-or 8-position, 2-amino-6-methylaminopurine, O 6 -methylguanine, 4-thio-pyrimidine, 4-amino-pyrimidine, 4-dimethylhydrazine-pyrimidine and O 4 -alkyl-pyrimidine; U.S. Pat. No. 5,378,825 and PCT No. WO 93/13121). For general discussion, see The Biochemistry of the Nucleic Acids 5-36, adams et al, 11 th edition, 1992). The nucleic acid may comprise one or more "abasic" residues, wherein the backbone does not comprise a nitrogenous base at the polymer position (U.S. Pat. No. 5,585,481). The nucleic acid may comprise only conventional RNA or DNA sugars, bases, and linkages, or may comprise conventional components with substitutions (e.g., conventional bases with 2' methoxy linkages, or polymers containing conventional bases and one or more base analogs). Nucleic acids include "locked nucleic acids" (LNA), analogs containing one or more LNA nucleotide monomers with a double cyclic furanose unit locked into RNA in a pseudo-glycoform that enhances the hybrid affinity for complementary RNA and DNA sequences (Vester and Wengel,2004,Biochemistry 43 (42): 13233-41). RNA and DNA have different sugar moieties and may differ in the presence of uracil or an analog thereof in RNA and thymine or an analog thereof in DNA.
"guide RNA," "gRNA," and simply "guide" are used interchangeably herein to refer to, for example, a guide that directs an RNA-guided DNA binding agent to a target DNA, and may be a single guide RNA or a combination of crRNA and trRNA (also referred to as tracrRNA). Exemplary grnas include modified or unmodified forms of class II Cas nuclease guide RNAs. crRNA and trRNA can be associated as a single RNA molecule (single guide RNA, sgRNA) or as two separate RNA strands (double guide RNA, dgRNA). "guide RNA" or "gRNA" refers to various types. the trRNA may be a naturally occurring sequence or a trRNA sequence having modifications or variations as compared to a naturally occurring sequence. As used herein, "guide sequence" refers to a sequence in a guide RNA that is complementary to a target sequence and is used to direct the guide RNA to the target sequence for binding or modification (e.g., cleavage) by an RNA-guided DNA binding agent. "guide sequences" may also be referred to as "targeting sequences" or "spacer sequences". The guide sequence may be 20 base pairs in length, for example in the case of streptococcus pyogenes (Streptococcus pyogenes) (i.e., spy Cas9 (SpCas 9)) and related Cas9 homologs/xenogenic homologs. Shorter or longer sequences can also be used as guides of, for example, 15, 16, 17, 18, 19, 21, 22, 23, 24 or 25 nucleotides in length. In some embodiments, the target sequence is in, for example, a gene or on a chromosome, and is complementary to the guide sequence. In some embodiments, the degree of complementarity or identity between a guide sequence and its corresponding target sequence may be about 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100%. In some embodiments, the guide sequence may be 100% complementary or identical to the target region. In other embodiments, the guide sequence may contain at least one mismatch with the target region. For example, the guide sequence and the target sequence may contain 1, 2, 3, or 4 mismatches, wherein the total length of the target sequence is at least 17, 18, 19, 20, or more base pairs. In some embodiments, the guide sequence and target region may contain 1 to 4 mismatches, wherein the guide sequence comprises at least 17, 18, 19, 20 or more nucleotides. In some embodiments, the guide sequence and target region may contain 1, 2, 3, or 4 mismatches, wherein the guide sequence comprises 20 nucleotides.
The target sequence of the RNA-guided DNA binding agent includes both the positive and negative strands of genomic DNA (i.e., the reverse complement of the given sequence and sequence) because the nucleic acid substrate of the RNA-guided DNA binding agent is a double-stranded nucleic acid. Thus, where the guide sequence is said to be "complementary to" the target sequence, it is understood that the guide sequence may bind the guide RNA to the reverse complement of the target sequence. Thus, in some embodiments, where the guide sequence binds to the reverse complement of the target sequence, the guide sequence has identity to certain nucleotides of the target sequence (e.g., a target sequence that does not comprise PAM) except that in the guide sequence U replaces T.
As used herein, "RNA-guided DNA binding agent" means a polypeptide or a complex of polypeptides having RNA and DNA binding activity, or a DNA binding subunit of such complex, wherein the DNA binding activity is sequence specific and dependent on the sequence of the RNA. Exemplary RNA-guided DNA binding agents include Cas lyase/nickase and inactive forms thereof ("dCas DNA binding agents"). As used herein, "Cas nuclease" is also referred to as "Cas protein" which encompasses Cas lyase, cas nickase, and dCas DNA binding agents. Cas lyase/nickase and dCas DNA binders include Csm or Cmr complexes of type III CRISPR systems, cas10, csm1 or Cmr2 subunits thereof, cascade complexes of type I CRISPR systems, cas3 subunits thereof, and class 2 Cas nucleases. As used herein, a "class 2 Cas nuclease" is a single-stranded polypeptide having RNA-guided DNA binding activity. Class 2 Cas nucleases include class 2 Cas lyases/nickases (e.g., H840A, D10A or N863A variants) that further have RNA-guided DNA lyase or nickase activity; and class 2 dCas DNA binders, wherein the lyase/nickase activity is not activated. Class 2 Cas nucleases include, for example, cas9, cpf1, C2, C2C3, HF Cas9 (e.g., N497A, R661A, Q695A, Q a variants), hypas 9 (e.g., N692A, M694A, Q695A, H698A variants), eSPCas9 (1.0) (e.g., K810A, K1003A, R1060A variants), and eSPCas9 (1.1) (e.g., K848A, K1003A, R a variants) proteins and modifications thereof. Cpf1 protein (Zetsche et al, cell,163:1-13 (2015)) is homologous to Cas9 and contains a RuvC-like nuclease domain. The Cpf1 sequence of Zetsche is incorporated by reference in its entirety. See, e.g., zetsche, tables S1 and S3. See, e.g., makarova et al, nat Rev Microbiol,13 (11): 722-36 (2015); shmakov et al, molecular Cell,60:385-397 (2015).
As used herein, the term "editing agent" means an agent comprising a polypeptide capable of modification within a DNA sequence. In some embodiments, the editing agent is a lyase, such as a Cas9 lyase. In some embodiments, the editing agent is capable of deaminating bases within a DNA molecule. In some embodiments, the editing agent is capable of deaminating cytosine (C) in DNA. In some embodiments, the editing agent is a fusion protein comprising an RNA-guided nicking enzyme fused to a cytidine deaminase. In some embodiments, the editing agent is a fusion protein comprising an RNA-guided nicking enzyme fused to apodec 3A deaminase (a 3A). In some embodiments, the editing agent comprises a Cas9 nickase fused to an apodec 3A deaminase (a 3A). In some embodiments, the editing agent is a fusion protein comprising an RNA-guided nicking enzyme fused to a cytidine deaminase domain and UGI. In some embodiments, the editing agent lacks UGI.
As used herein, "cytidine deaminase" means a polypeptide or polypeptide complex capable of having cytidine deaminase activity that catalyzes the hydrolytic deamination of cytidine or deoxycytidine, typically producing uridine or deoxyuridine. Cytidine deaminase encompasses enzymes in the cytidine deaminase superfamily, and in particular, enzymes of the apobic family (apobic 1, apobic 2, apobic 4 and apobic 3 subgroups of enzymes), activation-induced cytidine deaminase (AID or AICDA) and CMP deaminase. (see, e.g., conticello et al, mol. Biol. Evol.22:367-77,2005;Conticello,Genome Biol.9:229,2008;Muramatsu et al, J. Biol. Chem. 274:1870-6, 1999); carrington et al, cells 9:1690 (2020)).
As used herein, the term "apodec 3" refers to an apodec 3 protein, such as an apodec 3 protein expressed by any of the seven genes (A3 a-A3H) of the human apodec 3 locus. Apodec 3 may have catalytic DNA or RNA editing activity. The amino acid sequence of APOBEC3A (UniPROT accession ID: p 31941) has been described and is included herein as SEQ ID NO: 40. In some embodiments, the apodec 3 protein is a human apodec 3 protein and/or a wild-type protein. Variants include proteins having a sequence that differs from the wild-type apodec 3 protein by one or several mutations (i.e. substitutions, deletions, insertions), such as one or several single point substitutions. For example, a shortened apodec 3 sequence may be used, for example by deleting several N-terminal or C-terminal amino acids, preferably one to four amino acids at the C-terminal end of the sequence. As used herein, the term "variant" refers to allelic variants, splice variants, and natural or artificial mutants that are homologous to the apodec 3 reference sequence. Variants are "functional" in that they exhibit catalytic activity for DNA or RNA editing. In some embodiments, apodec 3 (such as human apodec 3A) has a wild-type amino acid position 57 (as numbered in the wild-type sequence). In some embodiments, apodec 3 (such as human apodec 3A) has an asparagine at amino acid position 57 (as numbered in the wild-type sequence).
As used herein, a "nicking enzyme" is an enzyme that produces a single-strand break (also referred to as a "nick") in double-stranded DNA, i.e., cleaves one strand of a DNA duplex but does not cleave the other strand. As used herein, "RNA-guided DNA nicking enzyme" means a polypeptide or complex of polypeptides having DNA nicking enzyme activity, wherein the DNA nicking enzyme activity is sequence specific and depends on the sequence of the RNA. Exemplary RNA-guided DNA nickases include Cas nickases. Cas nickases include the Csm or Cmr complex of a type III CRISPR system, the Cas10, csm1 or Cmr2 subunit thereof, the cascade complex of a type I CRISPR system, the Cas3 subunit thereof, and the nickase form of a class 2 Cas nuclease. Class 2 Cas nickases include variants in which only one of the two catalytic domains is inactivated, which has RNA-guided DNA nickase activity. Class 2 Cas nickases include, for example, cas9 (e.g., H840A, D a or N863A variants of SpyCas 9), cpf1, C2, C2C3, HF Cas9 (e.g., N497A, R661A, Q695A, Q a variants), hypas 9 (e.g., N692A, M694A, Q695A, H698A variants), eSPCas9 (1.0) (e.g., K810A, K A, R1060A variants), and eSPCas9 (1.1) (e.g., K848A, K1003A, R1060A variants) proteins and modifications thereof. Cpf1 protein (Zetsche et al, cell,163:1-13 (2015)) is homologous to Cas9 and contains a RuvC-like protein domain. The Cpf1 sequence of Zetsche is incorporated by reference in its entirety. See, e.g., zetsche, tables S1 and S3."Cas9" encompasses streptococcus pyogenes (Spy) Cas9, variants of Cas9 listed herein, and equivalents thereof. See, e.g., makarova et al, nat Rev Microbiol,13 (11): 722-36 (2015); shmakov et al, molecular Cell,60:385-397 (2015).
As used herein, the term "fusion protein" refers to a hybrid polypeptide comprising protein domains from at least two different proteins. A protein may be located at the amino-terminal (N-terminal) portion or the carboxy-terminal (C-terminal) protein of the fusion protein, thus forming an "amino-terminal fusion protein" or a "carboxy-terminal fusion protein", respectively. Any of the proteins provided herein can be produced by any method known in the art. For example, the proteins provided herein can be produced via recombinant protein expression and purification, which is particularly suitable for fusion proteins comprising a peptide linker. Methods of recombinant protein expression and purification are well known and include those described by Green and Sambrook, molecular Cloning: A Laboratory Manual (4 th edition, cold Spring Harbor Laboratory Press, cold Spring Harbor, n.y. (2012)), the entire contents of which are incorporated herein by reference.
As used herein, the term "linker" refers to a chemical group or molecule that connects two adjacent molecules or moieties. Typically, the linker is disposed between or flanked by two groups, molecules or other moieties and is linked to each other via a covalent bond. In some embodiments, the linker is an amino acid or multiple amino acids (e.g., a peptide or protein), such as a 16-amino acid residue "XTEN" linker or variant thereof (see, e.g., examples; and Schellenberger et al, A recombinant polypeptide extends the in vivo half-life of peptides and proteins in a tunable manner. Nat. Biotechnol.27,1186-1190 (2009)). In some embodiments, the XTEN linker comprises the sequence SGSETPGTSE SATPES (SEQ ID NO: 900), SGSETPGTSESA (SEQ ID NO: 901) or SGSETP GTSESATPEGGSGGS (SEQ ID NO: 902).
As used herein, the term "uracil glycosidase inhibitor" or "UGI" refers to a protein capable of inhibiting uracil-DNA glycosidase (UDG) base excision repair enzyme.
As used herein, an "open reading frame" or "ORF" of a gene refers to a sequence consisting of a series of codons specifying the amino acid sequence of the protein encoded by the gene. The ORF starts at a start codon (e.g., ATG in DNA or AUG in RNA) and ends with a stop codon, e.g., TAA, TAG or TGA in DNA or UAA, UAG or UGA in RNA.
As used herein, "ribonucleoprotein" (RNP) or "RNP complex" refers to guide RNA as well as RNA-guided DNA binding agents, such as Cas nucleases, e.g., cas lyase, cas nickase, or dCas DNA binding agents (e.g., cas 9). In some embodiments, the guide RNA directs an RNA-guided DNA binding agent, such as Cas9, to the target sequence, and the guide RNA hybridizes to the target sequence and the binding agent binds to the target sequence; in the case where the binding agent is a lyase or a nicking enzyme, the binding is followed by cleavage or nicking.
As used herein, a first sequence is considered to "comprise a sequence having at least X% identity to a second sequence" if an alignment of the first sequence to the second sequence reveals that X% or more of the position of the entire second sequence matches the first sequence. For example, the sequence AAGA comprises a sequence with 100% identity to the sequence AAG, since there is a match for all three positions of the second sequence, and thus an alignment will result in 100% identity. The difference between RNA and DNA (generally, uridine exchanged for thymidine or vice versa) and the presence of nucleoside analogues (such as modified uridine) does not result in a difference in identity or complementarity between polynucleotides, provided that the relevant nucleotides (such as thymidine, uridine or modified uridine) have the same complement (e.g. adenosine for thymidine, uridine or modified uridine as a whole; another example is cytosine and 5-methylcytosine, both having guanosine or modified guanosine as complement). Thus, for example, the sequence 5'-AXG (where X is any modified uridine such as pseudouridine, N1-methyl pseudouridine or 5-methoxyuridine) is considered 100% identical to AUG, since both are fully complementary to the same sequence (5' -CAU). Exemplary alignment algorithms are the Smith-Waterman (Smith-Waterman) and Needman-Wengsch (Needleman-Wunsch) algorithms well known in the art. Those skilled in the art will understand what algorithm and parameter settings are appropriate for the pair of test sequences to be aligned; the nidman-tumbler algorithm with default settings provided by the EBI at the www.ebi.ac.uk web site server is generally suitable for sequences having a generally similar length and >50% expected identity for amino acids or >75% expected identity for nucleotides.
"mRNA" is used herein to refer to a polynucleotide and includes an open reading frame that can be translated into a polypeptide (i.e., can serve as a substrate for translation by ribosomes and aminoacylates tRNA). The mRNA may comprise a phosphate-sugar backbone comprising ribose residues or analogs thereof, such as 2' -methoxy ribose residues. In some embodiments, the sugar of the mRNA phosphate-sugar backbone consists essentially of ribose residues, 2' -methoxy ribose residues, or combinations thereof.
As used herein, "insertion/deletion" refers to an insertion/deletion mutation consisting of a plurality of nucleotides, for example, insertion/deletion at a Double Strand Break (DSB) site in a target nucleic acid.
As used herein, "reducing or eliminating" expression of a protein on a cell refers to the partial or complete loss of expression of the protein relative to an unmodified cell. In some embodiments, the surface expression of a protein on a cell is measured by flow cytometry and has "reduced or eliminated" surface expression relative to an unmodified cell, as evidenced by a decrease in fluorescent signal after staining with the same antibody to the protein. Cells having "reduced or eliminated" protein surface expression relative to unmodified cells by flow cytometry can be referred to as "negative" for the expression of the protein, as evidenced by a fluorescent signal similar to cells stained with isotype control antibodies. "reduced or eliminated" protein expression is measured by other techniques known in the art using appropriate controls known to those skilled in the art.
As used herein, "knockdown" refers to a decrease in expression of a particular gene product (e.g., protein, mRNA, or both), e.g., as compared to the expression of an unedited target sequence. Protein knockdown can be measured by detecting the total cellular amount of protein from a sample, such as a tissue, body fluid, or cell population of interest. It can also be measured by measuring the surrogate, marker or activity of the protein. Methods for measuring knockdown of mRNA are known and include analysis of mRNA isolated from a sample of interest. In some embodiments, "knockdown" may refer to the lack of expression of some particular gene product, e.g., a decrease in the amount of transcribed mRNA or a decrease in the amount of protein expressed by a cell or population of cells (including in vivo populations such as those found in tissues).
As used herein, "knockout" refers to the lack of expression of a particular gene or a particular protein in a cell. Knock-out may result in expression falling below the level detected by the assay. Knock-out may be measured by detecting the total cell amount of protein in a cell, tissue or cell population.
As used herein, "target sequence" or "genomic target sequence" refers to a nucleic acid sequence in a target gene that has complementarity to the guide sequence of a gRNA. The interaction of the target sequence with the guide sequence directs the RNA-guided DNA binding agent to bind within the target sequence and possibly nick or cleave (depending on the activity of the agent).
As used herein, "treating" refers to administering or applying a therapeutic agent for a disease or disorder in a subject, and includes inhibiting the disease, suppressing its development, alleviating one or more symptoms of the disease, curing the disease, or preventing one or more symptoms of the disease, including symptom recurrence.
Reference will now be made in detail to certain embodiments of the invention, examples of which are illustrated in the accompanying drawings. While the invention will be described in conjunction with the illustrated embodiments, it will be understood that they are not intended to limit the invention to those embodiments. On the contrary, the invention is intended to cover all alternatives, modifications and equivalents, which may be included within the invention as defined by the appended claims and the included embodiments.
Before the present teachings are described in detail, it is to be understood that this disclosure is not limited to particular compositions or method steps, as such may vary. It should be noted that, as used in this specification and the appended claims, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to "a conjugate" includes a plurality of conjugates and reference to "a cell" includes a plurality of cells and the like.
Numerical ranges include numbers defining the ranges. In view of the significant digits and errors associated with measurements, measured values and measurable values are understood to be approximations. Furthermore, the use of "include/comprise", "contain/contain" and "include/include" is not intended to be limiting. It is to be understood that both the foregoing general description and the detailed description are exemplary and explanatory only and are not restrictive of the present teachings.
Unless specifically indicated in the specification, embodiments described herein as "comprising" various components are also contemplated as "consisting of" or "consisting essentially of" the recited components; embodiments described in this specification as "consisting of" various components are also contemplated as "comprising" or "consisting essentially of" the recited components; and embodiments in this specification that "consist essentially of the recited components are also considered to be" consisting of "or" comprising "the recited components (this interchangeability and inapplicability to the use of these terms in the claims). The term "or" is used in an inclusive sense, i.e., equal to "and/or (and/or)", unless the context clearly indicates otherwise.
The section headings used herein are for organizational purposes only and are not to be construed as limiting the required subject matter in any way. In the event that any material incorporated by reference contradicts any term defined in the specification or any other expression of the specification, the specification controls. While the teachings of the present disclosure are described in connection with various embodiments, it is not intended that the teachings of the present disclosure be limited to such embodiments. On the contrary, the present teachings encompass various alternatives, modifications, and equivalents, as will be appreciated by those of skill in the art.
Genetically modified cells
A. Engineered cell compositions
The present disclosure provides engineered cell compositions having reduced or eliminated MHC class II surface expression relative to unmodified cells. In some embodiments, the engineered cell composition comprises a genetic modification in the CIITA gene. In some embodiments, the engineered cell is an allogeneic cell. In some embodiments, the engineered cells with reduced MHC class II expression are suitable for adoptive cell transfer therapy. In some embodiments, the engineered cells comprise additional genetic modifications in the cell genome to produce cells required for allogeneic transplantation purposes.
As used herein, the term "within genomic coordinates" includes boundaries of a given genomic coordinate range. For example, if chr16:10895702-10895722 is given, the coordinates chr16:10895702 and chr16:10895722 are covered.
In some embodiments, for each given range of genomic coordinates, the range may encompass +/-10 nucleotides on either end of the specified coordinates. For each given range of genomic coordinates, a range may encompass +/-5 nucleotides on either end of the range. For example, if chr16:10895702-10895722 is given, in some embodiments, the genomic target sequence or genetic modification may fall within chr16:10895692-10895732.
Genetic modifications in the CIITA gene are further described herein. In some embodiments, the genetic modification in the CIITA gene comprises any one or more of an insertion, deletion, substitution, or deamination of at least one nucleotide in the target sequence.
In some embodiments, a given range of genomic coordinates may comprise a target sequence on both strands of DNA (i.e., the plus (+) strand and the minus (-) strand).
In some embodiments, an engineered cell having reduced or eliminated MHC class II surface expression relative to an unmodified cell is provided, comprising a genetic modification in a CIITA gene, wherein the genetic modification comprises at least one nucleotide of an exon within genomic coordinates chr16:10902662-chr16: 10923285.
Boundaries of exons in the CIITA gene are known based on the ENST00000618327 transcript and are provided in table 1 below. See https:// useast @ ensembl @ org/homo_sapiens/trans/Exonsdb =core; g=ensg 00000179583; r=16:10866222-10943021; t=enst 00000618327.
Table 1.CIITA region border (hg 38 transcript: CIITA-214ENST00000618327.4).
In some embodiments, an engineered cell having reduced or eliminated MHC class II surface expression relative to an unmodified cell is provided, comprising a genetic modification in a CIITA gene, wherein the genetic modification comprises at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, or at least 10 consecutive nucleotides within the genomic coordinates chr16:10902662-chr16: 10923285.
In some embodiments, an engineered cell having reduced or eliminated MHC class II surface expression relative to an unmodified cell is provided, comprising a genetic modification in a CIITA gene, wherein the genetic modification comprises at least 5 consecutive nucleotides within genomic coordinates chr16:10902662-chr16: 10923285.
In some embodiments, an engineered cell having reduced or eliminated MHC class II surface expression relative to an unmodified cell is provided, comprising a genetic modification in a CIITA gene, wherein the genetic modification comprises at least 6, at least 7, at least 8, at least 9, or at least 10 consecutive nucleotides within genomic coordinates chr16:10902662-chr16: 10923285. In some embodiments, an engineered cell having reduced or eliminated MHC class II surface expression relative to an unmodified cell is provided, comprising a genetic modification in a CIITA gene, wherein the genetic modification comprises at least 6 consecutive nucleotides within genomic coordinates chr16:10902662-chr16: 10923285. In some embodiments, an engineered cell having reduced or eliminated MHC class II surface expression relative to an unmodified cell is provided, comprising a genetic modification in a CIITA gene, wherein the genetic modification comprises at least 7 consecutive nucleotides within genomic coordinates chr16:10902662-chr16: 10923285. In some embodiments, an engineered cell having reduced or eliminated MHC class II surface expression relative to an unmodified cell is provided, comprising a genetic modification in a CIITA gene, wherein the genetic modification comprises at least 8 consecutive nucleotides within genomic coordinates chr16:10902662-chr16: 10923285. In some embodiments, an engineered cell having reduced or eliminated MHC class II surface expression relative to an unmodified cell is provided, comprising a genetic modification in a CIITA gene, wherein the genetic modification comprises at least 9 consecutive nucleotides within genomic coordinates chr16:10902662-chr16: 10923285. In some embodiments, an engineered cell having reduced or eliminated MHC class II surface expression relative to an unmodified cell is provided, comprising a genetic modification in a CIITA gene, wherein the genetic modification comprises at least 10 consecutive nucleotides within genomic coordinates chr16:10902662-chr16: 10923285.
In some embodiments, an engineered cell having reduced or eliminated MHC class II surface expression relative to an unmodified cell is provided comprising a genetic modification in a CIITA gene, wherein the genetic modification comprises a substitution of at least one C for T or at least one A for G within the genomic coordinates chr16:10902662-chr16: 10923285. In some embodiments, an engineered cell having reduced or eliminated MHC class II surface expression relative to an unmodified cell is provided, comprising a genetic modification in a CIITA gene, wherein the genetic modification comprises substitution of at least one C to T within the genomic coordinates chr16:10902662-chr16: 10923285. In some embodiments, an engineered cell having reduced or eliminated MHC class II surface expression relative to an unmodified cell is provided, comprising a genetic modification in a CIITA gene, wherein the genetic modification comprises substitution of at least one A to G within genomic coordinates chr16:10902662-chr16: 10923285.
In some embodiments, an engineered cell having reduced or eliminated MHC class II surface expression relative to an unmodified cell is provided, comprising a genetic modification in a CIITA gene, wherein the genetic modification comprises at least one nucleotide of an exon within genomic coordinates chr16:10906542-chr16: 10923285.
In some embodiments, an engineered cell having reduced or eliminated MHC class II surface expression relative to an unmodified cell is provided, comprising a genetic modification in a CIITA gene, wherein the genetic modification comprises at least one nucleotide of an exon within genomic coordinates chr16:10906542-chr16: 10908121.
In some embodiments, an engineered cell having reduced or eliminated MHC class II surface expression relative to an unmodified cell is provided, comprising a genetic modification in a CIITA gene, wherein the genetic modification comprises at least one nucleotide of an exon within genomic coordinates selected from the group consisting of: chr16:10916432-10916452, chr16:10922444-10922464, chr16:10907924-10907944, chr16:10906985-10907005, chr16:10908073-10908093, chr16:10907433-10907453, chr16:10907979-10907999, chr16:10907139-10907159, chr16:10922435-10922455, chr16:10907384-10907404, chr16:10907434-10907454, chr16:10907119-10907139, chr16:10907539-10907559, chr16:10907810-10907830, chr16:10907315-10907335, chr16:10916426-10916446, chr16:10909138-10909158, chr16:10908101-10908121, chr16:10907790-10907810, chr16:10907787-10907807, and chr16: 10907787-10907807.
In some embodiments, an engineered cell having reduced or eliminated MHC class II surface expression relative to an unmodified cell is provided, comprising a genetic modification in a CIITA gene, wherein the genetic modification comprises at least one nucleotide of an exon within genomic coordinates selected from the group consisting of: chr16:10907539-10907559, chr16:10916426-10916446, chr16:10906907-10906927, chr16:10895702-10895722, chr16:10907757-10907777, chr16:10907623-10907643, chr16:10915626-10915646, chr16:10906756-10906776, chr16:10907476-10907496, chr16:10907385-10907405 and chr16:10923265-10923285.
In some embodiments, an engineered cell having reduced or eliminated MHC class II surface expression relative to an unmodified cell is provided, comprising a genetic modification in a CIITA gene, wherein the genetic modification comprises at least one nucleotide of an exon within genomic coordinates selected from the group consisting of: chr16:10906853-10906873, chr16:10922444-10922464, chr16:10907924-10907944, chr16:10907315-10907335, chr16:10916432-10916452, chr16:10907932-10907952, chr16:10915626-10915646, chr16:10907586-10907606, chr16:10916426-10916446, chr16:10907476-10907496, chr16:10907787-10907807, chr16:10907979-10907999, chr16:10906904-10906924 and chr16:10909138-10909158.
In some embodiments, an engineered cell having reduced or eliminated MHC class II surface expression relative to an unmodified cell is provided, comprising a genetic modification in a CIITA gene, wherein the genetic modification comprises at least one nucleotide of an exon within genomic coordinates selected from the group consisting of: chr16:10895702-10895722, chr16:10916432-10916452, chr16:10907623-10907643, chr16:10907932-10907952, chr16:10906985-10907005, chr16:10915626-10915646, chr16:10907539-10907559, chr16:10916426-10916446, chr16:10907476-10907496, chr16:10907119-10907139, chr16:10907979-10907999 and chr16:10909138-10909158.
In some embodiments, an engineered cell having reduced or eliminated MHC class II surface expression relative to an unmodified cell is provided, comprising a genetic modification in a CIITA gene, wherein the genetic modification comprises at least one nucleotide of an exon within genomic coordinates selected from the group consisting of: chr16:10906853-10906873, chr16:10906757-10906777, chr16:10895302-10895322, chr16:10907539-10907559, chr16:10907730-10907750 and chr16:10895702-10895722.
In some embodiments, an engineered cell having reduced or eliminated MHC class II surface expression relative to an unmodified cell is provided, comprising a genetic modification in a CIITA gene, wherein the genetic modification comprises at least one nucleotide of an exon within genomic coordinates selected from the group consisting of: chr16:10906853-10906873, chr16:10922444-10922464 and chr16:10916432-10916452.
In some embodiments, an engineered cell having reduced or eliminated MHC class II surface expression relative to an unmodified cell is provided, comprising a genetic modification in a CIITA gene, wherein the genetic modification comprises at least one nucleotide of an exon within genomic coordinates chr16: 10906853-10906873. In some embodiments, an engineered cell having reduced or eliminated MHC class II surface expression relative to an unmodified cell is provided, comprising a genetic modification in a CIITA gene, wherein the genetic modification comprises at least one nucleotide of an exon within genomic coordinates chr16: 10922444-10922464. In some embodiments, an engineered cell having reduced or eliminated MHC class II surface expression relative to an unmodified cell is provided, comprising a genetic modification in a CIITA gene, wherein the genetic modification comprises at least one nucleotide of an exon within genomic coordinates chr16: 10916432-10916452. In some embodiments, an engineered cell having reduced or eliminated MHC class II surface expression relative to an unmodified cell is provided, comprising a genetic modification in a CIITA gene, wherein the genetic modification comprises at least one nucleotide of an exon within genomic coordinates chr16: 10906757-10906777. In some embodiments, an engineered cell having reduced or eliminated MHC class II surface expression relative to an unmodified cell is provided, comprising a genetic modification in a CIITA gene, wherein the genetic modification comprises at least one nucleotide of an exon within genomic coordinates chr16: 10895302-10895322. In some embodiments, an engineered cell having reduced or eliminated MHC class II surface expression relative to an unmodified cell is provided, comprising a genetic modification in a CIITA gene, wherein the genetic modification comprises at least one nucleotide of an exon within genomic coordinates chr16: 10907539-10907559. In some embodiments, an engineered cell having reduced or eliminated MHC class II surface expression relative to an unmodified cell is provided, comprising a genetic modification in a CIITA gene, wherein the genetic modification comprises at least one nucleotide of an exon within genomic coordinates chr16: 10907730-10907750. In some embodiments, an engineered cell having reduced or eliminated MHC class II surface expression relative to an unmodified cell is provided, comprising a genetic modification in a CII TA gene, wherein the genetic modification comprises at least one nucleotide of an exon within genomic coordinates chr16: 10895702-10895722. In some embodiments, an engineered cell having reduced or eliminated MHC class II surface expression relative to an unmodified cell is provided, comprising a genetic modification in a CIITA gene, wherein the genetic modification comprises at least one nucleotide of an exon within genomic coordinates chr16: 10907932-10907952. In some embodiments, an engineered cell having reduced or eliminated MHC class II surface expression relative to an unmodified cell is provided, comprising a genetic modification in a CIITA gene, wherein the genetic modification comprises at least one nucleotide of an exon within genomic coordinates chr16: 10907476-10907496. In some embodiments, an engineered cell having reduced or eliminated MHC class II surface expression relative to an unmodified cell is provided, comprising a genetic modification in a CIITA gene, wherein the genetic modification comprises at least one nucleotide of an exon within genomic coordinates chr16: 10909138-10909158.
In some embodiments, an engineered cell having reduced or eliminated MHC class II surface expression relative to an unmodified cell is provided, comprising a genetic modification in a CIITA gene, wherein the genetic modification comprises an insertion/deletion within genomic coordinates selected from the group consisting of: chr16: chr16: the chr16, chr16, 16, and 16 chr16:10907820-10907840, chr16:10907870-10907890, chr16:10907886-10907906, chr16:10907924-10907944, chr16:10907928-10907948, chr16:10907932-10907952, chr16:10907935-10907955, chr16:10907978-10907998, chr16:10907979-10907999, chr16:10908069-10908089, chr16:10908073-10908093, chr16:10908101-10908121, chr16:10909056-10909076, chr16:10909138-10909158, chr16:10910195-10910215, chr16:10910196-10910216, chr16:10915592-10915612, chr16:10915626-10915646, chr16:10916375-10916395, chr16:10916382-10916402, chr16:10916426-10916446, chr16:10916432-10916452, chr16:10918486-10918506, chr16:10918492-10918512, chr16:10918493-10918513, chr16:10922435-10922455, chr16:10922441-10922461, chr16:10922441-10922461, chr16:10922444-10922464, chr16:10922460-10922480, chr16:10923257-10923277 and chr16:10923265-10923285. In some embodiments, the genetic modification comprises at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, or at least 10 contiguous nucleotides within the genomic coordinates. In some embodiments, the genetic modification comprises at least 5 contiguous nucleotides within genomic coordinates. In some embodiments, the genetic modification comprises at least 6, 7, 8, 9, or 10 contiguous nucleotides within genomic coordinates. In some embodiments, the genetic modification comprises substitution of at least one C for T or substitution of at least one a for G within the genomic coordinates.
In some embodiments, an engineered cell having reduced or eliminated MHC class II surface expression relative to an unmodified cell is provided, comprising a genetic modification in a CIITA gene, wherein the genetic modification comprises an insertion/deletion within genomic coordinates selected from the group consisting of: chr16:10916432-10916452, chr16:10922444-10922464, chr16:10907924-10907944, chr16:10906985-10907005, chr16:10908073-10908093, chr16:10907433-10907453, chr16:10907979-10907999, chr16:10907139-10907159, chr16:10922435-10922455, chr16:10907384-10907404, chr16:10907434-10907454, chr16:10907119-10907139, chr16:10907539-10907559, chr16:10907810-10907830, chr16:10907315-10907335, chr16:10916426-10916446, chr16:10909138-10909158, chr16:10908101-10908121, chr16:10907790-10907810, chr16:10907787-10907807, and chr16: 10907787-10907807. In some embodiments, the genetic modification comprises at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, or at least 10 contiguous nucleotides within the genomic coordinates. In some embodiments, the genetic modification comprises at least 5 contiguous nucleotides within genomic coordinates. In some embodiments, the genetic modification comprises at least 6, 7, 8, 9, or 10 contiguous nucleotides within genomic coordinates. In some embodiments, the genetic modification comprises substitution of at least one C for T or substitution of at least one a for G within the genomic coordinates.
In some embodiments, an engineered cell having reduced or eliminated MHC class II surface expression relative to an unmodified cell is provided, comprising a genetic modification in a CIITA gene, wherein the genetic modification comprises an insertion/deletion within genomic coordinates selected from the group consisting of: chr16:10907539-10907559, chr16:10916426-10916446, chr16:10906907-10906927, chr16:10895702-10895722, chr16:10907757-10907777, chr16:10907623-10907643, chr16:10915626-10915646, chr16:10906756-10906776, chr16:10907476-10907496, chr16:10907385-10907405 and chr16:10923265-10923285. In some embodiments, the genetic modification comprises at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, or at least 10 contiguous nucleotides within the genomic coordinates. In some embodiments, the genetic modification comprises at least 5 contiguous nucleotides within genomic coordinates. In some embodiments, the genetic modification comprises at least 6, 7, 8, 9, or 10 contiguous nucleotides within genomic coordinates. In some embodiments, the genetic modification comprises substitution of at least one C for T or substitution of at least one a for G within the genomic coordinates.
In some embodiments, an engineered cell having reduced or eliminated MHC class II surface expression relative to an unmodified cell is provided, comprising a genetic modification in a CIITA gene, wherein the genetic modification comprises an insertion/deletion within genomic coordinates selected from the group consisting of: chr16:10906853-10906873, chr16:10922444-10922464, chr16:10907924-10907944, chr16:10907315-10907335, chr16:10916432-10916452, chr16:10907932-10907952, chr16:10915626-10915646, chr16:10907586-10907606, chr16:10916426-10916446, chr16:10907476-10907496, chr16:10907787-10907807, chr16:10907979-10907999 and chr16:10906904-10906924 and chr16:10909138-10909158. In some embodiments, the genetic modification comprises at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, or at least 10 contiguous nucleotides within the genomic coordinates. In some embodiments, the genetic modification comprises at least 5 contiguous nucleotides within genomic coordinates. In some embodiments, the genetic modification comprises at least 6, 7, 8, 9, or 10 contiguous nucleotides within genomic coordinates. In some embodiments, the genetic modification comprises substitution of at least one C for T or substitution of at least one a for G within the genomic coordinates.
In some embodiments, an engineered cell having reduced or eliminated MHC class II surface expression relative to an unmodified cell is provided, comprising a genetic modification in a CIITA gene, wherein the genetic modification comprises an insertion/deletion within genomic coordinates selected from the group consisting of: chr16:10895702-10895722, chr16:10916432-10916452, chr16:10907623-10907643, chr16:10907932-10907952, chr16:10906985-10907005, chr16:10915626-10915646, chr16:10907539-10907559, chr16:10916426-10916446, chr16:10907476-10907496, chr16:10907119-10907139, chr16:10907979-10907999 and chr16:10909138-10909158. In some embodiments, the genetic modification comprises at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, or at least 10 contiguous nucleotides within the genomic coordinates. In some embodiments, the genetic modification comprises at least 5 contiguous nucleotides within genomic coordinates. In some embodiments, the genetic modification comprises at least 6, 7, 8, 9, or 10 contiguous nucleotides within genomic coordinates. In some embodiments, the genetic modification comprises substitution of at least one C for T or substitution of at least one a for G within the genomic coordinates.
In some embodiments, an engineered cell having reduced or eliminated MHC class II surface expression relative to an unmodified cell is provided, comprising a genetic modification in a CIITA gene, wherein the genetic modification comprises an insertion/deletion within genomic coordinates selected from the group consisting of: chr16:10906853-10906873, chr16:10906757-10906777, chr16:10895302-10895322, chr16:10907539-10907559, chr16:10907730-10907750 and chr16:10895702-10895722. In some embodiments, the genetic modification comprises at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, or at least 10 contiguous nucleotides within the genomic coordinates. In some embodiments, the genetic modification comprises at least 5 contiguous nucleotides within genomic coordinates. In some embodiments, the genetic modification comprises at least 6, 7, 8, 9, or 10 contiguous nucleotides within genomic coordinates. In some embodiments, the genetic modification comprises substitution of at least one C for T or substitution of at least one a for G within the genomic coordinates.
In some embodiments, an engineered cell having reduced or eliminated MHC class II surface expression relative to an unmodified cell is provided, comprising a genetic modification in a CIITA gene, wherein the genetic modification comprises an insertion/deletion within genomic coordinates selected from the group consisting of: chr16:10906853-10906873, chr16:10922444-10922464 and chr16:10916432-10916452. In some embodiments, the genetic modification comprises at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, or at least 10 contiguous nucleotides within the genomic coordinates. In some embodiments, the genetic modification comprises at least 5 contiguous nucleotides within genomic coordinates. In some embodiments, the genetic modification comprises at least 6, 7, 8, 9, or 10 contiguous nucleotides within genomic coordinates. In some embodiments, the genetic modification comprises substitution of at least one C for T or substitution of at least one a for G within the genomic coordinates.
In some embodiments, an engineered cell having reduced or eliminated MHC class II surface expression relative to an unmodified cell is provided comprising a genetic modification in a CIITA gene, wherein the genetic modification comprises an insertion/deletion within genomic coordinates chr16:10906853-10906873, a substitution of C for T, or a substitution of a for G. In some embodiments, an engineered cell having reduced or eliminated MHC class II surface expression relative to an unmodified cell is provided comprising a genetic modification in a CIITA gene, wherein the genetic modification comprises an insertion/deletion within genomic coordinates chr16:10922444-10922464, a substitution of C for T, or a substitution of a for G. In some embodiments, an engineered cell having reduced or eliminated MHC class II surface expression relative to an unmodified cell is provided comprising a genetic modification in a CIITA gene, wherein the genetic modification comprises an insertion/deletion within genomic coordinates chr16:10916432-10916452, a substitution of C for T, or a substitution of a for G. In some embodiments, an engineered cell having reduced or eliminated MHC class II surface expression relative to an unmodified cell is provided comprising a genetic modification in a CIITA gene, wherein the genetic modification comprises an insertion/deletion within genomic coordinates chr16:10906757-10906777, a substitution of C for T, or a substitution of a for G. In some embodiments, an engineered cell having reduced or eliminated MHC class II surface expression relative to an unmodified cell is provided comprising a genetic modification in a CIITA gene, wherein the genetic modification comprises an insertion/deletion within genomic coordinates chr16:10895302-10895322, a substitution of C for T, or a substitution of a for G. In some embodiments, an engineered cell having reduced or eliminated MHC class II surface expression relative to an unmodified cell is provided comprising a genetic modification in a CIITA gene, wherein the genetic modification comprises an insertion/deletion within genomic coordinates chr16:10907539-10907559, a substitution of C for T, or a substitution of a for G. In some embodiments, an engineered cell having reduced or eliminated MHC class II surface expression relative to an unmodified cell is provided comprising a genetic modification in a CIITA gene, wherein the genetic modification comprises an insertion/deletion within genomic coordinates chr16:10907730-10907750, a substitution of C for T, or a substitution of a for G. In some embodiments, an engineered cell having reduced or eliminated MHC class II surface expression relative to an unmodified cell is provided comprising a genetic modification in a CIITA gene, wherein the genetic modification comprises an insertion/deletion within genomic coordinates chr16:10895702-10895722, a substitution of C for T, or a substitution of a for G. In some embodiments, an engineered cell having reduced or eliminated MHC class II surface expression relative to an unmodified cell is provided comprising a genetic modification in a CIITA gene, wherein the genetic modification comprises an insertion/deletion within genomic coordinates chr16:10907932-10907952, a substitution of C for T, or a substitution of a for G. In some embodiments, an engineered cell having reduced or eliminated MHC class II surface expression relative to an unmodified cell is provided comprising a genetic modification in a CIITA gene, wherein the genetic modification comprises an insertion/deletion within genomic coordinates chr16:10907476-10907496, a substitution of C for T, or a substitution of a for G. In some embodiments, an engineered cell having reduced or eliminated MHC class II surface expression relative to an unmodified cell is provided comprising a genetic modification in a CIITA gene, wherein the genetic modification comprises an insertion/deletion within genomic coordinates chr16:10909138-10909158, a substitution of C for T, or a substitution of a for G. In some embodiments, the genetic modification comprises at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, or at least 10 contiguous nucleotides within the genomic coordinates. In some embodiments, the genetic modification comprises at least 5 contiguous nucleotides within genomic coordinates. In some embodiments, the genetic modification comprises at least 6, 7, 8, 9, or 10 contiguous nucleotides within genomic coordinates. In some embodiments, the genetic modification comprises substitution of at least one C for T or substitution of at least one a for G within the genomic coordinates.
In some embodiments, an engineered cell is provided in which MHC class II expression is reduced or eliminated by a gene editing system that binds to a CIITA genomic target sequence comprising at least 5 contiguous nucleotides within genomic coordinates selected from the group consisting of: chr16: chr16: the chr16, chr16, 16, and 16 chr16:10907820-10907840, chr16:10907870-10907890, chr16:10907886-10907906, chr16:10907924-10907944, chr16:10907928-10907948, chr16:10907932-10907952, chr16:10907935-10907955, chr16:10907978-10907998, chr16:10907979-10907999, chr16:10908069-10908089, chr16:10908073-10908093, chr16:10908101-10908121, chr16:10909056-10909076, chr16:10909138-10909158, chr16:10910195-10910215, chr16:10910196-10910216, chr16:10915592-10915612, chr16:10915626-10915646, chr16:10916375-10916395, chr16:10916382-10916402, chr16:10916426-10916446, chr16:10916432-10916452, chr16:10918486-10918506, chr16:10918492-10918512, chr16:10918493-10918513, chr16:10922435-10922455, chr16:10922441-10922461, chr16:10922441-10922461, chr16:10922444-10922464, chr16:10922460-10922480, chr16:10923257-10923277, chr16:10923265-10923285. In some embodiments, the CIITA genomic target sequence comprises at least 10 contiguous nucleotides within genomic coordinates. In some embodiments, the CIITA genomic target sequence comprises at least 15 contiguous nucleotides within genomic coordinates. In some embodiments, the gene editing system comprises an RNA-guided DNA binding agent. In some embodiments, the RNA-guided DNA binding agent comprises a Cas9 protein, such as streptococcus pyogenes Cas9. In some embodiments, the RNA-guided DNA binding agent comprises apodec 3A deaminase (a 3A) and RNA-guided nicking enzyme.
In some embodiments, an engineered cell is provided in which MHC class II expression is reduced or eliminated by a gene editing system that binds to a CIITA genomic target sequence comprising at least 5 contiguous nucleotides within genomic coordinates selected from the group consisting of: chr16: chr16: the chr16, chr16, 16, and 16 chr16:10908101-10908121, chr16:10909056-10909076, chr16:10909138-10909158, chr16:10910195-10910215, chr16:10910196-10910216, chr16:10915592-10915612, chr16:10915626-10915646, chr16:10916375-10916395, chr16:10916382-10916402, chr16:10916426-10916446, chr16:10916432-10916452, chr16:10918486-10918506, chr16:10918492-10918512, chr16:10918493-10918513, chr16:10922435-10922455, chr16:10922441-10922461, chr16:10922441-10922461, chr16:10922444-10922464, chr16:10922460-10922480, chr16:10923257-10923277, chr16:10923265-10923285. In some embodiments, the CII TA genomic target sequence comprises at least 10 contiguous nucleotides within genomic coordinates. In some embodiments, the CIITA genomic target sequence comprises at least 15 contiguous nucleotides within genomic coordinates. In some embodiments, the gene editing system comprises an RNA-guided DNA binding agent. In some embodiments, the RNA-guided DNA binding agent comprises a Cas9 protein, such as streptococcus pyogenes Cas9. In some embodiments, the RNA-guided DNA binding agent comprises apodec 3A deaminase (a 3A) and RNA-guided nicking enzyme.
In some embodiments, an engineered cell is provided in which MHC class II expression is reduced or eliminated by a gene editing system that binds to a CIITA genomic target sequence comprising at least 5 contiguous nucleotides within genomic coordinates selected from the group consisting of: chr16: chr16: the chr16, chr16, 16, and 16 chr16:10908101-10908121. In some embodiments, the CIITA genomic target sequence comprises at least 10 contiguous nucleotides within genomic coordinates. In some embodiments, the CIITA genomic target sequence comprises at least 15 contiguous nucleotides within genomic coordinates. In some embodiments, the gene editing system comprises an RNA-guided DNA binding agent. In some embodiments, the RNA-guided DNA binding agent comprises a Cas9 protein, such as streptococcus pyogenes Cas9. In some embodiments, the RNA-guided DNA binding agent comprises apodec 3A deaminase (a 3A) and RNA-guided nicking enzyme.
In some embodiments, an engineered cell is provided in which MHC class II expression is reduced or eliminated by a gene editing system that binds to a CIITA genomic target sequence comprising at least 5 contiguous nucleotides within genomic coordinates selected from the group consisting of: chr16:10916432-10916452, chr16:10922444-10922464, chr16:10907924-10907944, chr16:10906985-10907005, chr16:10908073-10908093, chr16:10907433-10907453, chr16:10907979-10907999, chr16:10907139-10907159, chr16:10922435-10922455, chr16:10907384-10907404, chr16:10907434-10907454, chr16:10907119-10907139, chr16:10907539-10907559, chr16:10907810-10907830, chr16:10907315-10907335, chr16:10916426-10916446, chr16:10909138-10909158, chr16:10908101-10908121, chr16:10907790-10907810, chr16:10907787-10907807, and chr16: 10907787-10907807. In some embodiments, the CIITA genomic target sequence comprises at least 10 contiguous nucleotides within genomic coordinates. In some embodiments, the CIITA genomic target sequence comprises at least 15 contiguous nucleotides within genomic coordinates. In some embodiments, the gene editing system comprises an RNA-guided DNA binding agent. In some embodiments, the RNA-guided DNA binding agent comprises a Cas9 protein, such as streptococcus pyogenes Cas9. In some embodiments, the RNA-guided DNA binding agent comprises apodec 3A deaminase (a 3A) and RNA-guided nicking enzyme.
In some embodiments, an engineered cell is provided in which MHC class II expression is reduced or eliminated by a gene editing system that binds to a CIITA genomic target sequence comprising at least 5 contiguous nucleotides within genomic coordinates selected from the group consisting of: chr16:10907539-10907559, chr16:10916426-10916446, chr16:10906907-10906927, chr16:10895702-10895722, chr16:10907757-10907777, chr16:10907623-10907643, chr16:10915626-10915646, chr16:10906756-10906776, chr16:10907476-10907496, chr16:10907385-10907405 and chr16:10923265-10923285. In some embodiments, the CIITA genomic target sequence comprises at least 10 contiguous nucleotides within genomic coordinates. In some embodiments, the CIITA genomic target sequence comprises at least 15 contiguous nucleotides within genomic coordinates. In some embodiments, the gene editing system comprises an RNA-guided DNA binding agent. In some embodiments, the RNA-guided DN a binding agent comprises a Cas9 protein, such as streptococcus pyogenes Cas9.
In some embodiments, an engineered cell is provided in which MHC class II expression is reduced or eliminated by a gene editing system that binds to a CIITA genomic target sequence comprising at least 5 contiguous nucleotides within genomic coordinates selected from the group consisting of: chr16:10907539-10907559, chr16:10916426-10916446, chr16:10906907-10906927, chr16:10895702-10895722, chr16:10907757-10907777, chr16:10907623-10907643, chr16:10915626-10915646, chr16:10906756-10906776, chr16:10907476-10907496, chr16:10907385-10907405 and chr16:10923265-10923285. In some embodiments, the CIITA genomic target sequence comprises at least 10 contiguous nucleotides within genomic coordinates. In some embodiments, the CIITA genomic target sequence comprises at least 15 contiguous nucleotides within genomic coordinates. In some embodiments, the gene editing system comprises an RNA-guided DNA binding agent. In some embodiments, the RNA-guided DN a binding agent comprises apodec 3A deaminase (a 3A) and an RNA-guided nicking enzyme.
In some embodiments, an engineered cell is provided in which MHC class II expression is reduced or eliminated by a gene editing system that binds to a CIITA genomic target sequence comprising at least 5 contiguous nucleotides within genomic coordinates selected from the group consisting of: chr16:10906853-10906873, chr16:10922444-10922464, chr16:10907924-10907944, chr16:10907315-10907335, chr16:10916432-10916452, chr16:10907932-10907952, chr16:10915626-10915646, chr16:10907586-10907606, chr16:10916426-10916446, chr16:10907476-10907496, chr16:10907787-10907807, chr16:10907979-10907999, chr16:10906904-10906924 and chr16:10909138-10909158. In some embodiments, the CIITA genomic target sequence comprises at least 10 contiguous nucleotides within genomic coordinates. In some embodiments, the CIITA genomic target sequence comprises at least 15 contiguous nucleotides within genomic coordinates. In some embodiments, the gene editing system comprises an RNA-guided DNA binding agent. In some embodiments, the RNA-guided DNA binding agent comprises a Cas9 protein, such as streptococcus pyogenes Cas9.
In some embodiments, an engineered cell is provided in which MHC class II expression is reduced or eliminated by a gene editing system that binds to a CIITA genomic target sequence comprising at least 5 contiguous nucleotides within genomic coordinates selected from the group consisting of: chr16:10895702-10895722, chr16:10916432-10916452, chr16:10907623-10907643, chr16:10907932-10907952, chr16:10906985-10907005, chr16:10915626-10915646, chr16:10907539-10907559, chr16:10916426-10916446, chr16:10907476-10907496, chr16:10907119-10907139, chr16:10907979-10907999 and chr16:10909138-10909158. In some embodiments, the CIITA genomic target sequence comprises at least 10 contiguous nucleotides within genomic coordinates. In some embodiments, the CIITA genomic target sequence comprises at least 15 contiguous nucleotides within genomic coordinates. In some embodiments, the gene editing system comprises an RNA-guided DNA binding agent. In some embodiments, the RNA-guided DNA binding agent comprises apodec 3A deaminase (a 3A) and RNA-guided nicking enzyme.
In some embodiments, an engineered cell is provided in which MHC class II expression is reduced or eliminated by a gene editing system that binds to a CIITA genomic target sequence comprising at least 5 contiguous nucleotides within genomic coordinates selected from the group consisting of: chr16:10906853-10906873, chr16:10906757-10906777, chr16:10895302-10895322, chr16:10907539-10907559, chr16:10907730-10907750 and chr16:10895702-10895722. In some embodiments, the CIITA genomic target sequence comprises at least 10 contiguous nucleotides within genomic coordinates. In some embodiments, the CIITA genomic target sequence comprises at least 15 contiguous nucleotides within genomic coordinates. In some embodiments, the gene editing system comprises an RNA-guided DNA binding agent. In some embodiments, the RNA-guided DNA binding agent comprises a Cas9 protein, such as streptococcus pyogenes Cas9. In some embodiments, the RNA-guided DNA binding agent comprises apodec 3A deaminase (a 3A) and RNA-guided nicking enzyme.
In some embodiments, an engineered cell is provided in which MHC class II expression is reduced or eliminated by a gene editing system that binds to a CIITA genomic target sequence comprising at least 5 contiguous nucleotides within genomic coordinates selected from the group consisting of: chr16:10906853-10906873, chr16:10922444-10922464 and chr16:10916432-10916452. In some embodiments, the CIITA genomic target sequence comprises at least 10 contiguous nucleotides within genomic coordinates. In some embodiments, the CIITA genomic target sequence comprises at least 15 contiguous nucleotides within genomic coordinates. In some embodiments, the gene editing system comprises an RNA-guided DNA binding agent. In some embodiments, the RNA-guided DNA binding agent comprises a Cas9 protein, such as streptococcus pyogenes Cas9.
In some embodiments, an engineered cell is provided in which MHC class II expression is reduced or eliminated by a gene editing system that binds to a CIITA genomic target sequence comprising at least 5 contiguous nucleotides within genomic coordinates chr16: 10906853-10906873. In some embodiments, an engineered cell is provided in which MHC class II expression is reduced or eliminated by a gene editing system that binds to a CIITA genomic target sequence comprising at least 5 contiguous nucleotides within genomic coordinates chr16: 10922444-10922464. In some embodiments, an engineered cell is provided in which MHC class II expression is reduced or eliminated by a gene editing system that binds to a CIITA genomic target sequence comprising at least 5 contiguous nucleotides within genomic coordinates chr16: 10906757-10906777. In some embodiments, an engineered cell is provided in which MHC class II expression is reduced or eliminated by a gene editing system that binds to a CIITA genomic target sequence comprising at least 5 contiguous nucleotides within genomic coordinates chr16: 10895302-10895322. In some embodiments, an engineered cell is provided in which MHC class II expression is reduced or eliminated by a gene editing system that binds to a CIITA genomic target sequence comprising at least 5 contiguous nucleotides within genomic coordinates chr16: 10907539-10907559. In some embodiments, an engineered cell is provided in which MHC class II expression is reduced or eliminated by a gene editing system that binds to a CIITA genomic target sequence comprising at least 5 contiguous nucleotides within genomic coordinates chr16: 10907730-10907750. In some embodiments, an engineered cell is provided in which MHC class II expression is reduced or eliminated by a gene editing system that binds to a CIITA genomic target sequence comprising at least 5 contiguous nucleotides within genomic coordinates chr16: 10895702-10895722. In some embodiments, an engineered cell is provided in which MHC class II expression is reduced or eliminated by a gene editing system that binds to a CIITA genomic target sequence comprising at least 5 contiguous nucleotides within genomic coordinates chr16: 10907932-10907952. In some embodiments, an engineered cell is provided in which MHC class II expression is reduced or eliminated by a gene editing system that binds to a CIITA genomic target sequence comprising at least 5 contiguous nucleotides within genomic coordinates chr16: 10907476-10907496. In some embodiments, an engineered cell is provided in which MHC class II expression is reduced or eliminated by a gene editing system that binds to a CIITA genomic target sequence comprising at least 5 contiguous nucleotides within genomic coordinates chr16: 10909138-10909158. In some embodiments, the CIITA genomic target sequence comprises at least 15 contiguous nucleotides within genomic coordinates. In some embodiments, the gene editing system comprises an RNA-guided DNA binding agent. In some embodiments, the RNA-guided DNA binding agent comprises a Cas9 protein, such as streptococcus pyogenes Cas9. In some embodiments, the RNA-guided DNA binding agent comprises apodec 3A deaminase (a 3A) and RNA-guided nicking enzyme.
In some embodiments, an engineered cell having reduced or eliminated MHC class II surface expression relative to an unmodified cell is provided, comprising a genetic modification in a CIITA gene as described herein, and wherein the cell further has reduced or eliminated HLA-A surface expression. In some embodiments, the engineered cell comprises a genetic modification in an HLA-A gene. In some embodiments, the engineered cell comprises a genetic modification in an HLA-A gene, and wherein the cell is homozygous for HLa-B and homozygous for HLa-C. In some embodiments, the engineered cell comprises a genetic modification that eliminates expression of MHC class I proteins on the surface of the engineered cell.
The engineered human cells described herein may comprise a genetic modification in any HLA-A allele of the HLA-A gene. The HLA genes are located in a genomic region called the HLA superlocus in chromosome 6; hundreds of HLA-A alleles have been reported in the art (see, e.g., shiina et al, nature 54:15-39 (2009). Sequences of HLA-A alleles are available in the art (see, e.g., IPD-IMGT/HLA database https:// www.ebi.ac.uk/IPD/IMGT/HLa/ole. Html for retrieval of sequences of specific HLA-A alleles).
In any of the above embodiments, an engineered cell having reduced or eliminated MHC class II surface expression relative to an unmodified cell is provided, comprising a genetic modification in a CIITA gene, wherein the modification comprises at least one nucleotide of an exon within genomic coordinates chr16:10902662-chr16:10923285, further comprising a genetic modification in an HLA-A gene, and wherein the genetic modification in the HLA-A gene comprises at least one nucleotide within genomic coordinates selected from the group consisting of: chr6: 29942854-chr6: 29942913 and chr6: 29943518-chr6: 29943619. In some embodiments, the cell comprises a genetic modification in the HLA-A gene, and wherein the genetic modification in the HLA-A gene comprises at least one nucleotide within genomic coordinates selected from the group consisting of: chr6:29942864 to chr6:29942903. In some embodiments, the cell comprises a genetic modification in the HLA-A gene, and wherein the genetic modification in the HLA-A gene comprises at least one nucleotide within genomic coordinates selected from the group consisting of: chr6:29943528 to chr6:29943609. In some embodiments, the cell comprises a genetic modification in the HLA-A gene, and wherein the genetic modification in the HLA-A gene comprises at least one nucleotide within genomic coordinates selected from the group consisting of: chr6:29942864-29942884; chr6:29942868-29942888; chr6:29942876-29942896; chr6:29942877-29942897; chr6:29942883-29942903; chr6:29943126-29943146; chr6:29943528-29943548; chr6:29943529-29943549; chr6:29943530-29943550; chr6:29943537-29943557; chr6:29943549-29943569; chr6:29943589-29943609; and chr6:29944026-29944046. In some embodiments, the cell comprises a genetic modification in an HLA-A gene, and wherein the genetic modification in the HLA-A gene comprises an insertion/deletion within genomic coordinates selected from the group consisting of: chr6:29942864-29942884; chr6:29942868-29942888; chr6:29942876-29942896; chr6:29942877-29942897; chr6:29942883-29942903; chr6:29943126-29943146; chr6:29943528-29943548; chr6:29943529-29943549; chr6:29943530-29943550; chr6:29943537-29943557; chr6:29943549-29943569; chr6:29943589-29943609; and chr6:29944026-29944046. In some embodiments, HLA-A expression of a cell is reduced or eliminated by a gene editing system that binds to an HLA-A genomic target sequence comprising at least 5 contiguous nucleotides within genomic coordinates selected from the group consisting of: chr6:29942864-29942884; chr6:29942868-29942888; chr6:29942876-29942896; chr6:29942877-29942897; chr6:29942883-29942903; chr6:29943126-29943146; chr6:29943528-29943548; chr6:29943529-29943549; chr6:29943530-29943550; chr6:29943537-29943557; chr6:29943549-29943569; chr6:29943589-29943609; and chr6:29944026-29944046. In some embodiments, the cells are homozygous for HLA-B and homozygous for HLA-C.
In any of the above embodiments, there is provided an engineered cell having reduced or eliminated MHC class II surface expression relative to an unmodified cell, comprising a genetic modification in a CIITA gene, wherein the genetic modification comprises at least one nucleotide of an exon within genomic coordinates chr16:10906542-chr16:10923285, and wherein the cell further comprises a genetic modification in an HLA-A gene, and wherein the genetic modification in an HLA-A gene comprises at least one nucleotide within genomic coordinates selected from the group consisting of: chr6: 29942854-chr6: 29942913 and chr6: 29943518-chr6: 29943619. In some embodiments, the cell comprises a genetic modification in the HLA-A gene, and wherein the genetic modification in the HLA-A gene comprises at least one nucleotide within genomic coordinates selected from the group consisting of: chr6:29942864 to chr6:29942903. In some embodiments, the cell comprises a genetic modification in the HLA-A gene, and wherein the genetic modification in the HLA-A gene comprises at least one nucleotide within genomic coordinates selected from the group consisting of: chr6:29943528 to chr6:29943609. In some embodiments, the cell comprises a genetic modification in the HLA-A gene, and wherein the genetic modification in the HLA-A gene comprises at least one nucleotide within genomic coordinates selected from the group consisting of: chr6:29942864-29942884; chr6:29942868-29942888; chr6:29942876-29942896; chr6:29942877-29942897; chr6:29942883-29942903; chr6:29943126-29943146; chr6:29943528-29943548; chr6:29943529-29943549; chr6:29943530-29943550; chr6:29943537-29943557; chr6:29943549-29943569; chr6:29943589-29943609; and chr6:29944026-29944046. In some embodiments, the cell comprises a genetic modification in an HLA-A gene, and wherein the genetic modification in the HLA-A gene comprises an insertion/deletion within genomic coordinates selected from the group consisting of: chr6:29942864-29942884; chr6:29942868-29942888; chr6:29942876-29942896; chr6:29942877-29942897; chr6:29942883-29942903; chr6:29943126-29943146; chr6:29943528-29943548; chr6:29943529-29943549; chr6:29943530-29943550; chr6:29943537-29943557; chr6:29943549-29943569; chr6:29943589-29943609; and chr6:29944026-29944046. In some embodiments, HLA-A expression of a cell is reduced or eliminated by a gene editing system that binds to an HLA-A genomic target sequence comprising at least 5 contiguous nucleotides within genomic coordinates selected from the group consisting of: chr6:29942864-29942884; chr6:29942868-29942888; chr6:29942876-29942896; chr6:29942877-29942897; chr6:29942883-29942903; chr6:29943126-29943146; chr6:29943528-29943548; chr6:29943529-29943549; chr6:29943530-29943550; chr6:29943537-29943557; chr6:29943549-29943569; chr6:29943589-29943609; and chr6:29944026-29944046. In some embodiments, the cells are homozygous for HLA-B and homozygous for HLA-C.
In any of the above embodiments, there is provided an engineered cell having reduced or eliminated MHC class II surface expression relative to an unmodified cell, comprising a genetic modification in a CIITA gene, wherein the genetic modification comprises at least one nucleotide of an exon within genomic coordinates chr16:10906542-chr16:10908121, and wherein the cell further comprises a genetic modification in an HLA-A gene, and wherein the genetic modification in an HLA-A gene comprises at least one nucleotide within genomic coordinates selected from the group consisting of: chr6: 29942854-chr6: 29942913 and chr6: 29943518-chr6: 29943619. In some embodiments, the cell comprises a genetic modification in the HLA-A gene, and wherein the genetic modification in the HLA-A gene comprises at least one nucleotide within genomic coordinates selected from the group consisting of: chr6:29942864 to chr6:29942903. In some embodiments, the cell comprises a genetic modification in the HLA-A gene, and wherein the genetic modification in the HLA-A gene comprises at least one nucleotide within genomic coordinates selected from the group consisting of: chr6:29943528 to chr6:29943609. In some embodiments, the cell comprises a genetic modification in the HLA-A gene, and wherein the genetic modification in the HLA-A gene comprises at least one nucleotide within genomic coordinates selected from the group consisting of: chr6:29942864-29942884; chr6:29942868-29942888; chr6:29942876-29942896; chr6:29942877-29942897; chr6:29942883-29942903; chr6:29943126-29943146; chr6:29943528-29943548; chr6:29943529-29943549; chr6:29943530-29943550; chr6:29943537-29943557; chr6:29943549-29943569; chr6:29943589-29943609; and chr6:29944026-29944046. In some embodiments, the cell comprises a genetic modification in an HLA-A gene, and wherein the genetic modification in the HLA-A gene comprises an insertion/deletion within genomic coordinates selected from the group consisting of: chr6:29942864-29942884; chr6:29942868-29942888; chr6:29942876-29942896; chr6:29942877-29942897; chr6:29942883-29942903; chr6:29943126-29943146; chr6:29943528-29943548; chr6:29943529-29943549; chr6:29943530-29943550; chr6:29943537-29943557; chr6:29943549-29943569; chr6:29943589-29943609; and chr6:29944026-29944046. In some embodiments, HLA-A expression of a cell is reduced or eliminated by a gene editing system that binds to an HLA-A genomic target sequence comprising at least 5 contiguous nucleotides within genomic coordinates selected from the group consisting of: chr6:29942864-29942884; chr6:29942868-29942888; chr6:29942876-29942896; chr6:29942877-29942897; chr6:29942883-29942903; chr6:29943126-29943146; chr6:29943528-29943548; chr6:29943529-29943549; chr6:29943530-29943550; chr6:29943537-29943557; chr6:29943549-29943569; chr6:29943589-29943609; and chr6:29944026-29944046. In some embodiments, the cells are homozygous for HLA-B and homozygous for HLA-C.
In some embodiments, an engineered cell having reduced or eliminated MHC class II surface expression relative to an unmodified cell is provided, comprising a genetic modification in a CIITA gene, wherein the genetic modification comprises at least one nucleotide of an exon within genomic coordinates selected from the group consisting of: chr16:10916432-10916452, chr16:10922444-10922464, chr16:10907924-10907944, chr16:10906985-10907005, chr16:10908073-10908093, chr16:10907433-10907453, chr16:10907979-10907999, chr16:10907139-10907159, chr16:10922435-10922455, chr16:10907384-10907404, chr16:10907434-10907454, chr16:10907119-10907139, chr16:10907539-10907559, chr16:10907810-10907830, chr16:10907315-10907335, chr16:10916426-10916446, chr16:10909138-10909158, chr16:10908101-10908121, chr16:10907790-10907810, chr16:10907787-10907807, and HLa-37, and the gene of the gene is modified by the gene in the gene or the combination of the gene or the genes or for the genes or for chror for chr or for chr16 or for chr16 or for or 16 or for or 16 or 16 or for or 16 or for or for or-or-or: chr6: 29942854-chr6: 29942913 and chr6: 29943518-chr6: 29943619. In some embodiments, the cell comprises a genetic modification in the HLA-A gene, and wherein the genetic modification in the HLA-A gene comprises at least one nucleotide within genomic coordinates selected from the group consisting of: chr6:29942864 to chr6:29942903. In some embodiments, the cell comprises a genetic modification in the HLA-A gene, and wherein the genetic modification in the HLA-A gene comprises at least one nucleotide within genomic coordinates selected from the group consisting of: chr6:29943528 to chr6:29943609. In some embodiments, the cell comprises a genetic modification in the HLA-A gene, and wherein the genetic modification in the HLA-A gene comprises at least one nucleotide within genomic coordinates selected from the group consisting of: chr6:29942864-29942884; chr6:29942868-29942888; chr6:29942876-29942896; chr6:29942877-29942897; chr6:29942883-29942903; chr6:29943126-29943146; chr6:29943528-29943548; chr6:29943529-29943549; chr6:29943530-29943550; chr6:29943537-29943557; chr6:29943549-29943569; chr6:29943589-29943609; and chr6:29944026-29944046. In some embodiments, the cell comprises a genetic modification in an HLA-A gene, and wherein the genetic modification in the HLA-A gene comprises an insertion/deletion within genomic coordinates selected from the group consisting of: chr6:29942864-29942884; chr6:29942868-29942888; chr6:29942876-29942896; chr6:29942877-29942897; chr6:29942883-29942903; chr6:29943126-29943146; chr6:29943528-29943548; chr6:29943529-29943549; chr6:29943530-29943550; chr6:29943537-29943557; chr6:29943549-29943569; chr6:29943589-29943609; and chr6:29944026-29944046. In some embodiments, HLA-A expression of a cell is reduced or eliminated by a gene editing system that binds to an HLA-A genomic target sequence comprising at least 5 contiguous nucleotides within genomic coordinates selected from the group consisting of: chr6:29942864-29942884; chr6:29942868-29942888; chr6:29942876-29942896; chr6:29942877-29942897; chr6:29942883-29942903; chr6:29943126-29943146; chr6:29943528-29943548; chr6:29943529-29943549; chr6:29943530-29943550; chr6:29943537-29943557; chr6:29943549-29943569; chr6:29943589-29943609; and chr6:29944026-29944046. In some embodiments, the cells are homozygous for HLA-B and homozygous for HLA-C.
In some embodiments, an engineered cell having reduced or eliminated MHC class II surface expression relative to an unmodified cell is provided, comprising a genetic modification in a CIITA gene, wherein the genetic modification comprises at least one nucleotide of an exon within genomic coordinates selected from the group consisting of: chr16:10907539-10907559, chr16:10916426-10916446, chr16:10906907-10906927, chr16:10895702-10895722, chr16:10907757-10907777, chr16:10907623-10907643, chr16:10915626-10915646, chr16:10906756-10906776, chr16:10907476-10907496, chr16:10907385-10907405 and chr16:10923265-10923285, and wherein the cell further comprises a genetic modification in the HLA-A gene, and wherein the genetic modification in the HLA-A gene comprises at least one nucleotide within genomic coordinates selected from the group consisting of: chr6: 29942854-chr6: 29942913 and chr6: 29943518-chr6: 29943619. In some embodiments, the cell comprises a genetic modification in the HLA-A gene, and wherein the genetic modification in the HLA-A gene comprises at least one nucleotide within genomic coordinates selected from the group consisting of: chr6:29942864 to chr6:29942903. In some embodiments, the cell comprises a genetic modification in the HLA-A gene, and wherein the genetic modification in the HLA-A gene comprises at least one nucleotide within genomic coordinates selected from the group consisting of: chr6:29943528 to chr6:29943609. In some embodiments, the cell comprises a genetic modification in the HLA-A gene, and wherein the genetic modification in the HLA-A gene comprises at least one nucleotide within genomic coordinates selected from the group consisting of: chr6:29942864-29942884; chr6:29942868-29942888; chr6:29942876-29942896; chr6:29942877-29942897; chr6:29942883-29942903; chr6:29943126-29943146; chr6:29943528-29943548; chr6:29943529-29943549; chr6:29943530-29943550; chr6:29943537-29943557; chr6:29943549-29943569; chr6:29943589-29943609; and chr6:29944026-29944046. In some embodiments, the cell comprises a genetic modification in an HLA-A gene, and wherein the genetic modification in the HLA-A gene comprises an insertion/deletion within genomic coordinates selected from the group consisting of: chr6:29942864-29942884; chr6:29942868-29942888; chr6:29942876-29942896; chr6:29942877-29942897; chr6:29942883-29942903; chr6:29943126-29943146; chr6:29943528-29943548; chr6:29943529-29943549; chr6:29943530-29943550; chr6:29943537-29943557; chr6:29943549-29943569; chr6:29943589-29943609; and chr6:29944026-29944046. In some embodiments, HLA-A expression of a cell is reduced or eliminated by a gene editing system that binds to an HLA-A genomic target sequence comprising at least 5 contiguous nucleotides within genomic coordinates selected from the group consisting of: chr6:29942864-29942884; chr6:29942868-29942888; chr6:29942876-29942896; chr6:29942877-29942897; chr6:29942883-29942903; chr6:29943126-29943146; chr6:29943528-29943548; chr6:29943529-29943549; chr6:29943530-29943550; chr6:29943537-29943557; chr6:29943549-29943569; chr6:29943589-29943609; and chr6:29944026-29944046. In some embodiments, the cells are homozygous for HLA-B and homozygous for HLA-C.
In some embodiments, an engineered cell having reduced or eliminated MHC class II surface expression relative to an unmodified cell is provided, comprising a genetic modification in a CIITA gene, wherein the genetic modification comprises at least one nucleotide of an exon within genomic coordinates selected from the group consisting of: chr16:10906853-10906873, chr16:10906757-10906777, chr16:10895302-10895322, chr16:10907539-10907559, chr16:10907730-10907750, chr16:10895702-10895722, and wherein the cell further comprises a genetic modification in the HLA-A gene, and wherein the genetic modification in the HLA-A gene comprises at least one nucleotide within genomic coordinates selected from the group consisting of: chr6:29942854 to chr6: 29942913 and chr6:29943518 to chr6:29943619. In some embodiments, the cell comprises a genetic modification in the HLA-A gene, and wherein the genetic modification in the HLA-A gene comprises at least one nucleotide within genomic coordinates selected from the group consisting of: chr6:29942864 to chr6:29942903. In some embodiments, the cell comprises a genetic modification in the HLA-A gene, and wherein the genetic modification in the HLA-A gene comprises at least one nucleotide within genomic coordinates selected from the group consisting of: chr6:29943528 to chr6:29943609. In some embodiments, the cell comprises a genetic modification in the HLA-A gene, and wherein the genetic modification in the HLA-A gene comprises at least one nucleotide within genomic coordinates selected from the group consisting of: chr6:29942864-29942884; chr6:29942868-29942888; chr6:29942876-29942896; chr6:29942877-29942897; chr6:29942883-29942903; chr6:29943126-29943146; chr6:29943528-29943548; chr6:29943529-29943549; chr6:29943530-29943550; chr6:29943537-29943557; chr6:29943549-29943569; chr6:29943589-29943609; and chr6:29944026-29944046. In some embodiments, the cell comprises a genetic modification in an HLA-A gene, and wherein the genetic modification in the HLA-A gene comprises an insertion/deletion within genomic coordinates selected from the group consisting of: chr6:29942864-29942884; chr6:29942868-29942888; chr6:29942876-29942896; chr6:29942877-29942897; chr6:29942883-29942903; chr6:29943126-29943146; chr6:29943528-29943548; chr6:29943529-29943549; chr6:29943530-29943550; chr6:29943537-29943557; chr6:29943549-29943569; chr6:29943589-29943609; and chr6:29944026-29944046. In some embodiments, HLA-A expression of a cell is reduced or eliminated by a gene editing system that binds to an HLA-A genomic target sequence comprising at least 5 contiguous nucleotides within genomic coordinates selected from the group consisting of: chr6:29942864-29942884; chr6:29942868-29942888; chr6:29942876-29942896; chr6:29942877-29942897; chr6:29942883-29942903; chr6:29943126-29943146; chr6:29943528-29943548; chr6:29943529-29943549; chr6:29943530-29943550; chr6:29943537-29943557; chr6:29943549-29943569; chr6:29943589-29943609; and chr6:29944026-29944046. In some embodiments, the cells are homozygous for HLA-B and homozygous for HLA-C.
In some embodiments, an engineered cell having reduced or eliminated MHC class II surface expression relative to an unmodified cell is provided, comprising a genetic modification in a CIITA gene as described herein, and wherein the cell further has reduced or eliminated MHC class I surface expression. In some embodiments, the engineered cell comprises a genetic modification in the beta-2-microglobulin (B2M) gene. In some embodiments, the engineered cell comprises a genetic modification in the beta-2-microglobulin (B2M) gene and insertion of an exogenous nucleic acid encoding an NK cell inhibitor molecule. In some embodiments, the engineered cell comprises a genetic modification that eliminates MHC class I protein expression on the surface of the engineered cell.
In some embodiments, an engineered cell having reduced or eliminated MHC class II surface expression relative to an unmodified cell is provided, comprising a genetic modification in a CIITA gene, wherein the genetic modification comprises at least one nucleotide of an exon within genomic coordinates chr16:10902662-chr16:10923285, and wherein the cell further comprises an exogenous nucleic acid. In some embodiments, the exogenous nucleic acid encodes a targeting receptor expressed on the surface of the engineered cell. In some embodiments, the targeting receptor is a Chimeric Antigen Receptor (CAR). In some embodiments, the targeted receptor is a universal CAR (UniCar). In some embodiments, the targeting receptor is a T Cell Receptor (TCR). In some embodiments, the targeting receptor is a WT1 TCR. In some embodiments, the targeted receptor is a hybrid CAR/TCR. In some embodiments, the targeting receptor comprises an antigen recognition domain (e.g., a cancer antigen recognition domain and a subunit of a TCR). In some embodiments, the targeting receptor is a cytokine receptor. In some embodiments, the targeting receptor is a chemokine receptor. In some embodiments, the targeting receptor is a B Cell Receptor (BCR). In some embodiments, the exogenous nucleic acid encodes a polypeptide (i.e., a soluble polypeptide) secreted by the engineered cell. In some embodiments, the exogenous nucleic acid encodes a therapeutic polypeptide. In some embodiments, the exogenous nucleic acid encodes an antibody. In some embodiments, the exogenous nucleic acid encodes an enzyme. In some embodiments, the exogenous nucleic acid encodes a cytokine. In some embodiments, the exogenous nucleic acid encodes a chemokine. In some embodiments, the exogenous nucleic acid encodes a fusion protein.
In some embodiments, an engineered cell having reduced or eliminated MHC class II surface expression relative to an unmodified cell is provided, comprising a genetic modification in a CIITA gene, wherein the modification comprises at least one nucleotide of an exon within genomic coordinates chr16:10902662-chr16:10923285, wherein the cell further has reduced or eliminated MHC class I surface expression, and wherein the cell further comprises an exogenous nucleic acid. In some embodiments, the engineered cell comprises a genetic modification in the beta-2-microglobulin (B2M) gene. In some embodiments, the engineered cell comprises a genetic modification that reduces expression of an MHC class I protein on the surface of the engineered cell. In some embodiments, the exogenous nucleic acid encodes a targeting receptor expressed on the surface of the engineered cell. In some embodiments, the targeting receptor is a Chimeric Antigen Receptor (CAR). In some embodiments, the targeted receptor is a universal CAR (UniCar). In some embodiments, the targeting receptor is a T Cell Receptor (TCR). In some embodiments, the targeting receptor is a WT1 TCR. In some embodiments, the targeted receptor is a hybrid CAR/TCR. In some embodiments, the targeting receptor comprises an antigen recognition domain (e.g., a cancer antigen recognition domain and a subunit of a TCR). In some embodiments, the targeting receptor is a cytokine receptor. In some embodiments, the targeting receptor is a chemokine receptor. In some embodiments, the targeting receptor is a B Cell Receptor (BCR). In some embodiments, the exogenous nucleic acid encodes a polypeptide (i.e., a soluble polypeptide) secreted by the engineered cell. In some embodiments, the exogenous nucleic acid encodes a therapeutic polypeptide. In some embodiments, the exogenous nucleic acid encodes an antibody. In some embodiments, the exogenous nucleic acid encodes an enzyme. In some embodiments, the exogenous nucleic acid encodes a cytokine. In some embodiments, the exogenous nucleic acid encodes a chemokine. In some embodiments, the exogenous nucleic acid encodes a fusion protein.
In some embodiments, an engineered cell having reduced or eliminated MHC class II surface expression relative to an unmodified cell is provided, comprising a genetic modification in a CIITA gene, wherein the modification comprises at least one nucleotide of an exon within genomic coordinates chr16:10902662-chr16:10923285, wherein the cell further has reduced or eliminated HLA-A surface expression, and wherein the cell further comprises an exogenous nucleic acid. In some embodiments, the engineered cell comprises a genetic modification in an HLA-A gene. In some embodiments, the engineered cell comprises a genetic modification that reduces expression of an HLA-A protein on the surface of the engineered cell. In some embodiments, the exogenous nucleic acid encodes a targeting receptor expressed on the surface of the engineered cell. In some embodiments, the targeting receptor is a Chimeric Antigen Receptor (CAR). In some embodiments, the targeted receptor is a universal CAR (UniCar). In some embodiments, the targeting receptor is a T Cell Receptor (TCR). In some embodiments, the targeting receptor is a WT1 TCR. In some embodiments, the targeted receptor is a hybrid CAR/TCR. In some embodiments, the targeting receptor comprises an antigen recognition domain (e.g., a cancer antigen recognition domain and a subunit of a TCR). In some embodiments, the targeting receptor is a cytokine receptor. In some embodiments, the targeting receptor is a chemokine receptor. In some embodiments, the targeting receptor is a B Cell Receptor (BCR). In some embodiments, the exogenous nucleic acid encodes a polypeptide (i.e., a soluble polypeptide) secreted by the engineered cell. In some embodiments, the exogenous nucleic acid encodes a therapeutic polypeptide. In some embodiments, the exogenous nucleic acid encodes an antibody. In some embodiments, the exogenous nucleic acid encodes an enzyme. In some embodiments, the exogenous nucleic acid encodes a cytokine. In some embodiments, the exogenous nucleic acid encodes a chemokine. In some embodiments, the exogenous nucleic acid encodes a fusion protein.
In some embodiments, an engineered cell having reduced or eliminated MHC class II surface expression relative to an unmodified cell is provided, comprising a genetic modification in a CIITA gene, wherein the modification comprises at least one nucleotide of an exon within genomic coordinates chr16:10902662-chr16:10923285, wherein the cell further has reduced or eliminated endogenous TCR protein expression relative to an unmodified cell. In some embodiments, an engineered cell having reduced or eliminated MHC class II surface expression relative to an unmodified cell is provided, comprising a genetic modification in a CIITA gene, wherein the modification comprises at least one nucleotide of an exon within genomic coordinates chr16:10902662-chr16:10923285, wherein the cell further comprises an exogenous nucleic acid, and further has reduced or eliminated endogenous TCR protein expression relative to an unmodified cell. In some embodiments, an engineered cell having reduced or eliminated MHC class II surface expression relative to an unmodified cell is provided, comprising a genetic modification in a CIITA gene, wherein the modification comprises at least one nucleotide of an exon within genomic coordinates chr16:10902662-chr16:10923285, wherein the cell further has reduced or eliminated MHC class I surface expression, and wherein the cell further has reduced or eliminated endogenous TCR protein expression relative to an unmodified cell.
In some embodiments, an engineered cell having reduced or eliminated MHC class II surface expression relative to an unmodified cell is provided, comprising a genetic modification in a CIITA gene, wherein the modification comprises at least one nucleotide of an exon within genomic coordinates chr16:10902662-chr16:10923285, and wherein the cell further comprises an exogenous nucleic acid, and wherein the cell further has reduced or eliminated MHC class I surface expression, and wherein the cell further has reduced or eliminated endogenous TCR protein expression relative to an unmodified cell. In some embodiments, the engineered cells have reduced or eliminated expression of the TRAC protein relative to unmodified cells. In some embodiments, the engineered cells have reduced or eliminated expression of the TRBC protein relative to the unmodified cells. In some embodiments, the engineered cell comprises a genetic modification in the beta-2-microglobulin (B2M) gene. In some embodiments, the engineered cell comprises a genetic modification that reduces expression of an MHC class I protein on the surface of the engineered cell. In some embodiments, the exogenous nucleic acid encodes a targeting receptor expressed on the surface of the engineered cell. In some embodiments, the targeting receptor is a Chimeric Antigen Receptor (CAR). In some embodiments, the targeted receptor is a universal CAR (UniCar). In some embodiments, the targeting receptor is a T Cell Receptor (TCR). In some embodiments, the targeting receptor is a WT1 TCR. In some embodiments, the targeted receptor is a hybrid CAR/TCR. In some embodiments, the targeting receptor comprises an antigen recognition domain (e.g., a cancer antigen recognition domain and a subunit of a TCR). In some embodiments, the targeting receptor is a cytokine receptor. In some embodiments, the targeting receptor is a chemokine receptor. In some embodiments, the targeting receptor is a B Cell Receptor (BCR). In some embodiments, the exogenous nucleic acid encodes a polypeptide (i.e., a soluble polypeptide) secreted by the engineered cell. In some embodiments, the exogenous nucleic acid encodes a therapeutic polypeptide. In some embodiments, the exogenous nucleic acid encodes an antibody. In some embodiments, the exogenous nucleic acid encodes an enzyme. In some embodiments, the exogenous nucleic acid encodes a cytokine. In some embodiments, the exogenous nucleic acid encodes a chemokine. In some embodiments, the exogenous nucleic acid encodes a fusion protein.
In some embodiments, an engineered cell having reduced or eliminated MHC class II surface expression relative to an unmodified cell is provided, comprising a genetic modification in a CIITA gene, wherein the modification comprises at least one nucleotide of an exon within genomic coordinates chr16:10902662-chr16:10923285, and wherein the cell further comprises an exogenous nucleic acid, and wherein the cell further has reduced or eliminated HLA-A surface expression, and wherein the cell further has reduced or eliminated endogenous TCR protein expression relative to an unmodified cell. In some embodiments, the engineered cells have reduced or eliminated expression of the TRAC protein relative to unmodified cells. In some embodiments, the engineered cells have reduced or eliminated expression of the TRBC protein relative to the unmodified cells. In some embodiments, the engineered cell comprises a genetic modification in an HLA-A gene. In some embodiments, the engineered cell comprises a genetic modification that reduces expression of an HLA-A protein on the surface of the engineered cell. In some embodiments, the exogenous nucleic acid encodes a targeting receptor expressed on the surface of the engineered cell. In some embodiments, the targeting receptor is a Chimeric Antigen Receptor (CAR). In some embodiments, the targeted receptor is a universal CAR (UniCar). In some embodiments, the targeting receptor is a T Cell Receptor (TCR). In some embodiments, the targeting receptor is a WT1 TCR. In some embodiments, the targeted receptor is a hybrid CAR/TCR. In some embodiments, the targeting receptor comprises an antigen recognition domain (e.g., a cancer antigen recognition domain and a subunit of a TCR). In some embodiments, the targeting receptor is a cytokine receptor. In some embodiments, the targeting receptor is a chemokine receptor. In some embodiments, the targeting receptor is a B Cell Receptor (BCR). In some embodiments, the exogenous nucleic acid encodes a polypeptide (i.e., a soluble polypeptide) secreted by the engineered cell. In some embodiments, the exogenous nucleic acid encodes a therapeutic polypeptide. In some embodiments, the exogenous nucleic acid encodes an antibody. In some embodiments, the exogenous nucleic acid encodes an enzyme. In some embodiments, the exogenous nucleic acid encodes a cytokine. In some embodiments, the exogenous nucleic acid encodes a chemokine. In some embodiments, the exogenous nucleic acid encodes a fusion protein.
The engineered cell may be any of the exemplary cell types disclosed herein. In some embodiments, the engineered cell is an immune cell. In some embodiments, the engineered cell is a Hematopoietic Stem Cell (HSC). In some embodiments, the engineered cell is an Induced Pluripotent Stem Cell (iPSC). In some embodiments, the engineered cell is a monocyte, macrophage, mast cell, dendritic cell, or granulocyte. In some embodiments, the engineered cell is a monocyte. In some embodiments, the engineered cell is a macrophage. In some embodiments, the engineered cell is a mast cell. In some embodiments, the engineered cell is a dendritic cell.
In some embodiments, the engineered cell is a granulocyte. In some embodiments, the engineered cell is a lymphocyte. In some embodiments, the engineered cell is a T cell. In some embodiments, the engineered cell is a cd4+ T cell. In some embodiments, the engineered cell is a cd8+ T cell. In some embodiments, the engineered cell is a memory T cell. In some embodiments, the engineered cell is a B cell. In some embodiments, the engineered cell is a plasma B cell. In some embodiments, the engineered cell is a memory B cell.
In some embodiments, the engineered cells are homozygous for HLA-B and homozygous for HLA-C. In some embodiments, the HLA-B allele is selected from any one of the following HLA-B alleles: HLA-B.times.07:02; HLA-B08:01; HLA-B44:02; HLA-B35:01; HLA-B40:01; HLA-B57:01; HLA-B14:02; HLA-B15:01; HLA-B13:02; HLA-B44:03; HLA-B38:01; HLA-B18:01; HLA-B44:03; HLA-B51:01; HLA-B49:01; HLA-B15:01; HLA-B18:01; HLA-B27:05; HLA-B35:03; HLA-B18:01; HLA-B52:01; HLA-B51:01; HLA-B37:01; HLA-B53:01; HLA-B55:01; HLA-B44:02; HLA-B44:03; HLA-B35:02; HLA-B15:01; and HLA-B40:02.
In some embodiments, the HLA-C allele is selected from any one of the following HLA-C alleles: HLA-C.times.07:02; HLA-C.times.07:01; HLA-C05:01; HLA-C.times.04:01HLA-C.times.03:04; HLA-C06:02; HLA-C08:02; HLA-C.times.03:03; HLA-C06:02; HLA-C16:01; HLA-C12:03; HLA-C.times.07:01; HLA-C04:01; HLA-C15:02; HLA-C.times.07:01; HLA-C03:04; HLA-C12:03; HLA-C02:02; HLA-C04:01; HLA-C05:01; HLA-C12:02; HLA-C14:02; HLA-C06:02; HLA-C04:01; HLA-C.times.03:03; HLA-C07:04; HLA-C.times.07:01; HLA-C04:01; HLA-C04:01; and HLA-C02:02.
In some embodiments, the HLA-B allele is selected from any one of the following HLA-B alleles: HLA-B.times.07:02; HLA-B08:01; HLA-B44:02; HLA-B35:01; HLA-B40:01; HLA-B57:01; HLA-B14:02; HLA-B15:01; HLA-B13:02; HLA-B44:03; HLA-B38:01; HLA-B18:01; HLA-B44:03; HLA-B51:01; HLA-B49:01; HLA-B15:01; HLA-B18:01; HLA-B27:05; HLA-B35:03; HLA-B18:01; HLA-B52:01; HLA-B51:01; HLA-B37:01; HLA-B53:01; HLA-B55:01; HLA-B44:02; HLA-B44:03; HLA-B35:02; HLA-B15:01; and HLA-B40:02; and the HLA-C allele is selected from any one of the following HLA-C alleles: HLA-C.times.07:02; HLA-C.times.07:01; HLA-C05:01; HLA-C.times.04:01HLA-C.times.03:04; HLA-C06:02; HLA-C08:02; HLA-C.times.03:03; HLA-C06:02; HLA-C16:01; HLA-C12:03; HLA-C.times.07:01; HLA-C04:01; HLA-C15:02; HLA-C.times.07:01; HLA-C03:04; HLA-C12:03; HLA-C02:02; HLA-C04:01; HLA-C05:01; HLA-C12:02; HLA-C14:02; HLA-C06:02; HLA-C04:01; HLA-C.times.03:03; HLA-C07:04; HLA-C.times.07:01; HLA-C04:01; HLA-C04:01; and HLA-C02:02.
In some embodiments, the engineered cells are homozygous for HLA-B and homozygous for HLA-C, and the HLA-B and HLA-C alleles are selected from any one of the following HLA-B and HLA-C alleles: HLA-B.times.07:02 and HLA-C.times.07:02; HLA-B.times.08:01 and HLA-C.times.07:01; HLA-B44:02 and HLA-C05:01; HLA-B35:01 and HLA-C04:01; HLA-B.times.40:01 and HLA-C.times.03:04; HLA-B57:01 and HLA-C06:02; HLA-B14:02 and HLA-C08:02; HLA-B15:01 and HLA-C03:03; HLA-B13:02 and HLA-C06:02; HLA-B44:03 and HLA-C16:01; HLA-B.times.38:01 and HLA-C.times.12:03; HLA-B18:01 and HLA-C07:01; HLA-B44:03 and HLA-C04:01; HLA-B.times.51:01 and HLA-C.times.15:02; HLA-B.times.49:01 and HLA-C.times.07:01; HLA-B15:01 and HLA-C03:04; HLA-B18:01 and HLA-C12:03; HLA-B.times.27:05 and HLA-C.times.02:02; HLA-B35:03 and HLA-C04:01; HLA-B18:01 and HLA-C05:01; HLA-B52:01 and HLA-C12:02; HLA-B.times.51:01 and HLA-C.times.14:02; HLA-B37:01 and HLA-C06:02; HLA-B53:01 and HLA-C04:01; HLA-B55:01 and HLA-C03:03; HLA-B44:02 and HLA-C07:04; HLA-B44:03 and HLA-C07:01; HLA-B35:02 and HLA-C04:01; HLA-B15:01 and HLA-C04:01; and HLA-B.times.40:02 and HLA-C.times.02:02. In some embodiments, the cells are homozygous for HLA-B and homozygous for HLA-C, and the HLA-B and HLA-C alleles are HLA-B.times.07:02 and HLA-C.times.07:02. In some embodiments, the cells are homozygous for HLA-B and homozygous for HLA-C, and the HLA-B and HLA-C alleles are HLA-B08:01 and HLA-C07:01. In some embodiments, the cells are homozygous for HLA-B and homozygous for HLA-C, and the HLA-B and HLA-C alleles are HLA-B44:02 and HLA-C05:01. In some embodiments, the cells are homozygous for HLA-B and homozygous for HLA-C, and the HLA-B and HLA-C alleles are HLA-B35:01 and HLA-C04:01.
In some embodiments, the present disclosure provides a pharmaceutical composition comprising any one of the engineered cells disclosed herein. In some embodiments, the pharmaceutical composition comprises a population of any of the engineered cells disclosed herein. In some embodiments, the pharmaceutical composition comprises an engineered cell population that is at least 65% negative as measured by flow cytometry. In some embodiments, the pharmaceutical composition comprises an engineered cell population that is at least 70% negative as measured by flow cytometry. In some embodiments, the pharmaceutical composition comprises an engineered cell population that is at least 80% negative as measured by flow cytometry. In some embodiments, the pharmaceutical composition comprises an engineered cell population that is at least 90% negative as measured by flow cytometry. In some embodiments, the pharmaceutical composition comprises an engineered cell population that is at least 91% negative as measured by flow cytometry. In some embodiments, the pharmaceutical composition comprises an engineered cell population that is at least 92% negative as measured by flow cytometry. In some embodiments, the pharmaceutical composition comprises an engineered cell population that is at least 93% negative as measured by flow cytometry. In some embodiments, the pharmaceutical composition comprises an engineered cell population that is at least 94% negative as measured by flow cytometry. In some embodiments, the pharmaceutical composition comprises an engineered cell population that is negative for at least 95% of endogenous TCR proteins as measured by flow cytometry. In some embodiments, the pharmaceutical composition comprises at least 97% of the population of engineered cells that are negative for endogenous TCR proteins as measured by flow cytometry. In some embodiments, the pharmaceutical composition comprises an engineered cell population that is at least 98% negative for endogenous TCR proteins as measured by flow cytometry. In some embodiments, the pharmaceutical composition comprises an engineered cell population that is negative for at least 99% of endogenous TCR proteins as measured by flow cytometry.
In some embodiments, methods are provided for administering an engineered cell or pharmaceutical composition disclosed herein to a subject in need thereof. In some embodiments, methods of administering an engineered cell or pharmaceutical composition disclosed herein to a subject as ACT therapy are provided. In some embodiments, methods are provided for administering an engineered cell or pharmaceutical composition disclosed herein to a subject as a cancer treatment. In some embodiments, methods of administering an engineered cell or pharmaceutical composition disclosed herein to a subject as a treatment for an autoimmune disease are provided. In some embodiments, methods of administering an engineered cell or pharmaceutical composition disclosed herein to a subject as a treatment for an infectious disease are provided.
B. Methods and compositions for reducing or eliminating MHC class II surface expression
The present disclosure provides methods and compositions for reducing or eliminating the surface expression of MHC class II proteins on a cell relative to an unmodified cell by genetically modifying the CIITA gene. The resulting genetically modified cells are also referred to herein as engineered cells. In some embodiments, the genetically modified (or engineered) cell may be a starting cell that is further genetically modified using the methods or compositions provided herein. In some embodiments, the cell is an allogeneic cell. In some embodiments, cells with reduced MHC class II expression are suitable for adoptive cell transfer therapy. In some embodiments, editing of the CIITA gene is combined with additional genetic modifications to produce cells required for allograft purposes.
In some embodiments, a method comprises reducing or eliminating surface expression of an MHC class II protein on the surface of a cell, the method comprising contacting the cell with a composition comprising a CIITA guide RNA comprising i) a guide sequence selected from the group consisting of SEQ ID NOs 1-117; ii) at least 17, 18, 19 or 20 consecutive nucleotides of the sequence selected from SEQ ID NO. 1-117; iii) A leader sequence having at least 95%, 90% or 85% identity to a sequence selected from the group consisting of SEQ ID NOS.1-117; iv) a sequence of 10 consecutive nucleotides + -10 nucleotides comprising the genomic coordinates listed in table 2; v) at least 17, 18, 19 or 20 consecutive nucleotides of the sequence from (iv); or vi) a leader sequence having at least 95%, 90% or 85% identity to a sequence selected from (v). In some embodiments, the method further comprises contacting the cell with an RNA-guided DNA binding agent or a nucleic acid encoding an RNA-guided DNA binding agent. In some embodiments, the RNA-guided DNA binding agent is Cas9. In some embodiments, the RNA-guided DNA binding agent is streptococcus pyogenes Cas9. In some embodiments, the CIITA guide RNA is a streptococcus pyogenes Cas9 guide RNA. In some embodiments, the RNA-guided DNA binding agent further comprises a deaminase domain. In some embodiments, the RNA-guided DNA binding agent comprises apodec 3A deaminase (a 3A) and RNA-guided nicking enzyme. In some embodiments, the expression of MHC class II proteins on the surface of a cell (i.e., an engineered cell) is thereby reduced.
In some embodiments, a method comprises making an engineered cell having reduced or eliminated surface expression of an MHC class II protein relative to an unmodified cell, the method comprising contacting the cell with a composition comprising a CIITA guide RNA comprising i) a guide sequence selected from the group consisting of SEQ ID NOs 1-117; ii) at least 17, 18, 19 or 20 consecutive nucleotides of the sequence selected from SEQ ID NO. 1-117; iii) A leader sequence having at least 95%, 90% or 85% identity to a sequence selected from the group consisting of SEQ ID NOS.1-117; iv) a sequence of 10 consecutive nucleotides + -10 nucleotides comprising the genomic coordinates listed in table 2; v) at least 17, 18, 19 or 20 consecutive nucleotides of the sequence from (iv); or vi) a leader sequence having at least 95%, 90% or 85% identity to a sequence selected from (v). In some embodiments, the method further comprises contacting the cell with an RNA-guided DNA binding agent or a nucleic acid encoding an RNA-guided DNA binding agent. In some embodiments, the RNA-guided DNA binding agent is Cas9. In some embodiments, the RNA-guided DNA binding agent is streptococcus pyogenes Cas9. In some embodiments, the CIITA guide RNA is a streptococcus pyogenes Cas9 guide RNA. In some embodiments, the RNA-guided DNA binding agent further comprises a deaminase region. In some embodiments, the RNA-guided DNA binding agent comprises apodec 3A deaminase (a 3A) and RNA-guided nicking enzyme. In some embodiments, the expression of MHC class II proteins on the surface of a cell (i.e., an engineered cell) is thereby reduced.
In some embodiments, a method comprises genetically modifying a cell to reduce or eliminate surface expression of an MHC class II protein, the method comprising contacting the cell with a composition comprising a CIITA guide RNA comprising i) a guide sequence selected from the group consisting of SEQ ID NOs 1-117; ii) at least 17, 18, 19 or 20 consecutive nucleotides of the sequence selected from SEQ ID NO. 1-117; iii) A leader sequence having at least 95%, 90% or 85% identity to a sequence selected from the group consisting of SEQ ID NOS.1-117; iv) a sequence of 10 consecutive nucleotides + -10 nucleotides comprising the genomic coordinates listed in table 2; v) at least 17, 18, 19 or 20 consecutive nucleotides of the sequence from (iv); or vi) a leader sequence having at least 95%, 90% or 85% identity to a sequence selected from (v). In some embodiments, the method further comprises contacting the cell with an RNA-guided DNA binding agent or a nucleic acid encoding an RNA-guided DNA binding agent. In some embodiments, the RNA-guided DNA binding agent is Cas9. In some embodiments, the RNA-guided DNA binding agent is streptococcus pyogenes Cas9. In some embodiments, the CIITA guide RNA is a streptococcus pyogenes Cas9 guide RNA. In some embodiments, the RNA-guided DNA binding agent further comprises a deaminase region. In some embodiments, the RNA-guided DNA binding agent comprises apodec 3A deaminase (a 3A) and RNA-guided nicking enzyme. In some embodiments, the expression of MHC class II proteins on the surface of a cell (i.e., an engineered cell) is thereby reduced.
In some embodiments, a method of reducing expression of MHC class II proteins on the surface of a cell comprises contacting the cell with any one or more of the CIITA guide RNAs disclosed herein. In some embodiments, the CIITA guide RNA comprises a guide sequence selected from SEQ ID NOS.1-117.
In some embodiments, compositions are provided comprising CIITA guide RNA comprising i) a guide sequence selected from the group consisting of SEQ ID NOs 1-117; ii) at least 17, 18, 19 or 20 consecutive nucleotides of the sequence selected from SEQ ID NO. 1-117; iii) A leader sequence having at least 95%, 90% or 85% identity to a sequence selected from the group consisting of SEQ ID NOS.1-117; iv) a sequence of 10 consecutive nucleotides + -10 nucleotides comprising the genomic coordinates listed in table 2; v) at least 17, 18, 19 or 20 consecutive nucleotides of the sequence from (iv); or vi) a leader sequence having at least 95%, 90% or 85% identity to a sequence selected from (v). In some embodiments, the composition further comprises an RNA-guided DNA binding agent or a nucleic acid encoding an RNA-guided DNA binding agent. In some embodiments, the composition comprises an RNA-guided DNA binding agent that is Cas9. In some embodiments, the RNA-guided DNA binding agent is streptococcus pyogenes Cas9. In some embodiments, the CIITA guide RNA is a streptococcus pyogenes Cas9 guide RNA. In some embodiments, the RNA-guided DNA binding agent further comprises a deaminase region. In some embodiments, the RNA-guided DNA binding agent comprises apodec 3A deaminase (a 3A) and RNA-guided nicking enzyme.
In any of the preceding embodiments, the guide sequence is selected from i) SEQ ID NOs 32, 64, 67, 68, 74, 76, 84, 86, 90, 91 and 115; ii) at least 17, 18, 19 or 20 consecutive nucleotides of a sequence selected from the group consisting of SEQ ID NOs 32, 64, 67, 68, 74, 76, 84, 86, 90, 91 and 115; iii) A guide sequence having at least 95%, 90% or 85% identity to a sequence selected from the group consisting of SEQ ID NOs 32, 64, 67, 68, 74, 76, 84, 86, 90, 91 and 115.
In some embodiments, the composition further comprises a Uracil Glycosidase Inhibitor (UGI). In some embodiments, the composition comprises an RNA-guided DNA binding agent that produces a conversion of cytosine (C) to thymine (T) by a CIITA genomic target sequence. In some embodiments, the composition comprises an RNA-guided DNA binding agent that produces a conversion of adenosine (a) to guanine (G) by a CIITA genomic target sequence.
In some embodiments, engineered cells produced by the methods described herein are provided. In some embodiments, the engineered cells produced by the methods and compositions described herein are allogeneic cells. In some embodiments, the methods result in compositions comprising engineered cells having reduced MHC class II expression. In some embodiments, the methods result in compositions comprising engineered cells having reduced expression of CIITA proteins. In some embodiments, the methods result in a composition comprising an engineered cell having reduced CIITA levels in the nucleus. In some embodiments, the methods result in compositions comprising engineered cells expressing truncated forms of CIITA proteins. In some embodiments, the methods produce a composition comprising engineered cells that do not produce detectable CIITA proteins. In some embodiments, the engineered cells have reduced MHC class II expression, reduced CIITA protein, and/or reduced CIITA levels in the nucleus as compared to the unmodified cells. In some embodiments, the engineered cells produced by the methods disclosed herein have a reduced response elicited from cd4+ T cells as measured in an in vitro cell culture assay containing cd4+ T cells as compared to unmodified cells.
In some embodiments, the compositions disclosed herein further comprise a pharmaceutically acceptable carrier. In some embodiments, cells produced from a composition disclosed herein comprising a pharmaceutically acceptable carrier are provided. In some embodiments, compositions comprising the cells disclosed herein are provided.
CIITA guide RNA
The methods and compositions provided herein disclose CIITA guide RNAs useful for reducing expression of MHC class II proteins on a cell surface. In some embodiments, such guide RNAs direct RNA-guided DNA-binding agents to CIITA genomic target sequences, and may be referred to herein as "CIITA guide RNAs. In some embodiments, the CIITA guide RNA directs the RNA-guided DNA binding agent to a human CIITA genomic target sequence. In some embodiments, the CIITA guide RNA comprises a guide sequence selected from SEQ ID NOS.1-117.
In some embodiments, a composition is provided comprising CIITA guide RNAs and RNA-guided DNA binding agents or nucleic acids encoding RNA-guided DNA binding agents described herein.
In some embodiments, CIITA single guide RNA (sgRNA) comprising a guide sequence selected from SEQ ID NOS: 1-117 is provided. In some embodiments, a composition is provided comprising CIITA single guide RNA (sgRNA) comprising a guide sequence selected from the group consisting of SEQ ID NOS: 1-117. In some embodiments, a composition is provided comprising CIITA sgrnas and RNA-guided DNA binding agents or nucleic acids encoding RNA-guided DNA binding agents described herein.
In some embodiments, CIITA double guide RNA (dgRNA) comprising a guide sequence selected from SEQ ID NOS: 1-117 is provided. In some embodiments, a composition is provided comprising CIITA double guide RNA (dgRNA) comprising a guide sequence selected from SEQ ID NOS: 1-117. In some embodiments, a composition is provided comprising CIITA dgrnas and RNA-guided DNA binding agents or nucleic acids encoding RNA-guided DNA binding agents described herein.
Exemplary CIITA guide sequences are shown in Table 2 below (SEQ ID NOS: 1-117 and corresponding guide RNA sequences SEQ ID NOS: 218-334 and 335-426).
Table 2. Exemplary CIITA boot sequences.
/>
/>
/>
/>
/>
/>
/>
/>
/>
/>
/>
/>
/>
/>
/>
/>
/>
/>
/>
/>
/>
/>
/>
/>
/>
/>
/>
/>
/>
/>
/>
/>
/>
/>
/>
The terms "mA", "mC", "mU" or "mgs" may be used to indicate 2' -O-Me modified nucleotides.
In some embodiments, the CIITA guide RNA comprises a guide sequence selected from SEQ ID NOS.1-117. In some embodiments, the CIITA guide RNA comprises a guide sequence of at least 17, 18, 19 or 20 consecutive nucleotides of a sequence selected from the group consisting of SEQ ID NOS: 1-117. In some embodiments, the CIITA guide RNA comprises a guide sequence having at least 95%, 90% or 85% identity to a sequence selected from SEQ ID NOS: 1-117. In some embodiments, the CIITA guide RNA comprises a guide sequence that is at least 95% identical to a sequence selected from the group consisting of SEQ ID NOS: 1-117. In some embodiments disclosed herein, the guide sequence is (i) the guide sequence of SEQ ID NO 32, 64, 67, 68, 74, 76, 84, 86, 90, 91, or 115; ii) at least 17, 18, 19 or 20 consecutive nucleotides of a sequence selected from the group consisting of SEQ ID NOs 32, 64, 67, 68, 74, 76, 84, 86, 90, 91 and 115; iii) A guide sequence having at least 95%, 90% or 85% identity to a sequence selected from the group consisting of SEQ ID NOs 32, 64, 67, 68, 74, 76, 84, 86, 90, 91 and 115.
In some embodiments, the CIITA guide RNA comprises a guide sequence comprising at least 10 consecutive nucleotides ± 10 nucleotides of the genomic coordinates listed in table 2. As used herein, at least 10 contiguous nucleotides ± 10 nucleotides of a genomic coordinate means, for example, at least 10 contiguous nucleotides within the genomic coordinate, wherein the genomic coordinate comprises 10 nucleotides in the 5 'direction and 10 nucleotides in the 3' direction of the ranges listed in table 2. For example, CIITA guide RNA may comprise 10 consecutive nucleotides within genomic coordinates chr16:10877360-10877380 or chr16:10877350-10877390, including the boundary nucleotides of these ranges. In some embodiments, the CIITA guide RNA comprises a guide sequence that is at least 17, 18, 19 or 20 consecutive nucleotides of a sequence comprising 10 consecutive nucleotides ± 10 nucleotides of the genomic coordinates listed in table 2. In some embodiments, the CIITA guide RNA comprises a guide sequence that is at least 95%, 90% or 85% identical to a sequence selected from the group consisting of 17, 18, 19 or 20 consecutive nucleotides comprising a sequence of 10 consecutive nucleotides±10 nucleotides of the genomic coordinates listed in table 2.
In some embodiments, the CIITA guide RNA comprises a guide sequence comprising at least 15 contiguous nucleotides ± 10 nucleotides of the genomic coordinates listed in table 2. In some embodiments, the CIITA guide RNA comprises a guide sequence comprising at least 20 contiguous nucleotides ± 10 nucleotides of the genomic coordinates listed in table 2.
In some embodiments, the CIITA guide RNA comprises SEQ ID NO. 1. In some embodiments, the CIITA guide RNA comprises SEQ ID NO. 2. In some embodiments, the CIITA guide RNA comprises SEQ ID NO. 3. In some embodiments, the CIITA guide RNA comprises SEQ ID NO. 4. In some embodiments, the CIITA guide RNA comprises SEQ ID NO. 5. In some embodiments, the CIITA guide RNA comprises SEQ ID NO. 6. In some embodiments, the CIITA guide RNA comprises SEQ ID NO. 7. In some embodiments, the CIITA guide RNA comprises SEQ ID NO. 8. In some embodiments, the CIITA guide RNA comprises SEQ ID NO. 9. In some embodiments, the CIITA guide RNA comprises SEQ ID NO. 10. In some embodiments, the CIITA guide RNA comprises SEQ ID NO. 11. In some embodiments, the CIITA guide RNA comprises SEQ ID NO. 12. In some embodiments, the CIITA guide RNA comprises SEQ ID NO. 13. In some embodiments, the CIITA guide RNA comprises SEQ ID NO. 14. In some embodiments, the CIITA guide RNA comprises SEQ ID NO. 15. In some embodiments, the CIITA guide RNA comprises SEQ ID NO. 16. In some embodiments, the CIITA guide RNA comprises SEQ ID NO. 17. In some embodiments, the CIITA guide RNA comprises SEQ ID NO. 18. In some embodiments, the CIITA guide RNA comprises SEQ ID NO. 19. In some embodiments, the CIITA guide RNA comprises SEQ ID NO. 20. In some embodiments, the CIITA guide RNA comprises SEQ ID NO. 21. In some embodiments, the CIITA guide RNA comprises SEQ ID NO. 22. In some embodiments, the CIITA guide RNA comprises SEQ ID NO. 23. In some embodiments, the CIITA guide RNA comprises SEQ ID NO. 24. In some embodiments, the CIITA guide RNA comprises SEQ ID NO. 25. In some embodiments, the CIITA guide RNA comprises SEQ ID NO. 26. In some embodiments, the CIITA guide RNA comprises SEQ ID NO. 27. In some embodiments, the CIITA guide RNA comprises SEQ ID NO. 28. In some embodiments, the CIITA guide RNA comprises SEQ ID NO. 29. In some embodiments, the CIITA guide RNA comprises SEQ ID NO. 30. In some embodiments, the CIITA guide RNA comprises SEQ ID NO. 31. In some embodiments, the CIITA guide RNA comprises SEQ ID NO. 32. In some embodiments, the CIITA guide RNA comprises SEQ ID NO. 33. In some embodiments, the CIITA guide RNA comprises SEQ ID NO. 34. In some embodiments, the CIITA guide RNA comprises SEQ ID NO. 35. In some embodiments, the CIITA guide RNA comprises SEQ ID NO. 36. In some embodiments, the CIITA guide RNA comprises SEQ ID NO. 37. In some embodiments, the CIITA guide RNA comprises SEQ ID NO. 38. In some embodiments, the CIITA guide RNA comprises SEQ ID NO 39. In some embodiments, the CIITA guide RNA comprises SEQ ID NO. 40. In some embodiments, the CIITA guide RNA comprises SEQ ID NO. 41. In some embodiments, the CIITA guide RNA comprises SEQ ID NO. 42. In some embodiments, the CIITA guide RNA comprises SEQ ID NO. 43. In some embodiments, the CIITA guide RNA comprises SEQ ID NO. 44. In some embodiments, the CIITA guide RNA comprises SEQ ID NO. 45. In some embodiments, the CIITA guide RNA comprises SEQ ID NO. 46. In some embodiments, the CIITA guide RNA comprises SEQ ID NO. 47. In some embodiments, the CIITA guide RNA comprises SEQ ID NO. 48. In some embodiments, the CIITA guide RNA comprises SEQ ID NO. 49. In some embodiments, the CIITA guide RNA comprises SEQ ID NO. 50. In some embodiments, the CIITA guide RNA comprises SEQ ID NO. 51. In some embodiments, the CIITA guide RNA comprises SEQ ID NO. 52. In some embodiments, the CIITA guide RNA comprises SEQ ID NO. 53. In some embodiments, the CIITA guide RNA comprises SEQ ID NO. 54. In some embodiments, the CIITA guide RNA comprises SEQ ID NO. 55. In some embodiments, the CIITA guide RNA comprises SEQ ID NO. 56. In some embodiments, the CIITA guide RNA comprises SEQ ID NO. 57. In some embodiments, the CIITA guide RNA comprises SEQ ID NO. 58. In some embodiments, the CIITA guide RNA comprises SEQ ID NO. 59. In some embodiments, the CIITA guide RNA comprises SEQ ID NO. 60. In some embodiments, the CIITA guide RNA comprises SEQ ID NO. 61. In some embodiments, the CIITA guide RNA comprises SEQ ID NO. 62. In some embodiments, the CIITA guide RNA comprises SEQ ID NO. 63. In some embodiments, the CIITA guide RNA comprises SEQ ID NO. 64. In some embodiments, the CIITA guide RNA comprises SEQ ID NO. 65. In some embodiments, the CIITA guide RNA comprises SEQ ID NO. 66. In some embodiments, the CIITA guide RNA comprises SEQ ID NO. 67. In some embodiments, the CIITA guide RNA comprises SEQ ID NO. 68. In some embodiments, the CIITA guide RNA comprises SEQ ID NO. 69. In some embodiments, the CIITA guide RNA comprises SEQ ID NO. 70. In some embodiments, the CIITA guide RNA comprises SEQ ID NO. 71. In some embodiments, the CIITA guide RNA comprises SEQ ID NO. 72. In some embodiments, the CIITA guide RNA comprises SEQ ID NO. 73. In some embodiments, the CIITA guide RNA comprises SEQ ID NO. 74. In some embodiments, the CIITA guide RNA comprises SEQ ID NO. 75. In some embodiments, the CIITA guide RNA comprises SEQ ID NO. 76. In some embodiments, the CIITA guide RNA comprises SEQ ID NO. 77. In some embodiments, the CIITA guide RNA comprises SEQ ID NO. 78. In some embodiments, the CIITA guide RNA comprises SEQ ID NO. 79. In some embodiments, the CIITA guide RNA comprises SEQ ID NO. 80. In some embodiments, the CIITA guide RNA comprises SEQ ID NO. 81. In some embodiments, the CIITA guide RNA comprises SEQ ID NO. 82. In some embodiments, the CIITA guide RNA comprises SEQ ID NO. 83. In some embodiments, the CIITA guide RNA comprises SEQ ID NO. 84. In some embodiments, the CIITA guide RNA comprises SEQ ID NO. 85. In some embodiments, the CIITA guide RNA comprises SEQ ID NO. 86. In some embodiments, the CIITA guide RNA comprises SEQ ID NO. 87. In some embodiments, the CIITA guide RNA comprises SEQ ID NO. 88. In some embodiments, the CIITA guide RNA comprises SEQ ID NO. 89. In some embodiments, the CIITA guide RNA comprises SEQ ID NO. 90. In some embodiments, the CIITA guide RNA comprises SEQ ID NO. 91. In some embodiments, the CIITA guide RNA comprises SEQ ID NO. 92. In some embodiments, the CIITA guide RNA comprises SEQ ID NO. 93. In some embodiments, the CIITA guide RNA comprises SEQ ID NO. 94. In some embodiments, the CIITA guide RNA comprises SEQ ID NO. 95. In some embodiments, the CIITA guide RNA comprises SEQ ID NO. 96. In some embodiments, the CIITA guide RNA comprises SEQ ID NO. 97. In some embodiments, the CIITA guide RNA comprises SEQ ID NO. 98. In some embodiments, the CIITA guide RNA comprises SEQ ID NO. 99. In some embodiments, the CIITA guide RNA comprises SEQ ID NO. 100. In some embodiments, the CIITA guide RNA comprises SEQ ID NO. 101. In some embodiments, the CIITA guide RNA comprises SEQ ID NO. 102. In some embodiments, the CIITA guide RNA comprises SEQ ID NO. 103. In some embodiments, the CIITA guide RNA comprises SEQ ID NO. 104. In some embodiments, the CIITA guide RNA comprises SEQ ID NO. 105. In some embodiments, the CIITA guide RNA comprises SEQ ID NO. 106. In some embodiments, the CIITA guide RNA comprises SEQ ID NO. 107. In some embodiments, the CIITA guide RNA comprises SEQ ID NO. 108. In some embodiments, the CIITA guide RNA comprises SEQ ID NO. 109. In some embodiments, the CIITA guide RNA comprises SEQ ID NO. 110. In some embodiments, the CIITA guide RNA comprises SEQ ID NO. 111. In some embodiments, the CIITA guide RNA comprises SEQ ID NO. 112. In some embodiments, the CIITA guide RNA comprises SEQ ID NO. 113. In some embodiments, the CIITA guide RNA comprises SEQ ID NO. 114. In some embodiments, the CIITA guide RNA comprises SEQ ID NO. 115. In some embodiments, the CIITA guide RNA comprises SEQ ID NO. 116. In some embodiments, the CIITA guide RNA comprises SEQ ID NO. 117.
Additional embodiments of CIITA guide RNAs are provided herein, including, for example, exemplary modifications to guide RNAs.
2. Genetic modification of CIITA
In some embodiments, the methods and compositions disclosed herein genetically modify at least one nucleotide of an exon in a CIITA gene in a cell. Since CIITA proteins regulate MHC class II expression, in some embodiments, genetic modification to CIITA alters CIITA protein production and thus reduces MHC class II protein expression on the surface of genetically modified cells (or engineered cells). Genetic modifications encompass the modified population resulting from contact with the gene editing system (e.g., the edited population resulting from Cas9 and CIITA guide RNAs, or the edited population resulting from BC22 and CIITA guide RNAs).
In some embodiments, the genetic modification includes at least one nucleotide of an exon within genomic coordinates chr16:10902662-chr16: 10923285. In some embodiments, the genetic modification includes at least one nucleotide of an exon within genomic coordinates chr16:10906542-chr16: 10923285. In some embodiments, the genetic modification includes at least one nucleotide of an exon within genomic coordinates chr16:10906542-chr16: 10908121. In some embodiments, the genetic modification comprises at least one nucleotide of an exon within genomic coordinates selected from the group consisting of: chr16:10916432-10916452, chr16:10922444-10922464, chr16:10907924-10907944, chr16:10906985-10907005, chr16:10908073-10908093, chr16:10907433-10907453, chr16:10907979-10907999, chr16:10907139-10907159, chr16:10922435-10922455, chr16:10907384-10907404, chr16:10907434-10907454, chr16:10907119-10907139, chr16:10907539-10907559, chr16:10907810-10907830, chr16:10907315-10907335, chr16:10916426-10916446, chr16:10909138-10909158, chr16:10908101-10908121, chr16:10907790-10907810, chr16:10907787-10907807, and chr16: 10907787-10907807. In some embodiments, the genetic modification comprises at least one nucleotide of an exon within genomic coordinates selected from the group consisting of: chr16:10907539-10907559, chr16:10916426-10916446, chr16:10906907-10906927, chr16:10895702-10895722, chr16:10907757-10907777, chr16:10907623-10907643, chr16:10915626-10915646, chr16:10906756-10906776, chr16:10907476-10907496, chr16:10907385-10907405 and chr16:10923265-10923285. In some embodiments, the genetic modification comprises at least one nucleotide of an exon within genomic coordinates selected from the group consisting of: chr16:10906853-10906873, chr16:10922444-10922464, chr16:10907924-10907944, chr16:10907315-10907335, chr16:10916432-10916452, chr16:10907932-10907952, chr16:10915626-10915646, chr16:10907586-10907606, chr16:10916426-10916446, chr16:10907476-10907496, chr16:10907787-10907807, chr16:10907979-10907999, chr16:10906904-10906924 and chr16:10909138-10909158. In some embodiments, the genetic modification comprises at least one nucleotide of an exon within genomic coordinates selected from the group consisting of: chr16:10895702-10895722, chr16:10916432-10916452, chr16:10907623-10907643, chr16:10907932-10907952, chr16:10906985-10907005, chr16:10915626-10915646, chr16:10907539-10907559, chr16:10916426-10916446, chr16:10907476-10907496, chr16:10907119-10907139, chr16:10907979-10907999 and chr16:10909138-10909158. In some embodiments, the genetic modification comprises at least one nucleotide of an exon within genomic coordinates selected from the group consisting of: chr16:10906853-10906873, chr16:10906757-10906777, chr16:10895302-10895322, chr16:10907539-10907559, chr16:10907730-10907750 and chr16:10895702-10895722. In some embodiments, the genetic modification comprises at least one nucleotide of an exon within genomic coordinates selected from the group consisting of: chr16:10906853-10906873, chr16:10922444-10922464, chr16:10916432-10916452. In some embodiments, the genetic modification includes at least one nucleotide of an exon within genomic coordinates chr16: 10906853-10906873. In some embodiments, the genetic modification includes at least one nucleotide of an exon within genomic coordinates chr16: 10922444-10922464. In some embodiments, the genetic modification includes at least one nucleotide of an exon within genomic coordinates chr16: 10906757-10906777. In some embodiments, the genetic modification includes at least one nucleotide of an exon within genomic coordinates chr16:10916432-10916452. In some embodiments, the genetic modification includes at least one nucleotide of an exon within genomic coordinates chr16:10895302-10895322. In some embodiments, the genetic modification includes at least one nucleotide of an exon within genomic coordinates chr16: 10907539-10907559. In some embodiments, the genetic modification includes at least one nucleotide of an exon within genomic coordinates chr16: 10907730-10907750. In some embodiments, the genetic modification includes at least one nucleotide of an exon within genomic coordinates chr16:10895702-10895722. In some embodiments, the genetic modification includes at least one nucleotide of an exon within genomic coordinates chr16: 10907932-10907952. In some embodiments, the genetic modification includes at least one nucleotide of an exon within genomic coordinates chr16: 10907476-10907496. In some embodiments, the genetic modification includes at least one nucleotide of an exon within genomic coordinates chr16:10909138-10909158.
In some embodiments, the genetic modification comprises at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, or at least 10 contiguous nucleotides within the genomic coordinates selected from: chr16: chr16: the chr16, chr16, 16, and 16 chr16:10907886-10907906, chr16:10907924-10907944, chr16:10907928-10907948, chr16:10907932-10907952, chr16:10907935-10907955, chr16:10907978-10907998, chr16:10907979-10907999, chr16:10908069-10908089, chr16:10908073-10908093, chr16:10908101-10908121, chr16:10909056-10909076, chr16:10909138-10909158, chr16:10910195-10910215, chr16:10910196-10910216, chr16:10915592-10915612, chr16:10915626-10915646, chr16:10916375-10916395, chr16:10916382-10916402, chr16:10916426-10916446, chr16:10916432-10916452, chr16:10918486-10918506, chr16:10918492-10918512, chr16:10918493-10918513, chr16:10922435-10922455, chr16:10922441-10922461, chr16:10922441-10922461, chr16:10922444-10922464, chr16:10922460-10922480, chr16:10923257-10923277 and chr16:10923265-10923285. In some embodiments, the genetic modification comprises at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, or at least 10 contiguous nucleotides within the genomic coordinates selected from: chr16:10916432-10916452, chr16:10922444-10922464, chr16:10907924-10907944, chr16:10906985-10907005, chr16:10908073-10908093, chr16:10907433-10907453, chr16:10907979-10907999, chr16:10907139-10907159, chr16:10922435-10922455, chr16:10907384-10907404, chr16:10907434-10907454, chr16:10907119-10907139, chr16:10907539-10907559, chr16:10907810-10907830, chr16:10907315-10907335, chr16:10916426-10916446, chr16:10909138-10909158, chr16:10908101-10908121, chr16:10907790-10907810, chr16:10907787-10907807, and chr16: 10907787-10907807. In some embodiments, the genetic modification comprises at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, or at least 10 nucleotides of an exon within the genomic coordinates selected from: chr16:10907539-10907559, chr16:10916426-10916446, chr16:10906907-10906927, chr16:10895702-10895722, chr16:10907757-10907777, chr16:10907623-10907643, chr16:10915626-10915646, chr16:10906756-10906776, chr16:10907476-10907496, chr16:10907385-10907405 and chr16:10923265-10923285. In some embodiments, the genetic modification comprises at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, or at least 10 nucleotides of an exon within the genomic coordinates selected from: chr16:10906853-10906873, chr16:10922444-10922464, chr16:10907924-10907944, chr16:10907315-10907335, chr16:10916432-10916452, chr16:10907932-10907952, chr16:10915626-10915646, chr16:10907586-10907606, chr16:10916426-10916446, chr16:10907476-10907496, chr16:10907787-10907807, chr16:10907979-10907999, chr16:10906904-10906924 and chr16:10909138-10909158. In some embodiments, the genetic modification comprises at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, or at least 10 nucleotides of an exon within the genomic coordinates selected from: chr16:10895702-10895722, chr16:10916432-10916452, chr16:10907623-10907643, chr16:10907932-10907952, chr16:10906985-10907005, chr16:10915626-10915646, chr16:10907539-10907559, chr16:10916426-10916446, chr16:10907476-10907496, chr16:10907119-10907139, chr16:10907979-10907999 and chr16:10909138-10909158. In some embodiments, the genetic modification comprises at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, or at least 10 nucleotides of an exon within the genomic coordinates selected from: chr16:10906853-10906873, chr16:10906757-10906777, chr16:10895302-10895322, chr16:10907539-10907559, chr16:10907730-10907750 and chr16:10895702-10895722. In some embodiments, the genetic modification comprises at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, or at least 10 nucleotides of an exon within the genomic coordinates selected from: chr16:10906853-10906873, chr16:10922444-10922464, chr16:10916432-10916452. In some embodiments, the genetic modification comprises at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, or at least 10 nucleotides of an exon within genomic coordinates chr16: 10906853-10906873. In some embodiments, the genetic modification comprises at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, or at least 10 nucleotides of an exon within genomic coordinates chr16: 10922444-10922464. In some embodiments, the genetic modification comprises at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, or at least 10 nucleotides of an exon within genomic coordinates chr16: 10906757-10906777. In some embodiments, the genetic modification comprises at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, or at least 10 nucleotides of an exon within genomic coordinates chr16:10916432-10916452. In some embodiments, the genetic modification comprises at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, or at least 10 nucleotides of an exon within genomic coordinates chr16:10895302-10895322. In some embodiments, the genetic modification comprises at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, or at least 10 nucleotides of an exon within genomic coordinates chr16: 10907539-10907559. In some embodiments, the genetic modification comprises at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, or at least 10 nucleotides of an exon within genomic coordinates chr16: 10907730-10907750. In some embodiments, the genetic modification comprises at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, or at least 10 nucleotides of an exon within genomic coordinates chr16:10895702-10895722. In some embodiments, the genetic modification comprises at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, or at least 10 nucleotides of an exon within genomic coordinates chr16: 10907932-10907952. In some embodiments, the genetic modification comprises at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, or at least 10 nucleotides of an exon within genomic coordinates chr16: 10907476-10907496. In some embodiments, the genetic modification comprises at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, or at least 10 nucleotides of an exon within genomic coordinates chr16:10909138-10909158.
In some embodiments, the genetic modification comprises at least 5 consecutive nucleotides within genomic coordinates selected from the group consisting of: chr16: chr16: the chr16, chr16, 16, and 16 chr16:10907886-10907906, chr16:10907924-10907944, chr16:10907928-10907948, chr16:10907932-10907952, chr16:10907935-10907955, chr16:10907978-10907998, chr16:10907979-10907999, chr16:10908069-10908089, chr16:10908073-10908093, chr16:10908101-10908121, chr16:10909056-10909076, chr16:10909138-10909158, chr16:10910195-10910215, chr16:10910196-10910216, chr16:10915592-10915612, chr16:10915626-10915646, chr16:10916375-10916395, chr16:10916382-10916402, chr16:10916426-10916446, chr16:10916432-10916452, chr16:10918486-10918506, chr16:10918492-10918512, chr16:10918493-10918513, chr16:10922435-10922455, chr16:10922441-10922461, chr16:10922441-10922461, chr16:10922444-10922464, chr16:10922460-10922480, chr16:10923257-10923277 and chr16:10923265-10923285. In some embodiments, the genetic modification comprises at least 5 consecutive nucleotides within genomic coordinates selected from the group consisting of: chr16:10916432-10916452, chr16:10922444-10922464, chr16:10907924-10907944, chr16:10906985-10907005, chr16:10908073-10908093, chr16:10907433-10907453, chr16:10907979-10907999, chr16:10907139-10907159, chr16:10922435-10922455, chr16:10907384-10907404, chr16:10907434-10907454, chr16:10907119-10907139, chr16:10907539-10907559, chr16:10907810-10907830, chr16:10907315-10907335, chr16:10916426-10916446, chr16:10909138-10909158, chr16:10908101-10908121, chr16:10907790-10907810, chr16:10907787-10907807, and chr16: 10907787-10907807. In some embodiments, the genetic modification comprises at least 5 nucleotides of an exon within genomic coordinates selected from the group consisting of: chr16:10907539-10907559, chr16:10916426-10916446, chr16:10906907-10906927, chr16:10895702-10895722, chr16:10907757-10907777, chr16:10907623-10907643, chr16:10915626-10915646, chr16:10906756-10906776, chr16:10907476-10907496, chr16:10907385-10907405 and chr16:10923265-10923285. In some embodiments, the genetic modification comprises at least 5 nucleotides of an exon within genomic coordinates selected from the group consisting of: chr16:10906853-10906873, chr16:10922444-10922464, chr16:10907924-10907944, chr16:10907315-10907335, chr16:10916432-10916452, chr16:10907932-10907952, chr16:10915626-10915646, chr16:10907586-10907606, chr16:10916426-10916446, chr16:10907476-10907496, chr16:10907787-10907807, chr16:10907979-10907999, chr16:10906904-10906924 and chr16:10909138-10909158. In some embodiments, the genetic modification comprises at least 5 nucleotides of an exon within genomic coordinates selected from the group consisting of: chr16:10895702-10895722, chr16:10916432-10916452, chr16:10907623-10907643, chr16:10907932-10907952, chr16:10906985-10907005, chr16:10915626-10915646, chr16:10907539-10907559, chr16:10916426-10916446, chr16:10907476-10907496, chr16:10907119-10907139, chr16:10907979-10907999 and chr16:10909138-10909158. In some embodiments, the genetic modification comprises at least 5 nucleotides of an exon within genomic coordinates selected from the group consisting of: chr16:10906853-10906873, chr16:10906757-10906777, chr16:10895302-10895322, chr16:10907539-10907559, chr16:10907730-10907750 and chr16:10895702-10895722. In some embodiments, the genetic modification comprises at least 5 nucleotides of an exon within genomic coordinates selected from the group consisting of: chr16:10906853-10906873, chr16:10922444-10922464, chr16:10916432-10916452. In some embodiments, the genetic modification comprises at least 5 nucleotides of an exon within genomic coordinates chr16: 10906853-10906873. In some embodiments, the genetic modification comprises at least 5 nucleotides of an exon within genomic coordinates chr16: 10922444-10922464. In some embodiments, the genetic modification comprises at least 5 nucleotides of an exon within genomic coordinates chr16: 10906757-10906777. In some embodiments, the genetic modification comprises at least 5 nucleotides of an exon within genomic coordinates chr16:10916432-10916452. In some embodiments, the genetic modification comprises at least 5 nucleotides of an exon within genomic coordinates chr16:10895302-10895322. In some embodiments, the genetic modification comprises at least 5 nucleotides of an exon within genomic coordinates chr16: 10907539-10907559. In some embodiments, the genetic modification comprises at least 5 nucleotides of an exon within genomic coordinates chr16: 10907730-10907750. In some embodiments, the genetic modification comprises at least 5 nucleotides of an exon within genomic coordinates chr16:10895702-10895722. In some embodiments, the genetic modification comprises at least 5 nucleotides of an exon within genomic coordinates chr16: 10907932-10907952. In some embodiments, the genetic modification comprises at least 5 nucleotides of an exon within genomic coordinates chr16: 10907476-10907496. In some embodiments, the genetic modification comprises at least 5 nucleotides of an exon within genomic coordinates chr16:10909138-10909158.
In some embodiments, the genetic modification comprises at least 10 consecutive nucleotides within genomic coordinates selected from the group consisting of: chr16: chr16: the chr16, chr16, 16, and 16 chr16:10907886-10907906, chr16:10907924-10907944, chr16:10907928-10907948, chr16:10907932-10907952, chr16:10907935-10907955, chr16:10907978-10907998, chr16:10907979-10907999, chr16:10908069-10908089, chr16:10908073-10908093, chr16:10908101-10908121, chr16:10909056-10909076, chr16:10909138-10909158, chr16:10910195-10910215, chr16:10910196-10910216, chr16:10915592-10915612, chr16:10915626-10915646, chr16:10916375-10916395, chr16:10916382-10916402, chr16:10916426-10916446, chr16:10916432-10916452, chr16:10918486-10918506, chr16:10918492-10918512, chr16:10918493-10918513, chr16:10922435-10922455, chr16:10922441-10922461, chr16:10922441-10922461, chr16:10922444-10922464, chr16:10922460-10922480, chr16:10923257-10923277 and chr16:10923265-10923285. In some embodiments, the genetic modification comprises at least 10 consecutive nucleotides within genomic coordinates selected from the group consisting of: chr16:10916432-10916452, chr16:10922444-10922464, chr16:10907924-10907944, chr16:10906985-10907005, chr16:10908073-10908093, chr16:10907433-10907453, chr16:10907979-10907999, chr16:10907139-10907159, chr16:10922435-10922455, chr16:10907384-10907404, chr16:10907434-10907454, chr16:10907119-10907139, chr16:10907539-10907559, chr16:10907810-10907830, chr16:10907315-10907335, chr16:10916426-10916446, chr16:10909138-10909158, chr16:10908101-10908121, chr16:10907790-10907810, chr16:10907787-10907807, and chr16: 10907787-10907807. In some embodiments, the genetic modification comprises at least 10 nucleotides of an exon within genomic coordinates selected from the group consisting of: chr16:10907539-10907559, chr16:10916426-10916446, chr16:10906907-10906927, chr16:10895702-10895722, chr16:10907757-10907777, chr16:10907623-10907643, chr16:10915626-10915646, chr16:10906756-10906776, chr16:10907476-10907496, chr16:10907385-10907405 and chr16:10923265-10923285. In some embodiments, the genetic modification comprises at least 10 nucleotides of an exon within genomic coordinates selected from the group consisting of: chr16:10906853-10906873, chr16:10922444-10922464, chr16:10907924-10907944, chr16:10907315-10907335, chr16:10916432-10916452, chr16:10907932-10907952, chr16:10915626-10915646, chr16:10907586-10907606, chr16:10916426-10916446, chr16:10907476-10907496, chr16:10907787-10907807, chr16:10907979-10907999, chr16:10906904-10906924 and chr16:10909138-10909158. In some embodiments, the genetic modification comprises at least 10 nucleotides of an exon within genomic coordinates selected from the group consisting of: chr16:10895702-10895722, chr16:10916432-10916452, chr16:10907623-10907643, chr16:10907932-10907952, chr16:10906985-10907005, chr16:10915626-10915646, chr16:10907539-10907559, chr16:10916426-10916446, chr16:10907476-10907496, chr16:10907119-10907139, chr16:10907979-10907999 and chr16:10909138-10909158. In some embodiments, the genetic modification comprises at least 10 nucleotides of an exon within genomic coordinates selected from the group consisting of: chr16:10906853-10906873, chr16:10906757-10906777, chr16:10895302-10895322, chr16:10907539-10907559, chr16:10907730-10907750 and chr16:10895702-10895722. In some embodiments, the genetic modification comprises at least 10 nucleotides of an exon within genomic coordinates selected from the group consisting of: chr16:10906853-10906873, chr16:10922444-10922464 and chr16:10916432-10916452. In some embodiments, the genetic modification comprises at least 10 nucleotides of an exon within genomic coordinates chr16: 10906853-10906873. In some embodiments, the genetic modification comprises at least 10 nucleotides of an exon within genomic coordinates chr16: 10922444-10922464. In some embodiments, the genetic modification comprises at least 10 nucleotides of an exon within genomic coordinates chr16: 10906757-10906777. In some embodiments, the genetic modification comprises at least 10 nucleotides of an exon within genomic coordinates chr16:10916432-10916452. In some embodiments, the genetic modification comprises at least 10 nucleotides of an exon within genomic coordinates chr16:10895302-10895322. In some embodiments, the genetic modification comprises at least 10 nucleotides of an exon within genomic coordinates chr16: 10907539-10907559. In some embodiments, the genetic modification comprises at least 10 nucleotides of an exon within genomic coordinates chr16: 10907730-10907750. In some embodiments, the genetic modification comprises at least 10 nucleotides of an exon within genomic coordinates chr16:10895702-10895722. In some embodiments, the genetic modification comprises at least 10 nucleotides of an exon within genomic coordinates chr16: 10907932-10907952. In some embodiments, the genetic modification comprises at least 10 nucleotides of an exon within genomic coordinates chr16: 10907476-10907496. In some embodiments, the genetic modification comprises at least 10 nucleotides of an exon within genomic coordinates chr16:10909138-10909158.
In some embodiments, the genetic modification comprises a substitution of at least one C for T or a substitution of at least one a for G within the genomic coordinates selected from: chr16: chr16: the chr16, chr16, 16, and 16 chr16:10907886-10907906, chr16:10907924-10907944, chr16:10907928-10907948, chr16:10907932-10907952, chr16:10907935-10907955, chr16:10907978-10907998, chr16:10907979-10907999, chr16:10908069-10908089, chr16:10908073-10908093, chr16:10908101-10908121, chr16:10909056-10909076, chr16:10909138-10909158, chr16:10910195-10910215, chr16:10910196-10910216, chr16:10915592-10915612, chr16:10915626-10915646, chr16:10916375-10916395, chr16:10916382-10916402, chr16:10916426-10916446, chr16:10916432-10916452, chr16:10918486-10918506, chr16:10918492-10918512, chr16:10918493-10918513, chr16:10922435-10922455, chr16:10922441-10922461, chr16:10922441-10922461, chr16:10922444-10922464, chr16:10922460-10922480, chr16:10923257-10923277 and chr16:10923265-10923285. In some embodiments, the genetic modification comprises a substitution of at least one C for T or a substitution of at least one a for G within the genomic coordinates selected from: chr16:10916432-10916452, chr16:10922444-10922464, chr16:10907924-10907944, chr16:10906985-10907005, chr16:10908073-10908093, chr16:10907433-10907453, chr16:10907979-10907999, chr16:10907139-10907159, chr16:10922435-10922455, chr16:10907384-10907404, chr16:10907434-10907454, chr16:10907119-10907139, chr16:10907539-10907559, chr16:10907810-10907830, chr16:10907315-10907335, chr16:10916426-10916446, chr16:10909138-10909158, chr16:10908101-10908121, chr16:10907790-10907810, chr16:10907787-10907807, and chr16: 10907787-10907807. In some embodiments, the genetic modification comprises a substitution of at least one C for T or a substitution of at least one a for G within the genomic coordinates selected from: chr16:10907539-10907559, chr16:10916426-10916446, chr16:10906907-10906927, chr16:10895702-10895722, chr16:10907757-10907777, chr16:10907623-10907643, chr16:10915626-10915646, chr16:10906756-10906776, chr16:10907476-10907496, chr16:10907385-10907405 and chr16:10923265-10923285. In some embodiments, the genetic modification comprises a substitution of at least one C for T or a substitution of at least one a for G within the genomic coordinates selected from: chr16:10906853-10906873, chr16:10922444-10922464, chr16:10907924-10907944, chr16:10907315-10907335, chr16:10916432-10916452, chr16:10907932-10907952, chr16:10915626-10915646, chr16:10907586-10907606, chr16:10916426-10916446, chr16:10907476-10907496, chr16:10907787-10907807, chr16:10907979-10907999, chr16:10906904-10906924 and chr16:10909138-10909158. In some embodiments, the genetic modification comprises a substitution of at least one C for T or a substitution of at least one a for G within the genomic coordinates selected from: chr16:10895702-10895722, chr16:10916432-10916452, chr16:10907623-10907643, chr16:10907932-10907952, chr16:10906985-10907005, chr16:10915626-10915646, chr16:10907539-10907559, chr16:10916426-10916446, chr16:10907476-10907496, chr16:10907119-10907139, chr16:10907979-10907999 and chr16:10909138-10909158. In some embodiments, the genetic modification comprises a substitution of at least one C for T or a substitution of at least one a for G within the genomic coordinates selected from: chr16:10906853-10906873, chr16:10906757-10906777, chr16:10895302-10895322, chr16:10907539-10907559, chr16:10907730-10907750 and chr16:10895702-10895722. In some embodiments, the genetic modification comprises a substitution of at least one C for T or a substitution of at least one a for G within the genomic coordinates selected from: chr16:10906853-10906873, chr16:10922444-10922464, chr16:10916432-10916452. In some embodiments, the genetic modification includes substitution of at least one C for T or substitution of at least one A for G within genomic coordinates chr16: 10906853-10906873. In some embodiments, the genetic modification includes substitution of at least one C for T or substitution of at least one A for G within genomic coordinates chr16: 10922444-10922464. In some embodiments, the genetic modification includes substitution of at least one C for T or substitution of at least one A for G within genomic coordinates chr16: 10906757-10906777. In some embodiments, the genetic modification includes substitution of at least one C for T or substitution of at least one A for G within genomic coordinates chr16:10916432-10916452. In some embodiments, the genetic modification includes substitution of at least one C for T or substitution of at least one A for G within genomic coordinates chr16:10895302-10895322. In some embodiments, the genetic modification includes substitution of at least one C for T or substitution of at least one A for G within genomic coordinates chr16: 10907539-10907559. In some embodiments, the genetic modification includes substitution of at least one C for T or substitution of at least one A for G within genomic coordinates chr16: 10907730-10907750. In some embodiments, the genetic modification includes substitution of at least one C for T or substitution of at least one A for G within genomic coordinates chr16:10895702-10895722. In some embodiments, the genetic modification includes substitution of at least one C for T or substitution of at least one A for G within genomic coordinates chr16: 10907932-10907952. In some embodiments, the genetic modification includes substitution of at least one C for T or substitution of at least one A for G within genomic coordinates chr16: 10907476-10907496. In some embodiments, the genetic modification includes substitution of at least one C for T or substitution of at least one A for G within genomic coordinates chr16:10909138-10909158.
In some embodiments, the modification to the CIITA gene comprises any one or more of an insertion, deletion, substitution, or deamination of at least one nucleotide in the target sequence. In some embodiments, the modification to CIITA comprises insertion of 1, 2, 3, 4 or 5 or more nucleotides in the target sequence. In some embodiments, the modification to CIITA comprises a deletion of 1, 2, 3, 4 or 5 or more nucleotides in the target sequence. In other embodiments, modifications to CIITA include insertion of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, or 25 or more nucleotides in the target sequence. In other embodiments, modifications to CIITA include deletions of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, or 25 or more nucleotides in the target sequence. In some embodiments, modifications to CIITA include insertions/deletions, which are generally defined in the art as insertions or deletions of less than 1000 base pairs (bp). In some embodiments, the modification to CIITA comprises an insertion/deletion that causes an in-frame transfer mutation in the target sequence. In some embodiments, modifications to CIITA include substitutions of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, or 25 or more nucleotides in the target sequence. In some embodiments, the modifications to CIITA include one or more of nucleotide insertions, deletions, or substitutions resulting from incorporation of the template nucleic acid. In some embodiments, the modification to CIITA comprises insertion of a donor nucleic acid in the target sequence. In some embodiments, modifications to CIITA are not transient.
In some embodiments, genetic modification to CIITA results in the use of an out-of-frame stop codon. In some embodiments, the genetic modification to CIITA results in reduced expression of CIITA protein by the cell. In some embodiments, the genetic modification to CIITA results in a decrease in CIITA in the nucleus. In some embodiments, the modification to CIITA results in reduced expression of MHC class II proteins on the cell surface.
In some embodiments, genetic modification to CIITA results in truncated forms of CIITA protein. In some embodiments, the truncated CIITA protein does not bind to GTP. In some embodiments, the truncated CIITA protein does not localize to the nucleus. In some embodiments, the activity of the CIITA protein (e.g., a truncated form of the CIITA protein) is impaired compared to the activity of the wild-type CIITA protein associated with modulating MHC class II expression. In some embodiments, MHC class II expression on the cell surface is reduced due to impaired CIITA protein activity. In some embodiments, MHC class II expression on the cell surface is absent due to impaired CIITA protein activity.
Efficacy of CIITA guide RNA
Efficacy of CIITA guide RNAs can be determined by techniques available in the art that assess the efficiency of editing of guide RNAs, levels of CIITA proteins and/or mRNA, and/or levels of MHC class II in target cells.
In some embodiments, the efficacy of the CIITA guide RNA is determined by measuring the level of CIITA protein in the cell. Levels of CIITA protein can be detected, for example, by cell lysates and western blotting using anti-CIITA antibodies. In some embodiments, the efficacy of the CIITA guide RNA is determined by measuring the level of CIITA protein in the nucleus. In some embodiments, the efficacy of the CIITA guide RNA is determined by measuring the level of CIITA mRNA in the cell. The level of CIITA mRNA can be detected, for example, by RT-PCR. In some embodiments, a decrease in CIITA protein and/or CIITA mRNA levels in the target cell as compared to the unmodified cell is indicative of an effective CIITA guide RNA.
"unmodified cells" refers to control cells (control cells) of the same cell type in an experiment or test, wherein the "unmodified" control cells have not been contacted with the CIITA guide. Thus, an unmodified cell may be a cell that has not been contacted with a guide RNA, or a cell that has been contacted with a guide RNA that is not targeted to CIITA.
In some embodiments, the efficacy of CIITA guide RNAs is determined by measuring the reduction or elimination of MHC class II protein expression of target cells. The CIITA protein acts as a transductor activating MHC class II promoters and is critical for expression of MHC class II proteins. In some embodiments, MHC class II protein expression may be detected on the surface of a target cell. In some embodiments, MHC class II protein expression is measured by flow cytometry. In some embodiments, antibodies (e.g., anti-HLA-DR, -DQ, -DP) to MHC class II proteins may be used to detect MHC class II protein expression, e.g., by flow cytometry. In some embodiments, one or more antibodies (e.g., anti-HLA-DR, -DQ, -DP) to MHC class II proteins may be used to detect MHC class II protein expression, e.g., by flow cytometry. In some embodiments, the one or more antibodies to MHC class II proteins comprise one or more of an antibody to HLA-DR, an antibody to HLA-DQ, and an antibody to HLA-DP. In some embodiments, the one or more antibodies to MHC class II proteins comprise an antibody to HLA-DR, an antibody to HLA-DQ, and an antibody to HLA-DP. In some embodiments, the one or more antibodies to MHC class II proteins comprise antibodies to HLA-DR, HLA-DQ, and HLA-DP.
In some embodiments, a decrease or elimination of MHC class II protein on the surface of a cell (or population of cells) as compared to an unmodified cell (or population of unmodified cells) is indicative of an effective CIITA guide RNA. In some embodiments, cells (or cell populations) that have been contacted with a particular CIITA guide RNA that is negative for MHC class II proteins according to flow cytometry and an RNA-guided DNA binding agent are indicative of an effective CIITA guide RNA.
In some embodiments, MHC class II protein expression in a cell population is reduced or eliminated using the methods and compositions disclosed herein. In some embodiments, the cell population is enriched (e.g., by FACS or MACS) and is at least 65%, 70%, 80%, 90%, 91%, 92%, 93% or 94% MHC class II negative relative to the unmodified cell population, as measured by flow cytometry. In some embodiments, the cell population is not enriched (e.g., by FACS or MACS) and is at least 65%, 70%, 80%, 90%, 91%, 92%, 93% or 94% MHC class II negative relative to the unmodified cell population, as measured by flow cytometry.
In some embodiments, the cell population is at least 65% MHC class II negative as measured by flow cytometry relative to an unmodified cell population. In some embodiments, the cell population is at least 70% MHC class II negative as measured by flow cytometry relative to an unmodified cell population. In some embodiments, the cell population is at least 80% mhc class II negative as measured by flow cytometry relative to an unmodified cell population. In some embodiments, the cell population is at least 90% MHC class II negative as measured by flow cytometry relative to an unmodified cell population. In some embodiments, the cell population is at least 91% MHC class II negative as measured by flow cytometry relative to an unmodified cell population. In some embodiments, the cell population is at least 92% MHC class II negative as measured by flow cytometry relative to an unmodified cell population. In some embodiments, the cell population is at least 93% MHC class II negative as measured by flow cytometry relative to an unmodified cell population. In some embodiments, the cell population is at least 94% MHC class II negative as measured by flow cytometry relative to an unmodified cell population. In some embodiments, the cell population is at least 95% MHC class II negative as measured by flow cytometry relative to an unmodified cell population. In some embodiments, the cell population is at least 96% MHC class II negative as measured by flow cytometry relative to an unmodified cell population. In some embodiments, the cell population is at least 97% mhc class II negative as measured by flow cytometry relative to an unmodified cell population. In some embodiments, the cell population is at least 98% MHC class II negative as measured by flow cytometry relative to an unmodified cell population. In some embodiments, the cell population is at least 99% MHC class II negative as measured by flow cytometry relative to an unmodified cell population.
In some embodiments, the cell population is at least 65% MHC class II negative as measured by flow cytometry using one or more of an antibody to HLA-DR, an antibody to HLA-DQ, and an antibody to HLA-DP relative to an unmodified cell population. In some embodiments, the cell population is at least 65% MHC class II negative as measured by flow cytometry using antibodies to HLA-DR, antibodies to HLA-DQ, and antibodies to HLA-DP relative to an unmodified cell population. In some embodiments, the cell population is at least 65% MHC class II negative as measured by flow cytometry using antibodies against HLA-DR, HLA-DQ, and HLA-DP relative to an unmodified cell population. In some embodiments, the cell population is at least 70% MHC class II negative as measured by flow cytometry using one or more of an antibody to HLA-DR, an antibody to HLA-DQ, and an antibody to HLA-DP relative to an unmodified cell population. In some embodiments, the cell population is at least 70% MHC class II negative as measured by flow cytometry using antibodies to HLA-DR, antibodies to HLA-DQ, and antibodies to HLA-DP relative to an unmodified cell population. In some embodiments, the cell population is at least 70% MHC class II negative as measured by flow cytometry using antibodies against HLA-DR, HLA-DQ, and HLA-DP relative to an unmodified cell population. In some embodiments, the cell population is at least 80% MHC class II negative as measured by flow cytometry using one or more of an antibody to HLA-DR, an antibody to HLA-DQ, and an antibody to HLA-DP relative to an unmodified cell population. In some embodiments, the cell population is at least 80% MHC class II negative as measured by flow cytometry using antibodies to HLA-DR, antibodies to HLA-DQ, and antibodies to HLA-DP relative to an unmodified cell population. In some embodiments, the cell population is at least 80% MHC class II negative as measured by flow cytometry using antibodies against HLA-DR, HLA-DQ, and HLA-DP relative to an unmodified cell population. In some embodiments, the cell population is at least 90% MHC class II negative as measured by flow cytometry using one or more of an antibody to HLA-DR, an antibody to HLA-DQ, and an antibody to HLA-DP relative to an unmodified cell population. In some embodiments, the cell population is at least 90% MHC class II negative as measured by flow cytometry using antibodies to HLA-DR, antibodies to HLA-DQ, and antibodies to HLA-DP relative to an unmodified cell population. In some embodiments, the cell population is at least 90% MHC class II negative as measured by flow cytometry using antibodies against HLA-DR, HLA-DQ, and HLA-DP relative to an unmodified cell population. In some embodiments, the cell population is at least 92% MHC class II negative as measured by flow cytometry using one or more of an antibody to HLA-DR, an antibody to HLA-DQ, and an antibody to HLA-DP relative to an unmodified cell population. In some embodiments, the cell population is at least 92% MHC class II negative as measured by flow cytometry using antibodies to HLA-DR, antibodies to HLA-DQ, and antibodies to HLA-DP relative to an unmodified cell population. In some embodiments, the cell population is at least 92% mhc class II negative as measured by flow cytometry using antibodies against HLA-DR, HLA-DQ, and HLA-DP relative to an unmodified cell population. In some embodiments, the cell population is at least 93% MHC class II negative as measured by flow cytometry using one or more of an antibody to HLA-DR, an antibody to HLA-DQ, and an antibody to HLA-DP relative to an unmodified cell population. In some embodiments, the cell population is at least 93% MHC class II negative as measured by flow cytometry using antibodies to HLA-DR, antibodies to HLA-DQ, and antibodies to HLA-DP relative to an unmodified cell population. In some embodiments, the cell population is at least 93% MHC class II negative as measured by flow cytometry using antibodies against HLA-DR, HLA-DQ, and HLA-DP relative to an unmodified cell population. In some embodiments, the cell population is at least 94% MHC class II negative as measured by flow cytometry using one or more of an antibody to HLA-DR, an antibody to HLA-DQ, and an antibody to HLA-DP relative to an unmodified cell population. In some embodiments, the cell population is at least 94% MHC class II negative as measured by flow cytometry using antibodies to HLA-DR, antibodies to HLA-DQ, and antibodies to HLA-DP relative to an unmodified cell population. In some embodiments, the cell population is at least 94% MHC class II negative as measured by flow cytometry using antibodies against HLA-DR, HLA-DQ, and HLA-DP relative to an unmodified cell population. In some embodiments, the cell population is at least 95% MHC class II negative as measured by flow cytometry using one or more of an antibody to HLA-DR, an antibody to HLA-DQ, and an antibody to HLA-DP relative to an unmodified cell population. In some embodiments, the cell population is at least 95% MHC class II negative as measured by flow cytometry using antibodies to HLA-DR, antibodies to HLA-DQ, and antibodies to HLA-DP relative to an unmodified cell population. In some embodiments, the cell population is at least 95% MHC class II negative as measured by flow cytometry using antibodies against HLA-DR, HLA-DQ, and HLA-DP relative to an unmodified cell population. In some embodiments, the cell population is at least 96% MHC class II negative as measured by flow cytometry using one or more of an antibody to HLA-DR, an antibody to HLA-DQ, and an antibody to HLA-DP relative to an unmodified cell population. In some embodiments, the cell population is at least 96% MHC class II negative as measured by flow cytometry using antibodies to HLA-DR, antibodies to HLA-DQ, and antibodies to HLA-DP relative to an unmodified cell population. In some embodiments, the cell population is at least 96% MHC class II negative as measured by flow cytometry using antibodies against HLA-DR, HLA-DQ, and HLA-DP relative to an unmodified cell population. In some embodiments, the cell population is at least 97% mhc class II negative as measured by flow cytometry using one or more of an antibody to HLA-DR, an antibody to HLA-DQ, and an antibody to HLA-DP relative to an unmodified cell population. In some embodiments, the cell population is at least 97% MHC class II negative as measured by flow cytometry using antibodies to HLA-DR, antibodies to HLA-DQ, and antibodies to HLA-DP relative to an unmodified cell population. In some embodiments, the cell population is at least 97% MHC class II negative as measured by flow cytometry using antibodies against HLA-DR, HLA-DQ, and HLA-DP relative to an unmodified cell population. In some embodiments, the cell population is at least 98% MHC class II negative as measured by flow cytometry using one or more of an antibody to HLA-DR, an antibody to HLA-DQ, and an antibody to HLA-DP relative to an unmodified cell population. In some embodiments, the cell population is at least 98% MHC class II negative as measured by flow cytometry using antibodies to HLA-DR, antibodies to HLA-DQ, and antibodies to HLA-DP relative to an unmodified cell population. In some embodiments, the cell population is at least 98% MHC class II negative as measured by flow cytometry using antibodies against HLA-DR, HLA-DQ, and HLA-DP relative to an unmodified cell population. In some embodiments, the cell population is at least 99% MHC class II negative as measured by flow cytometry using one or more of an antibody to HLA-DR, an antibody to HLA-DQ, and an antibody to HLA-DP relative to an unmodified cell population. In some embodiments, the cell population is at least 99% mhc class II negative as measured by flow cytometry using antibodies to HLA-DR, antibodies to HLA-DQ, and antibodies to HLA-DP relative to an unmodified cell population. In some embodiments, the cell population is at least 99% mhc class II negative as measured by flow cytometry using antibodies against HLA-DR, HLA-DQ, and HLA-DP relative to an unmodified cell population.
In some embodiments, effective CIITA guide RNAs can be determined by measuring the response of immune cells (e.g., cd4+ T cells) to genetically modified target cells in vitro or in vivo. The cd4+ T cell response may be assessed by assays (e.g., flow cytometry, ELISA) that measure the activation response of cd4+ T cells (e.g., cd4+ T cell proliferation), expression of activation markers, and/or cytokine production (IL-2, IL-12, IFN- γ). The response of cd4+ T cells can be assessed in an in vitro cell culture assay in which genetically modified cells are co-cultured with cells comprising cd4+ T cells. For example, the genetically modified cells can be co-cultured, for example, with PBMCs, purified cd3+ T cells comprising cd4+ T cells, purified cd4+ T cells, or a cd4+ T cell line. The cd4+ T cell response elicited by genetically modified cells can be compared to the response elicited by unmodified cells. Decreased response of cd4+ T cells indicates effective CIITA guide RNAs.
The efficacy of CIITA guide RNAs can also be assessed by survival of cells after editing. In some embodiments, the cells survive at least one week to six weeks after editing. In some embodiments, the cells survive at least one week to twelve weeks after editing. In some embodiments, the cells survive at least two weeks after editing. In some embodiments, the cells survive at least three weeks after editing. In some embodiments, the cells survive at least four weeks after editing. In some embodiments, the cells survive at least five weeks after editing. In some embodiments, the cells survive at least six weeks after editing. Viability of the genetically modified cells can be measured using standard techniques, including, for example, by measuring cell death, by flow cytometry live/dead staining, or cell proliferation.
C. Methods and compositions for reducing or eliminating MHC class II and additional modifications
MHC class I knockout
In some embodiments, methods are provided for reducing or eliminating expression of MHC class II proteins on a cell surface by genetically modifying CIITA as disclosed herein, wherein the methods further provide for reducing or eliminating expression of MHC class I proteins on a cell surface relative to an unmodified cell. In one approach, MHC class I protein expression is reduced or eliminated by genetically modifying the B2M gene. In some embodiments, MHC class I protein expression is reduced or eliminated by contacting the cell with a B2M guide RNA. In another method, expression of MHC class I protein HLA-A is reduced or eliminated by genetically modifying HLA-A, thereby reducing or eliminating surface expression of HLA-A in human cells, wherein the human cells are homozygous for HLA-B and homozygous for HLA-C. Thus, in some embodiments, either HLA-A protein expression is reduced or eliminated by contacting a human cell with HLA-A guide RNA, wherein the human cell is homozygous for HLa-B and homozygous for HLa-C. In some embodiments, the resulting cell is an allogeneic cell.
In some embodiments, the method comprises reducing or eliminating surface expression of MHC class II proteins in an engineered cell relative to an unmodified cell, the method comprising contacting the cell with a composition comprising a CIITA guide RNA disclosed herein, the method further comprising contacting the cell with a B2M guide RNA. In some embodiments, the method further comprises contacting the cell with an RNA-guided DNA binding agent. In some embodiments, the method further comprises inducing DSB or SSB in the B2M target sequence. In some embodiments, B2M expression is thereby reduced by the cell. In some embodiments, MHC class I protein expression is thereby reduced or eliminated by the cell.
In some embodiments, the B2M guide RNA targets a human B2M gene.
In some embodiments, the B2M guide RNA comprises SEQ ID NO. 701. In some embodiments, the B2M guide RNA comprises a guide sequence of at least 17, 18, 19 or 20 consecutive nucleotides of SEQ ID NO. 701. In some embodiments, the B2M guide RNA comprises a guide sequence that is at least 95%, 90% or 85% identical to SEQ ID NO. 701.
Additional embodiments of B2M guide RNAs are provided herein, including, for example, exemplary modifications to the guide RNAs.
In some embodiments, the efficacy of the B2M guide RNA is determined by measuring the level of B2M protein in the cell relative to an unmodified cell. In some embodiments, the efficacy of the B2M guide RNA is determined by measuring the level of B2M protein expressed by the cell. In some embodiments, antibodies to B2M proteins (e.g., anti-B2M) may be used to detect the level of B2M protein by, for example, flow cytometry. In some embodiments, the efficacy of B2M guide RNA is determined by measuring the level of B2M mRNA in a cell (e.g., by RT-PCR). In some embodiments, a decrease or elimination of the level of B2M protein or B2M mRNA compared to the level of B2M protein in the unmodified cell is indicative of an effective B2M guide RNA. In some embodiments, cells (or cell populations) negative for B2M protein according to flow cytometry are indicative of effective B2M guide RNA as compared to unmodified cells (or unmodified cell populations). In some embodiments, cells (or cell populations) that have been contacted with a particular B2M guide RNA that is negative for MHC class I proteins according to flow cytometry and an RNA-guided DNA binding agent are indicative of an effective B2M guide RNA.
In some embodiments, the efficacy of B2M guide RNA is determined by measuring the level of MHC class I proteins on the cell surface. In some embodiments, MHC class I protein levels are measured by flow cytometry (e.g., with antibodies to HLA-A, HLA-B, or HLA-C). In some embodiments, the cell population is at least 65% MHC class I negative as measured by flow cytometry relative to an unmodified cell population. In some embodiments, the cell population is at least 70% MHC class I negative as measured by flow cytometry relative to an unmodified cell population. In some embodiments, the cell population is at least 80% MHC class I negative as measured by flow cytometry relative to an unmodified cell population. In some embodiments, the cell population is at least 90% MHC class I negative as measured by flow cytometry relative to an unmodified cell population. In some embodiments, the cell population is at least 95% MHC class I negative as measured by flow cytometry relative to an unmodified cell population. In some embodiments, the cell population is at least 100% MHC class I negative as measured by flow cytometry relative to an unmodified cell population.
In some embodiments, a method comprises reducing or eliminating surface expression of an MHC class II protein in an engineered cell relative to an unmodified cell, the method comprising contacting the cell with a composition comprising a CIITA guide RNA disclosed herein, the method further comprising reducing or eliminating HLA-A expression of the cell by a gene editing system that binds to an HLA-A genomic target sequence comprising at least 5 contiguous nucleotides within genomic coordinates selected from the group consisting of: chr6:29942864-29942884; chr6:29942868-29942888; chr6:29942876-29942896; chr6:29942877-29942897; chr6:29942883-29942903; chr6:29943126-29943146; chr6:29943528-29943548; chr6:29943529-29943549; chr6:29943530-29943550; chr6:29943537-29943557; chr6:29943549-29943569; chr6:29943589-29943609 and chr6:29944026-29944046. In some embodiments, a method comprises reducing or eliminating surface expression of an MHC class II protein in an engineered cell relative to an unmodified cell, the method comprising contacting the cell with a composition comprising a CIITA guide RNA disclosed herein, the method further comprising reducing or eliminating HLA-A expression of the cell by a gene editing system that binds to an HLA-A genomic target sequence comprising at least 10 contiguous nucleotides within genomic coordinates selected from the group consisting of: chr6:29942864-29942884; chr6:29942868-29942888; chr6:29942876-29942896; chr6:29942877-29942897; chr6:29942883-29942903; chr6:29943126-29943146; chr6:29943528-29943548; chr6:29943529-29943549; chr6:29943530-29943550; chr6:29943537-29943557; chr6:29943549-29943569; chr6:29943589-29943609 and chr6:29944026-29944046. In some embodiments, the HLA-A genomic coordinates are selected from chr6:29942864-29942884. In some embodiments, the HLA-A genomic coordinates are selected from chr6:29942868-29942888. In some embodiments, the HLA-A genomic coordinates are selected from chr6:29942876-29942896. In some embodiments, the HLA-A genomic coordinates are selected from chr6:29942877-29942897. In some embodiments, the HLA-A genomic coordinates are selected from chr6:29942883-29942903. In some embodiments, the HLA-A genomic coordinates are selected from chr6:29943126-29943146. In some embodiments, the HLA-A genomic coordinates are selected from chr6:29943528-29943548. In some embodiments, the HLA-A genomic coordinates are selected from chr6:29943529-29943549. In some embodiments, the HLA-A genomic coordinates are selected from chr6:29943530-29943550. In some embodiments, the HLA-A genomic coordinates are selected from chr6:29943537-29943557. In some embodiments, the HLA-A genomic coordinates are selected from chr6:29943549-29943569. In some embodiments, the HLA-A genomic coordinates are selected from chr6:29943589-29943609. In some embodiments, the HLA-A genomic coordinates are selected from chr6:29944026-29944046. In some embodiments, the gene editing system comprises an RNA-guided DNA binding agent. In some embodiments, the RNA-guided DNA binding agent comprises a Cas9 protein, such as streptococcus pyogenes Cas9. In some embodiments, the cells are homozygous for HLA-B and homozygous for HLA-C.
In some embodiments, a method comprises reducing or eliminating surface expression of MHC class II proteins in an engineered cell relative to an unmodified cell, the method comprising contacting the cell with a composition comprising a CIITA guide RNA disclosed herein, the method further comprising contacting the cell with an HLA-A guide RNA. In some embodiments, the HLA-A guide RNA comprises a guide sequence selected from SEQ ID NO:2001-2095 (see Table 3 below). In some embodiments, the method further comprises contacting the cell with an RNA-guided DNA binding agent. In some embodiments, the RNA-guided DNA binding agent comprises a Cas9 protein, such as streptococcus pyogenes Cas9. In some embodiments, the cells are homozygous for HLA-B and homozygous for HLA-C.
In some embodiments, methods are provided for making an engineered cell having reduced or eliminated MHC class II protein surface expression relative to an unmodified cell, comprising: a. contacting the cell with a CIITA guide RNA, wherein the guide RNA comprises a guide sequence selected from SEQ ID NOs 1-117; contacting the cell with HLA-A guide RNA, wherein the HLA-A guide RNA comprises a guide sequence selected from any one of SEQ ID NOs 2001-2095 (see table 3 below); optionally contacting the cell with an RNA-guided DNA binding agent or a nucleic acid encoding an RNA-guided DNA binding agent; wherein there is reduced or eliminated expression of the HLA-A surface in the cell relative to an unmodified cell. In some embodiments, the method comprises contacting the cell with an RNA-guided DNA binding agent or a nucleic acid encoding an RNA-guided DNA binding agent. In some embodiments, the RNA-guided DNA binding agent comprises streptococcus pyogenes Cas9. In some embodiments, the cells are homozygous for HLA-B and homozygous for HLA-C.
Exemplary HLA-A guide RNAs are provided in Table 3 (SEQ ID NOS: 2001-2095 and corresponding guide RNA sequences SEQ ID NOS: 427-521 and 603-697).
TABLE 3 exemplary HLA-A guide sequences
/>
/>
/>
/>
/>
/>
/>
/>
/>
/>
/>
/>
/>
/>
/>
/>
/>
/>
/>
/>
/>
/>
/>
/>
/>
/>
In some embodiments, the efficacy of HLA-A guide RNAs is determined by measuring the level of HLA-A protein on the cell surface. In some embodiments, HLA-A protein levels are measured by flow cytometry (e.g., using antibodies to HLA-A2 and/or HLA-A 3). In some embodiments, the cell population is at least 65% HLA-A negative as measured by flow cytometry relative to an unmodified cell population. In some embodiments, the cell population is at least 70% HLA-A negative as measured by flow cytometry relative to an unmodified cell population. In some embodiments, the cell population is at least 80% HLA-A negative as measured by flow cytometry relative to an unmodified cell population. In some embodiments, the cell population is at least 90% HLA-A negative as measured by flow cytometry relative to an unmodified cell population. In some embodiments, the cell population is at least 95% negative for HLA-A as measured by flow cytometry relative to an unmodified cell population. In some embodiments, the cell population is at least 100% HLA-A negative as measured by flow cytometry relative to an unmodified cell population.
In some embodiments, the efficacy of a B2M guide RNA or HLA-A guide can be determined by measuring the response of an immune cell (e.g., a cd8+ T cell) to a genetically modified target cell in vitro or in vivo as compared to an unmodified cell. For example, a decreased response of cd8+ T cells indicates a potent B2M guide RNA or HLA-A guide RNA. The cd8+ T cell response can be assessed by measuring cd8+ T cell activation response (e.g., cd8+ T cell proliferation), expression of activation markers, and/or assays of cytokine production (IL-2, IFN- γ, TNF- α) (e.g., flow cytometry, ELISA). Cd8+ T cell responses can be assessed in vitro or in vivo. In some embodiments, cd8+ T cell responses can be assessed by co-culturing genetically modified cells with cd8+ T cells in vitro. In some embodiments, cd8+ T cell activity may be assessed in an in vivo model, e.g., a rodent model. In vivo models, for example, genetically modified cells may be administered with cd8+ T cells; survival of the genetically modified cells indicates that cd8+ T cell lysis can be avoided. In some embodiments, the methods result in a composition comprising cells that survive in vivo in the presence of cd8+ T cells for more than 1, 2, 3, 4, 5, or 6 weeks or more. In some embodiments, the methods result in a composition comprising cells that survive in vivo in the presence of cd8+ T cells for at least one week to six weeks. In some embodiments, the methods result in a composition comprising cells that survive in vivo in the presence of cd8+ T cells for at least two to four weeks. In some embodiments, the methods result in a composition comprising cells that survive in vivo in the presence of cd8+ T cells for at least four to six weeks. In some embodiments, the methods result in a composition comprising cells that survive in vivo for more than six weeks in the presence of cd8+ T cells.
In some embodiments, the methods result in a composition comprising cells having reduced or eliminated MHC class II expression and reduced or eliminated MHC class I expression relative to unmodified cells. In some embodiments, the methods result in a composition comprising a cell having reduced or eliminated MHC class II protein expression, reduced or eliminated CIITA protein expression, and/or reduced or eliminated CIITA levels in the nucleus of the cell, and/or reduced or eliminated MHC class I protein expression. In some embodiments, the methods result in a composition comprising a cell having reduced or eliminated MHC class II protein expression, reduced or eliminated CIITA protein expression, and/or reduced or eliminated CIITA levels in the nucleus of the cell, and/or reduced B2M protein expression. In some embodiments, the methods result in a composition comprising a cell having reduced or eliminated MHC class II protein expression, reduced or eliminated CIITA protein expression, and/or reduced or eliminated CIITA levels in the nucleus of the cell, and reduced or eliminated B2M mRNA levels. In some embodiments, the cells elicit a reduced or eliminated response from cd8+ T cells.
In some embodiments, the methods result in a composition comprising cells having reduced or eliminated MHC class II expression and reduced or eliminated HLA-A expression relative to unmodified cells, wherein the cells are homozygous for HLa-B and homozygous for HLa-C. In some embodiments, the methods result in a composition comprising a cell having reduced or eliminated MHC class II protein expression, reduced or eliminated CIITA protein expression, and/or reduced or eliminated CIITA levels in the nucleus of the cell, and/or reduced or eliminated HLA-A protein expression. In some embodiments, the methods result in a composition comprising a cell having reduced or eliminated MHC class II protein expression, reduced or eliminated CIITA protein expression, and/or reduced or eliminated CIITA levels in the nucleus of the cell, and/or reduced HLA-A protein expression. In some embodiments, the cells elicit a reduced or eliminated response from cd8+ T cells.
In some embodiments, an engineered cell is provided, wherein the cell has reduced or eliminated expression of MHC class II and MHC class I proteins on the cell surface, wherein the cell comprises a genetic modification in CIITA, and wherein the cell comprises a modification in B2M. In some embodiments, the cells elicit a reduced response from cd4+ T cells and elicit a reduced response from cd8+ T cells.
In some embodiments, an engineered cell is provided, wherein the cell has reduced or eliminated expression of MHC class II and HLA-A proteins on the cell surface, wherein the cell comprises a genetic modification in CIITA, and wherein the cell comprises a genetic modification in HLA-A gene, wherein the cell is homozygous for HLa-B and homozygous for HLa-C. In some embodiments, an engineered cell is provided, wherein the cell has reduced or eliminated expression of MHC class II and HLA-A proteins on the cell surface, wherein the cell comprises a genetic modification in CIITA, and wherein the cell comprises a genetic modification in an HLA-A gene. In some embodiments, the cells are homozygous for HLA-B and HLAC. In some embodiments, the cells elicit a reduced response from cd4+ T cells and elicit a reduced response from cd8+ T cells.
2. Exogenous nucleic acid
In some embodiments, the present disclosure provides methods and compositions for reducing or eliminating expression of MHC class II proteins on a cell surface by genetically modifying CIITA as disclosed herein, wherein the methods and compositions further provide for expression of exogenous nucleic acids by an engineered cell.
a) NK cell inhibitor gene insertion
In some embodiments, the present disclosure provides methods for reducing or eliminating expression of MHC class II proteins on the surface of a cell by genetically modifying CIITA as disclosed herein, wherein the methods further provide for expression of an exogenous nucleic acid by the cell, wherein the exogenous nucleic acid encodes an NK cell inhibitor molecule. In some embodiments, the NK cell inhibitor molecule is expressed on the cell surface, thereby avoiding NK cell activity (e.g., lysis of the cell by NK cells). In some embodiments, the ability of the genetically modified cells to avoid NK cell lysis enables the cells to undergo adoptive cell transfer therapy. In some embodiments, the cell is an allogeneic cell.
In some embodiments, the method comprises reducing or eliminating expression of MHC class II proteins on the surface of a cell, comprising genetically modifying CIITA, comprising contacting the cell with a composition comprising a CIITA guide RNA disclosed herein, the method further comprising contacting the cell with a nucleic acid encoding an NK cell inhibitor molecule. In some embodiments, the method comprises reducing or eliminating expression of MHC class II proteins on the surface of a cell comprising modifying CIITA, the method comprising contacting the cell with a composition comprising the disclosed CIITA guide RNAs, the method further comprising contacting the cell with a nucleic acid encoding an NK cell inhibitor molecule and B2M guide RNA, thereby reducing or eliminating expression of MHC class I proteins on the surface of the cell. In some embodiments, the method further comprises contacting the cell with an RNA-guided DNA binding agent.
In some embodiments, the method comprises reducing or eliminating expression of MHC class II proteins on the surface of a cell, comprising genetically modifying the cell with one or more compositions comprising: the CIITA guide RNAs, B2M guide RNAs, nucleic acids encoding NK cell inhibitor molecules and RNA-guided DNA binding agents or nucleic acids encoding RNA-guided DNA binding agents disclosed herein.
In some embodiments, the method comprises inducing DSB or SSB in CIITA, the method further comprising contacting the cell with a composition comprising a CIITA guide RNA disclosed herein, the method further comprising contacting the cell with a nucleic acid encoding an NK cell inhibitor molecule. In some embodiments, the method comprises inducing DSB or SSB in CIITA, the method comprising contacting the cell with a composition comprising a CIITA guide RNA disclosed herein, the method further comprising contacting the cell with a nucleic acid encoding an NK cell inhibitor molecule and B2M guide RNA, thereby reducing expression of MHC class I proteins on the cell surface. In some embodiments, the method further comprises contacting the cell with an RNA-guided DNA binding agent.
In some embodiments, the method comprises reducing or eliminating expression of a CIITA protein in a cell, the method comprising delivering a composition comprising a CIITA guide RNA disclosed herein, the method further comprising contacting the cell with a nucleic acid encoding an NK cell inhibitor molecule. In some embodiments, the method comprises reducing expression of CIITA protein in a cell, the method comprising delivering a composition comprising a CIITA guide RNA disclosed herein, the method further comprising contacting the cell with a nucleic acid encoding an NK cell inhibitor molecule and B2M guide RNA, thereby reducing expression of MHC class I protein on the cell surface. In some embodiments, the method further comprises contacting the cell with an RNA-guided DNA binding agent.
In some embodiments, the NK cell inhibitor molecule binds to an inhibitory receptor on an NK cell. In some embodiments, the NK cell inhibitor molecule binds to an inhibitory receptor specific for MHC class I. In some embodiments, the NK cell inhibitor molecule binds to an inhibitory receptor that is not specific for MHC class I. NK cell inhibitory receptors include, for example, KIR (human), CD94-NKG2A heterodimer (human/mouse), ly49 (mouse), 2B4, SLAMF6, NKFP-B, TIGIT, KIR2DL4.
In some embodiments, the NK cell inhibitor molecule binds NKG 2A.
In some embodiments, the NK cell inhibitor molecule is an MHC class I molecule. In some embodiments, the NK cell inhibitor molecule is a classical MHC class I molecule. In some embodiments, the NK cell inhibitor molecule is a non-classical MHC class I molecule. In some embodiments, the NK cell inhibitor molecule is an HLA molecule. NK cell inhibitor molecules include, for example, HLA-C, HLA-E, HLA-G, cd1, CD48, SLAMF6, clr-b and CD155.
In some embodiments, the NK cell inhibitor molecule is HLA-E.
In some embodiments, the NK cell inhibitor molecule is a fusion protein. In some embodiments, the NK cell inhibitor molecule is a fusion protein comprising HLA-E. In some embodiments, the NK cell inhibitor molecule comprises B2M. In some embodiments, the NK cell inhibitor molecule comprises HLA-E and B2M. In some embodiments, the fusion protein includes a linker. In some embodiments, the HLA-E construct is provided in a vector. In some embodiments, the vector comprising the HLA-E construct is a lentiviral vector. In some embodiments, the HLA-E construct is delivered to the cell via lentiviral transduction.
In some embodiments, the NK cell inhibitor molecule is inserted into the genome of the target cell. In some embodiments, the NK cell inhibitor molecule is integrated into the genome of the target cell. In some embodiments, the NK cell inhibitor molecule is integrated into the genome of the target cell by Homologous Recombination (HR). In some embodiments, the NK cell inhibitor molecule is integrated into the genome of the target cell by blunt-ended insertion. In some embodiments, the NK cell inhibitor molecule is integrated into the genome of the target cell by non-homologous end joining. In some embodiments, the NK cell inhibitor molecule is integrated into a safe harbor locus in the cell genome. In some embodiments, the NK cell inhibitor molecule is integrated into one of the TRAC locus, the B2M locus, the AAVS1 locus, and/or the CIITA locus. In some embodiments, the NK cell inhibitor molecule is provided to the cell in a lipid nucleic acid assembly composition. In some embodiments, the lipid nucleic acid assembly composition is a Lipid Nanoparticle (LNP).
In some embodiments, the method produces an engineered cell that elicits a reduced response from an NK cell. NK cell response can be assessed in vitro or in vivo. In some embodiments, NK cell activity can be assessed by co-culturing genetically modified cells with NK cells in vitro. In some embodiments, NK cell activity can be assessed in an in vivo model, e.g., a rodent model. In vivo models, for example, genetically modified cells can be administered with NK cells; survival of genetically modified cells indicates that NK cell lysis can be avoided. In some embodiments, the method produces a composition comprising cells that survive in vivo in the presence of NK cells for more than 1, 2, 3, 4, 5, or 6 weeks or more. In some embodiments, the method produces a composition comprising cells that survive in vivo in the presence of NK cells for at least one to six weeks. In some embodiments, the methods result in a composition comprising cells that survive in vivo in the presence of NK cells for at least two to four weeks. In some embodiments, the method produces a composition comprising cells that survive in vivo in the presence of NK cells for at least four to six weeks. In some embodiments, the method produces a composition comprising cells that survive in vivo in the presence of NK cells for more than six weeks.
In some embodiments, the methods result in compositions comprising engineered cells having reduced or eliminated MHC class II expression and comprising a nucleic acid encoding an NK cell inhibitor molecule. In some embodiments, the methods result in compositions comprising engineered cells having reduced or eliminated MHC class II expression and NK cell inhibitor molecule expression on the cell surface. In some embodiments, the methods result in a composition comprising cells having reduced or eliminated MHC class II expression and eliciting a reduced response from NK cells. In some embodiments, the methods result in a composition comprising a cell having reduced or eliminated MHC class II expression, reduced or eliminated CIITA protein expression and/or reduced or eliminated CIITA levels in the nucleus of the cell, and eliciting a reduced response from NK cells, and having reduced or eliminated MHC class I protein expression. In some embodiments, the cells elicit a reduced response from cd4+ T cells, cd8+ T cells, and/or NK cells.
In some embodiments, allogeneic cells are provided, wherein the cells have reduced or eliminated MHC class II and MHC class I protein expression on the cell surface, wherein the cells comprise a modification in CIITA as disclosed herein, wherein the cells comprise a genetic modification in B2M, and wherein the cells comprise a nucleic acid encoding an NK cell inhibitor molecule. In some embodiments, the allogeneic cells elicit a reduced response from cd4+ T cells, cd8+ T cells, and/or NK cells.
b) Polypeptides that target receptor and other cell surface expression; secretion polypeptides
In some embodiments, the disclosure provides methods of reducing or eliminating expression of MHC class II proteins on a cell surface by genetically modifying CIITA as disclosed herein, wherein the methods further provide for expression of one or more exogenous nucleic acids (e.g., antibodies, chimeric Antigen Receptors (CARs), T Cell Receptors (TCRs), cytokines or cytokine receptors, chemokines or chemokine receptors, enzymes, fusion proteins, or other types of cell surface binding or soluble polypeptides). In some embodiments, the exogenous nucleic acid encodes a protein expressed on the surface of the cell. For example, in some embodiments, the exogenous nucleic acid encodes a targeting receptor expressed on the surface of a cell (described further herein). In some embodiments, the genetically modified cells can serve as a "cell factory" for expressing secreted polypeptides encoded by exogenous nucleic acids, including, for example, as a source for continuous production of polypeptides in vivo (as further described herein). In some embodiments, the cell is an allogeneic cell.
In some embodiments, the method comprises reducing or eliminating expression of MHC class II proteins on the surface of a cell, comprising genetically modifying CIITA, comprising contacting the cell with a composition comprising a CIITA guide RNA as disclosed herein, the method further comprising contacting the cell with an exogenous nucleic acid. In some embodiments, the method comprises reducing or eliminating expression of MHC class II proteins on the surface of a cell, comprising genetically modifying CIITA, comprising contacting the cell with a composition comprising CIITA guide RNAs as disclosed herein, the method further comprising contacting the cell with exogenous nucleic acid and B2M guide RNA, thereby reducing or eliminating expression of MHC class I proteins on the surface of the cell. In some embodiments, the method comprises reducing or eliminating expression of MHC class II proteins on the surface of a cell, including genetically modifying a CIITA gene, comprising contacting the cell with a composition comprising a CIITA guide RNA as disclosed herein, the method further comprising contacting the cell with an exogenous nucleic acid, a cell surface expressed (e.g., a targeting receptor) polypeptide or a soluble (e.g., a secretion) polypeptide, and B2M guide RNA, thereby reducing or eliminating expression of MHC class I proteins on the surface of the cell. In some embodiments, the method comprises contacting the cell with more than one exogenous nucleic acid. In some embodiments, the method further comprises contacting the cell with an RNA-guided DNA binding agent.
In some embodiments, the method comprises reducing or eliminating expression of MHC class II proteins on the surface of a cell, comprising genetically modifying CIITA, comprising contacting the cell with a composition comprising a CIITA guide RNA as disclosed herein, the method further comprising contacting the cell with an exogenous nucleic acid. In some embodiments, the method comprises reducing or eliminating expression of MHC class II proteins on the surface of a cell, comprising genetically modifying CIITA, comprising contacting the cell with a composition comprising CIITA guide RNAs as disclosed herein, the method further comprising contacting the cell with an exogenous nucleic acid and HLA-A guide RNA, thereby reducing or eliminating expression of HLA-A proteins on the surface of the cell. In some embodiments, the method comprises reducing or eliminating expression of MHC class II proteins on the surface of a cell, including genetically modifying a CIITA gene, comprising contacting the cell with a composition comprising a CIITA guide RNA as disclosed herein, the method further comprising contacting the cell with an exogenous nucleic acid, a cell surface expressed (e.g., a targeting receptor) polypeptide or a soluble (e.g., a secretion) polypeptide, and HLA-A guide RNA, thereby reducing or eliminating expression of HLA-A protein on the cell surface. In some embodiments, the method comprises contacting the cell with more than one exogenous nucleic acid. In some embodiments, the method further comprises contacting the cell with an RNA-guided DNA binding agent.
In some embodiments, the method comprises reducing or eliminating expression of MHC class II proteins on the surface of a cell, comprising genetically modifying the cell with one or more compositions comprising: the CIITA guide RNAs, B2M guide RNAs, exogenous nucleic acids encoding NK cell inhibitor molecules, exogenous nucleic acids encoding polypeptides (e.g., targeting receptors) and RNA-guided DNA binding agents or nucleic acids encoding RNA-guided DNA binding agents disclosed herein.
In some embodiments, the method comprises reducing or eliminating expression of MHC class II proteins and MHC class I proteins on the surface of a cell, comprising genetically modifying the cell with one or more compositions comprising: the CIITA guide RNAs, B2M guide RNAs, exogenous nucleic acids encoding NK cell inhibitor molecules, exogenous nucleic acids encoding polypeptides (e.g., targeting receptors) and RNA-guided DNA binding agents or nucleic acids encoding RNA-guided DNA binding agents disclosed herein.
In some embodiments, the method comprises reducing or eliminating expression of MHC class II proteins and HLA-A proteins on the surface of a cell, comprising genetically modifying the cell with one or more compositions comprising: the CIITA guide RNAs, B2M guide RNAs, exogenous nucleic acids encoding polypeptides (e.g., targeting receptors) and RNA-guided DNA binding agents or nucleic acids encoding RNA-guided DNA binding agents disclosed herein.
In some embodiments, the exogenous nucleic acid encodes a polypeptide expressed on the surface of the cell. In some embodiments, the exogenous nucleic acid encodes a soluble polypeptide. As used herein, "soluble" polypeptide refers to a polypeptide secreted by a cell. In some embodiments, the soluble polypeptide is a therapeutic polypeptide. In some embodiments, the soluble polypeptide is an antibody. In some embodiments, the soluble polypeptide is an enzyme. In some embodiments, the soluble polypeptide is a cytokine. In some embodiments, the soluble polypeptide is a chemokine. In some embodiments, the soluble polypeptide is a fusion protein.
In some embodiments, the exogenous nucleic acid encodes an antibody. In some embodiments, the exogenous nucleic acid encodes an antibody fragment (e.g., fab 2). In some embodiments, the exogenous nucleic acid encodes a full-length antibody. In some embodiments, the exogenous nucleic acid encodes a single chain antibody (e.g., scFv). In some embodiments, the antibody is IgG, igM, igD, igA or IgE. In some embodiments, the antibody is an IgG antibody. In some embodiments, the antibody is an IgG1 antibody. In some embodiments, the antibody is an IgG4 antibody. In some embodiments, the heavy chain constant region contains mutations known to reduce effector function. In some embodiments, the heavy chain constant region contains mutations known to enhance effector function. In some embodiments, the antibody is a bispecific antibody. In some embodiments, the antibody is a single domain antibody (e.g., a VH domain-only antibody).
In some embodiments, the exogenous nucleic acid encodes a neutralizing antibody. Neutralizing antibodies neutralize the activity of their target antigens. In some embodiments, the antibody is a neutralizing antibody to a viral antigen. In some embodiments, the antibody neutralizes the target viral antigen, blocking the ability of the virus to infect the cell. In some embodimentsIn, cell-based neutralization assays can be used to measure the neutralizing activity of antibodies. The specific cells and readings will depend on the target antigen of the neutralizing antibody. Half maximal effective concentration of antibody (EC 50 ) Can be measured in a cell-based neutralization assay, wherein lower EC 50 Indicating a more effective neutralizing antibody.
In some embodiments, the exogenous nucleic acid encodes an antibody that binds to an antigen associated with a disease or disorder (see, e.g., the diseases and disorders described in section IV).
In some embodiments, the exogenous nucleic acid encodes a polypeptide expressed on the cell surface (i.e., a cell surface binding protein). In some embodiments, the exogenous nucleic acid encodes a targeted receptor. A "targeted receptor" is a receptor that is present on the surface of a cell (e.g., a T cell) to allow the cell to bind to a target site, such as a particular cell or tissue in an organism. In some embodiments, the targeting receptor is a CAR. In some embodiments, the targeted receptor is a universal CAR (UniCAR). In some embodiments, the targeted receptor is a TCR. In some embodiments, the targeting receptor is TRuC. In some embodiments, the targeting receptor is a B Cell Receptor (BCR) (e.g., expressed on B cells). In some embodiments, the targeting receptor is a chemokine receptor. In some embodiments, the targeting receptor is a cytokine receptor.
In some embodiments, the targeting receptor comprises a Chimeric Antigen Receptor (CAR), a T Cell Receptor (TCR), and a cell surface molecule receptor operably linked via at least one transmembrane domain in an internal signaling domain capable of activating T cells upon binding of an extracellular receptor moiety. In some embodiments, CAR refers to an extracellular antigen recognition domain, e.g., scFv, VHH, nanobody; operably linked to an intracellular signaling domain that activates T cells upon antigen binding. CAR consists of four regions: an antigen recognition domain, an extracellular hinge region, a transmembrane domain, and an intracellular T cell signaling domain. Such receptors are well known in the art (see e.g. WO2020092057, WO2019191114, WO2019147805, WO 2018208837). Inverse universal CARs that promote binding of immune cells to target cells via adapter molecules are also contemplated (see, e.g., WO 2019238722). The CAR can target any antigen that can produce antibodies and is generally directed against a molecule that is presented on the surface of the cell or tissue to be targeted. In some embodiments, the targeting receptor comprises an antigen recognition domain (e.g., a cancer antigen recognition domain and a subunit of a TCR (e.g., TRuC) (see Baeuerle et al Nature Communications2087 (2019)).
In some embodiments, the exogenous nucleic acid encodes a TCR. In some embodiments, the exogenous nucleic acid encodes a genetically modified TCR. In some embodiments, the exogenous nucleic acid encodes a genetically modified TCR that is specific for a polypeptide expressed by a cancer cell. In some embodiments, the exogenous nucleic acid encodes a targeting receptor specific for Wilms' tumor gene (WT 1) antigen. In some embodiments, the exogenous nucleic acid encodes a WT 1-specific TCR (see, e.g., WO2020/081613A 1).
In some embodiments, the exogenous nucleic acid is inserted into the genome of the target cell. In some embodiments, the exogenous nucleic acid is integrated into the genome of the target cell. In some embodiments, the exogenous nucleic acid is integrated into the genome of the target cell by Homologous Recombination (HR). In some embodiments, the exogenous nucleic acid is integrated into the genome of the target cell by blunt-ended insertion. In some embodiments, the exogenous nucleic acid is integrated into the genome of the target cell by non-homologous end joining. In some embodiments, the exogenous nucleic acid is integrated into a safe harbor locus in the cell genome. In some embodiments, the exogenous nucleic acid is integrated into one of the TRAC locus, the B2M locus, the AAVS1 locus, and/or the CIITA locus. In some embodiments, the exogenous nucleic acid is provided to the cell in a lipid nucleic acid assembly composition. In some embodiments, the lipid nucleic acid assembly composition is a Lipid Nanoparticle (LNP).
In some embodiments, the methods result in compositions comprising engineered cells having reduced or eliminated MHC class II expression and comprising exogenous nucleic acid. In some embodiments, the methods result in compositions comprising engineered cells having reduced or eliminated MHC class II expression and secreting and/or expressing a polypeptide encoded by an exogenous nucleic acid integrated into the genome of the cell. In some embodiments, the methods result in compositions comprising engineered cells having reduced or eliminated MHC class II protein expression, reduced or eliminated CIITA protein expression, and/or reduced or eliminated CIITA levels in the nucleus, and eliciting a reduced response from NK cells, and having reduced MHC class I protein expression, and secreting and/or expressing a polypeptide encoded by an exogenous nucleic acid integrated into the genome of the cell. In some embodiments, the engineered cells elicit a reduced response from cd4+ T cells, cd8+ T cells, and/or NK cells.
In some embodiments, an allogeneic cell is provided, wherein the cell has reduced or eliminated expression of MHC class II and MHC class I proteins on the cell surface, wherein the cell comprises a modification in CIITA as disclosed herein, wherein the cell comprises a modification in B2M, wherein the cell comprises an exogenous nucleic acid encoding an NK cell inhibitor molecule, and wherein the cell further comprises an exogenous nucleic acid encoding a polypeptide (e.g., a targeting receptor). In some embodiments, the allogeneic cells elicit a reduced response from cd4+ T cells, cd8+ T cells, and/or NK cells, and further secrete and/or express the therapeutic agent.
In some embodiments, an allogeneic cell is provided, wherein the cell has reduced or eliminated MHC class II and HLA-A protein expression on the cell surface, wherein the cell comprises a modification in CIITA as disclosed herein, wherein the cell comprises a modification in an HLA-A gene, and wherein the cell further comprises an exogenous nucleic acid encoding a polypeptide (e.g., a targeting receptor). In some embodiments, the allogeneic cells elicit a reduced response from cd4+ T cells and/or cd8+ T cells.
In some embodiments, the present disclosure provides methods for reducing or eliminating expression of MHC class II proteins on a cell surface by genetically modifying CIITA as disclosed herein, wherein the methods further provide for reducing expression of one or more additional target genes (e.g., TRAC, TRBC). In some embodiments, additional genetic modifications provide additional advantages in using genetically modified cells for adoptive cell transfer applications. In some embodiments, the cell is an allogeneic cell.
In some embodiments, the method comprises reducing or eliminating expression of MHC class II proteins on the surface of a cell, including genetically modifying CIITA, comprising contacting the cell with a composition comprising CIITA guide RNAs as disclosed herein, the method further comprising contacting the cell with a guide RNA that directs RNA-guided DNA binding agents to target sequences located in another gene (e.g., a gene other than CIITA or B2M or HLA-A), thereby reducing or eliminating expression of the other gene. In some embodiments, the method comprises reducing or eliminating expression of MHC class II proteins on the surface of a cell, comprising genetically modifying CIITA, comprising contacting the cell with a composition comprising CIITA guide RNAs as disclosed herein, the method further comprising contacting the cell with a guide RNA that directs RNA-guided DNA binding agents to target sequences located in another gene and B2M guide RNA, thereby reducing or eliminating expression of MHC class I proteins on the surface of the cell. In some embodiments, the methods comprise reducing or eliminating expression of MHC class II proteins on the surface of a cell, including genetically modifying CIITA, comprising contacting the cell with a composition comprising CIITA guide RNAs as disclosed herein, the method further comprising contacting the cell with a guide RNA that directs an RNA-guided DNA binding agent to a target sequence located in another gene (thereby reducing or eliminating expression of the other gene) and an exogenous nucleic acid encoding a polypeptide (e.g., a targeting receptor).
In some embodiments, the method comprises reducing or eliminating expression of MHC class II proteins on the surface of a cell, including genetically modifying CIITA, comprising contacting the cell with a composition comprising CIITA guide RNAs as disclosed herein, the method further comprising contacting the cell with a guide RNA that directs RNA-guided DNA binding agents to target sequences located in another gene (e.g., a gene other than CIITA or B2M or HLA-A), thereby reducing or eliminating expression of the other gene. In some embodiments, the method comprises reducing or eliminating expression of MHC class II proteins on the surface of a cell, comprising genetically modifying CIITA, comprising contacting the cell with a composition comprising CIITA guide RNAs as disclosed herein, the method further comprising contacting the cell with a guide RNA that directs RNA-guided DNA binding agents to target sequences located in another gene and HLA-A guide RNA, thereby reducing or eliminating expression of HLA-A proteins on the surface of the cell. In some embodiments, the methods comprise reducing or eliminating expression of MHC class II proteins on the surface of a cell, including genetically modifying CIITA, comprising contacting the cell with a composition comprising CIITA guide RNAs as disclosed herein, the method further comprising contacting the cell with a guide RNA that directs an RNA-guided DNA binding agent to a target sequence located in another gene (thereby reducing or eliminating expression of the other gene) and an exogenous nucleic acid encoding a polypeptide (e.g., a targeting receptor).
In some embodiments, the method comprises reducing or eliminating expression of MHC class II proteins on the surface of a cell, comprising genetically modifying CIITA, comprising contacting the cell with a composition comprising CIITA guide RNAs as disclosed herein, the method further comprising contacting the cell with a guide RNA that directs an RNA-guided DNA binding agent to a target sequence located in another gene (thereby reducing expression of the other gene), B2M guide RNA (thereby reducing expression of MHC class I proteins on the surface of the cell), and an exogenous nucleic acid encoding an NK cell inhibitor.
In some embodiments, the method comprises reducing or eliminating expression of MHC class II proteins on the surface of a cell, comprising genetically modifying CIITA, comprising contacting the cell with a composition comprising CIITA guide RNAs as disclosed herein, the method further comprising contacting the cell with a guide RNA that directs an RNA-guided DNA binding agent to a target sequence located in another gene (thereby reducing expression of the other gene) and an HLA-A guide RNA (thereby reducing expression of HLA-A protein on the surface of the cell).
In some embodiments, the method comprises reducing or eliminating expression of MHC class II proteins on the surface of a cell, including genetically modifying CIITA, comprising contacting the cell with a composition comprising CIITA guide RNAs as disclosed herein, the method further comprising contacting the cell with a guide RNA that directs an RNA-guided DNA binding agent to a target sequence located in another gene (thereby reducing or eliminating expression of the other gene), B2M guide RNA (thereby reducing or eliminating expression of MHC class I proteins on the surface of the cell), and an exogenous nucleic acid encoding a polypeptide (e.g., a targeting receptor).
In some embodiments, the method comprises reducing or eliminating expression of MHC class II proteins on the surface of a cell, including genetically modifying CIITA, comprising contacting the cell with a composition comprising CIITA guide RNAs as disclosed herein, the method further comprising contacting the cell with a guide RNA that directs RNA-guided DNA binding agents to target sequences located in another gene, an exogenous nucleic acid encoding an NK cell inhibitor molecule, and an exogenous nucleic acid encoding a polypeptide (e.g., a targeting receptor).
In some embodiments, the methods comprise reducing or eliminating expression of MHC class II proteins on the surface of a cell, including genetically modifying CIITA, comprising contacting the cell with a composition comprising CIITA guide RNAs as disclosed herein, the method further comprising contacting the cell with a guide RNA that directs an RNA-guided DNA binding agent to a target sequence located in another gene (thereby reducing expression of the other gene), and HLA-A guide RNA (thereby reducing expression of HLA-A protein on the surface of the cell) and an exogenous nucleic acid encoding a polypeptide (e.g., a targeting receptor).
In some embodiments, the method comprises reducing or eliminating expression of MHC class II proteins on the surface of a cell, including genetically modifying CIITA, comprising contacting the cell with a composition comprising CIITA guide RNAs as disclosed herein, the method further comprising contacting the cell with a guide RNA that directs an RNA-guided DNA binding agent to a target sequence located in another gene (thereby reducing or eliminating expression of the other gene), B2M guide RNA (thereby reducing or eliminating expression of MHC class I proteins on the surface of the cell), an exogenous nucleic acid encoding an inhibitor molecule of an NK cell, and an exogenous nucleic acid encoding a polypeptide (e.g., a targeting receptor). In some embodiments, the method further comprises contacting the cell with an RNA-guided DNA binding agent.
In some embodiments, the method comprises reducing or eliminating expression of MHC class II proteins on the surface of a cell, comprising genetically modifying the cell with one or more compositions comprising CIITA guide RNAs, B2M guide RNAs, exogenous nucleic acids encoding NK cell inhibitor molecules, exogenous nucleic acids encoding polypeptides (e.g., targeting receptors), guide RNAs that direct RNA-guided DNA binding agents to target sequences located in another gene (thereby reducing or eliminating expression of another gene), and RNA-guided DNA binding agents or nucleic acids encoding RNA-guided DNA binding agents as disclosed herein.
In some embodiments, the methods comprise reducing or eliminating expression of MHC class II proteins on the surface of a cell, comprising genetically modifying the cell with one or more compositions comprising CIITA guide RNAs as disclosed herein, HLA-A guide RNAs, exogenous nucleic acids encoding polypeptides (e.g., targeting receptors), guide RNAs that direct RNA-guided DNA binding agents to target sequences located in another gene (thereby reducing or eliminating expression of another gene), and RNA-guided DNA binding agents or nucleic acids encoding RNA-guided DNA binding agents.
In some embodiments, the additional target gene is TRAC. In some embodiments, the additional target gene is TRBC.
D. Exemplary cell types
In some embodiments, the methods and compositions disclosed herein genetically modify a cell. In some embodiments, the cell is an allogeneic cell. In some embodiments, the cell is a human cell. In some embodiments, the genetically modified cell is referred to as an engineered cell. Engineered cells refer to cells (or cell progeny) that comprise an engineered genetic modification, e.g., have been contacted with and genetically modified by a gene editing system. The terms "engineered cell" and "genetically modified cell" are used interchangeably throughout. The engineered cell may be any of the exemplary cell types disclosed herein.
In some embodiments, the cell is an immune cell. As used herein, "immune cells" refers to cells of the immune system, including, for example, lymphocytes (e.g., T cells, B cells,Natural killer cells ("NK cells" and NKT cells or iNKT cells)), monocytes, macrophages, mast cells, dendritic cells or granulocytes (e.g., neutrophils, eosinophils and basophils). In some embodiments, the cell is a primary immune cell. In some embodiments, the immune system cells may be selected from CD3 + 、CD4 + And CD8 + T cells, regulatory T cells (tregs), B cells, NK cells, and Dendritic Cells (DCs). In some embodiments, the immune cells are allogeneic.
In some embodiments, the cell is a lymphocyte. In some embodiments, the cell is an adaptive immune cell. In some embodiments, the cell is a T cell. In some embodiments, the cell is a B cell. In some embodiments, the cell is an NK cell. In some embodiments, the lymphocytes are allogeneic.
As used herein, a T cell may be defined as a cell that expresses a T cell receptor ("TCR" or "αβ TCR" or "γδ TCR"), however, in some embodiments, the TCR of the T cell may be genetically modified to reduce its expression (e.g., by genetic modification of the TRAC or TRBC gene), and thus expression of the protein CD3 may be used as a marker to identify T cells by standard flow cytometry methods. CD3 is a multiple subunit signaling complex associated with TCRs. Thus, T cells may be referred to as cd3+. In some embodiments, the T cell is a cell that expresses a cd3+ marker and a cd4+ or cd8+ marker. In some embodiments, the T cells are allogeneic.
In some embodiments, the T cells express glycoprotein CD8 and are therefore cd8+ according to standard flow cytometry methods, and may be referred to as "cytotoxic" T cells. In some embodiments, the T cells express glycoprotein CD4 and are thus cd4+ according to standard flow cytometry methods, and may be referred to as "helper" T cells. Cd4+ T cells can differentiate into subpopulations and may be referred to as Th1 cells, th2 cells, th9 cells, th17 cells, th22 cells, T regulatory ("Treg") cells, or T follicular helper cells ("Tfh"). Each cd4+ subpopulation releases specific cytokines that may have pro-inflammatory or anti-inflammatory functions, survival or protective functions. T cells can be isolated from the subject by cd4+ or cd8+ selection methods.
In some embodiments, the T cell is a memory T cell. In vivo, memory T cells encounter antigens. Memory T cells may be located in secondary lymphoid organs (central memory T cells) or recently infected tissues (effector memory T cells). The memory T cells may be cd8+ T cells. The memory T cells may be cd4+ T cells.
As used herein, "central memory T cells" may be defined as T cells that undergo antigen, and may express CD62L and CD45RO, for example. Central memory T cells can be detected as CD62L+ and CD45RO+ by central memory T cells that also express CCR7, and thus CCR7+ by standard flow cytometry methods.
As used herein, "early stem cell memory T cells" (or "Tscm") may be defined as T cells expressing CD27 and CD45RA, and thus cd27+ and cd45ra+ by standard flow cytometry methods. Tscm does not express CD45 isoform CD45RO, so if this isoform is stained by standard flow cytometry methods, tscm will be further CD45RO-. Thus, CD45RO-CD27+ cells are also early stem cell memory T cells. Tscm cells further express CD62L and CCR7 and thus can be detected as cd62l+ and ccr7+ by standard flow cytometry methods. Early stem cell memory T cells have been shown to be associated with increased persistence and therapeutic efficacy of cell therapy products.
In some embodiments, the cell is a B cell. As used herein, "B cells" may be defined as cells expressing CD19 and/or CD20, and/or B cell maturation antigen ("BCMA"), and thus B cells are cd19+, and/or cd20+, and/or bcma+ by standard flow cytometry methods. B cells were further negative for CD3 and CD56 by standard flow cytometry methods. The B cells may be plasma cells. The B cells may be memory B cells. The B cells may be untreated B cells. B cells may be igm+, or B cell receptors with class switching (e.g., igg+ or iga+). In some embodiments, the B cells are allogeneic.
In some embodiments, the cells are monocytes, such as from bone marrow or peripheral blood. In some embodiments, the cells are peripheral blood mononuclear cells ("PBMCs"). In some embodiments, the cells are PBMCs, such as lymphocytes or monocytes. In some embodiments, the cells are peripheral blood lymphocytes ("PBLs"). In some embodiments, the monocytes are allogeneic.
Including cells used in ACT and/or tissue regeneration therapies, such as stem cells, progenitor cells, and primary cells. For example, stem cells include Pluripotent Stem Cells (PSCs); induced pluripotent stem cells (ipscs); embryonic Stem Cells (ESCs); mesenchymal stem cells (MSCs, e.g., isolated from Bone Marrow (BM), peripheral Blood (PB), placenta, umbilical Cord (UC), or fat); hematopoietic stem cells (HSC; e.g., isolated from BM or UC); neural Stem Cells (NSCs); tissue-specific progenitor stem cells (tsscs); and Limbal Stem Cells (LSCs). Progenitor and primary cells include monocytes (MNCs, e.g., isolated from BM or PB); endothelial progenitor cells (EPC, e.g., isolated from BM, PB, and UC); neural Progenitor Cells (NPCs); and tissue-specific primary cells or cells derived Therefrom (TSCs), including chondrocytes, myocytes, and keratinocytes. Also included are cells for organ or tissue transplantation, such as islet cells, cardiomyocytes, thyroid cells, thymocytes, neuronal cells, skin cells and retinal cells.
In some embodiments, the cell is a human cell, such as a cell isolated from a human subject. In some embodiments, the cells are isolated from human donor PBMC or white blood cell harvest (leukopak). In some embodiments, the cell is from a subject having a condition, disorder, or disease. In some embodiments, the cells are from a human donor with an epstein-barr virus (Epstein Barr Virus, "EBV").
In some embodiments, the method is performed ex vivo. As used herein, "ex vivo" refers to an in vitro method in which cells are capable of being transferred into a subject, for example as ACT therapy. In some embodiments, the ex vivo method is an in vitro method involving ACT therapy cells or cell populations.
In some embodiments, the cell is from a cell line. In some embodiments, the cell line is derived from a human subject. In some embodiments, the cell line is a lymphoblastoid cell line ("LCL"). Cells can be cryopreserved and thawed. Cells may not have been previously cryopreserved.
In some embodiments, the cells are from a cell bank. In some embodiments, the cells are genetically modified and subsequently transferred to a cell bank. In some embodiments, the cells are removed from the subject, genetically modified ex vivo, and transferred to a cell bank. In some embodiments, the genetically modified cell population is transferred to a cell bank. In some embodiments, the genetically modified immune cell population is transferred to a cell bank. In some embodiments, the population of genetically modified immune cells comprises a first subpopulation and a second subpopulation, wherein the first subpopulation and the second subpopulation have at least one common genetic modification and at least one different genetic modification is transferred into a cell bank.
In some embodiments, when the cell is homozygous for HLA-B, the HLA-B allele is selected from any one of the following HLA-B alleles: HLA-B.times.07:02; HLA-B08:01; HLA-B44:02; HLA-B35:01; HLA-B40:01; HLA-B57:01; HLA-B14:02; HLA-B15:01; HLA-B13:02; HLA-B44:03; HLA-B38:01; HLA-B18:01; HLA-B44:03; HLA-B51:01; HLA-B49:01; HLA-B15:01; HLA-B18:01; HLA-B27:05; HLA-B35:03; HLA-B18:01; HLA-B52:01; HLA-B51:01; HLA-B37:01; HLA-B53:01; HLA-B55:01; HLA-B44:02; HLA-B44:03; HLA-B35:02; HLA-B15:01 and HLA-B40:02.
In some embodiments, when the cell is homozygous for HLA-C, the HLA-C allele is selected from any one of the following HLA-C alleles: HLA-C.times.07:02; HLA-C.times.07:01; HLA-C05:01; HLA-C.times.04:01HLA-C.times.03:04; HLA-C06:02; HLA-C08:02; HLA-C.times.03:03; HLA-C06:02; HLA-C16:01; HLA-C12:03; HLA-C.times.07:01; HLA-C04:01; HLA-C15:02; HLA-C.times.07:01; HLA-C03:04; HLA-C12:03; HLA-C02:02; HLA-C04:01; HLA-C05:01; HLA-C12:02; HLA-C14:02; HLA-C06:02; HLA-C04:01; HLA-C.times.03:03; HLA-C07:04; HLA-C.times.07:01; HLA-C04:01; HLA-C.times.04:01 and HLA-C.times.02:02.
In some embodiments, the cell is homozygous for HLA-B and homozygous for HLA-C, and the HLA-B allele is selected from any one of the following HLA-B alleles: HLA-B.times.07:02; HLA-B08:01; HLA-B44:02; HLA-B35:01; HLA-B40:01; HLA-B57:01; HLA-B14:02; HLA-B15:01; HLA-B13:02; HLA-B44:03; HLA-B38:01; HLA-B18:01; HLA-B44:03; HLA-B51:01; HLA-B49:01; HLA-B15:01; HLA-B18:01; HLA-B27:05; HLA-B35:03; HLA-B18:01; HLA-B52:01; HLA-B51:01; HLA-B37:01; HLA-B53:01; HLA-B55:01; HLA-B44:02; HLA-B44:03; HLA-B35:02; HLA-B15:01 and HLA-B40:02; and the HLA-C allele is selected from any one of the following HLA-C alleles: HLA-C.times.07:02; HLA-C.times.07:01; HLA-C05:01; HLA-C.times.04:01HLA-C.times.03:04; HLA-C06:02; HLA-C08:02; HLA-C.times.03:03; HLA-C06:02; HLA-C16:01; HLA-C12:03; HLA-C.times.07:01; HLA-C04:01; HLA-C15:02; HLA-C.times.07:01; HLA-C03:04; HLA-C12:03; HLA-C02:02; HLA-C04:01; HLA-C05:01; HLA-C12:02; HLA-C14:02; HLA-C06:02; HLA-C04:01; HLA-C.times.03:03; HLA-C07:04; HLA-C.times.07:01; HLA-C04:01; HLA-C.times.04:01 and HLA-C.times.02:02.
In some embodiments, the cells are homozygous for HLA-B and homozygous for HLA-C, and the HLA-B and HLA-C alleles are selected from any one of the following HLA-B and HLA-C alleles: HLA-B.times.07:02 and HLA-C.times.07:02; HLA-B.times.08:01 and HLA-C.times.07:01; HLA-B44:02 and HLA-C05:01; HLA-B35:01 and HLA-C04:01; HLA-B.times.40:01 and HLA-C.times.03:04; HLA-B57:01 and HLA-C06:02; HLA-B14:02 and HLA-C08:02; HLA-B15:01 and HLA-C03:03; HLA-B13:02 and HLA-C06:02; HLA-B44:03 and HLA-C16:01; HLA-B.times.38:01 and HLA-C.times.12:03; HLA-B18:01 and HLA-C07:01; HLA-B44:03 and HLA-C04:01; HLA-B.times.51:01 and HLA-C.times.15:02; HLA-B.times.49:01 and HLA-C.times.07:01; HLA-B15:01 and HLA-C03:04; HLA-B18:01 and HLA-C12:03; HLA-B.times.27:05 and HLA-C.times.02:02; HLA-B35:03 and HLA-C04:01; HLA-B18:01 and HLA-C05:01; HLA-B52:01 and HLA-C12:02; HLA-B.times.51:01 and HLA-C.times.14:02; HLA-B37:01 and HLA-C06:02; HLA-B53:01 and HLA-C04:01; HLA-B55:01 and HLA-C03:03; HLA-B44:02 and HLA-C07:04; HLA-B44:03 and HLA-C07:01; HLA-B35:02 and HLA-C04:01; HLA-B15:01 and HLA-C04:01; and HLA-B.times.40:02 and HLA-C.times.02:02. In some embodiments, the cells are homozygous for HLA-B and homozygous for HLA-C, and the HLA-B and HLA-C alleles are HLA-B.times.07:02 and HLA-C.times.07:02. In some embodiments, the cells are homozygous for HLA-B and homozygous for HLA-C, and the HLA-B and HLA-C alleles are HLA-B08:01 and HLA-C07:01. In some embodiments, the cells are homozygous for HLA-B and homozygous for HLA-C, and the HLA-B and HLA-C alleles are HLA-B44:02 and HLA-C05:01. In some embodiments, the cells are homozygous for HLA-B and homozygous for HLA-C, and the HLA-B and HLA-C alleles are HLA-B35:01 and HLA-C04:01.
Details of Gene editing System
Various suitable gene editing systems can be used to make the engineered cells disclosed herein, including but not limited to CRISPR/Cas systems; a Zinc Finger Nuclease (ZFN) system; and transcription activator-like effector nuclease (TALEN) systems. In general, gene editing systems involve the use of engineered cleavage systems to induce Double Strand Breaks (DSBs) or nicks (e.g., single strand breaks or SSBs) in a target DNA sequence. Cleavage or nicking can occur via the use of specific nucleases such as engineered ZFNs, TALENs, or using CRISPR/Cas systems with engineered guide RNAs to guide specific cleavage or nicking of target DNA sequences. Furthermore, targeting nucleases were developed based on the Algu system (Argonaute system) (e.g. from Thermus thermophilus (T. Thermophilus), called 'TtAgo', see Swarts et al (2014) Nature 507 (7491): 258-261), which may also have potential for use in gene editing and gene therapy.
In some embodiments, the gene editing system is a TALEN system. Transcription activator-like effector nucleases (TALENs) are restriction enzymes that can be engineered to cleave specific sequences of DNA. It is made by fusing TAL effector DNA binding domain with DNA cleavage domain (nuclease that cleaves DNA strands). Transcription activator-like effector (TALE) can be engineered to bind to a desired DNA sequence to promote DNA cleavage at a specific location (see, e.g., boch,2011,Nature Biotech). Restriction enzymes can be introduced into cells for gene editing or for in situ gene editing, a technique known as gene editing with engineered nucleases. Such methods and compositions for use therein are known in the art. See, e.g., WO2019147805, WO2014040370, WO2018073393, the contents of which are hereby incorporated in their entirety.
In some embodiments, the gene editing system is a zinc finger system. Zinc Finger Nucleases (ZFNs) are artificial restriction enzymes produced by fusing a zinc finger DNA binding domain to a DNA cleavage domain. The zinc finger domains can be engineered to target specific desired DNA sequences, thereby enabling zinc finger nucleases to target unique sequences within complex genomes. The non-specific cleavage domain from type II restriction endonuclease fokl is typically used as the cleavage domain in ZFNs. Cleavage is repaired by endogenous DNA repair mechanisms, allowing ZFNs to precisely alter the genome of higher organisms. Such methods and compositions for use therein are known in the art. See, for example, WO2011091324, the contents of which are hereby incorporated in their entirety.
In some embodiments, the gene editing system is a CRISPR/Cas system, including, for example, CRISPR guide RNAs comprising a guide sequence and an RNA-guided DNA binding agent, and is further described herein.
CRISPR guide RNA
Provided herein are guide sequences suitable for modifying a target sequence, for example, using guide RNAs and RNA-guided DNA binding agents (e.g., CRISPR/Cas systems) comprising the disclosed guide sequences.
Each of the guide sequences disclosed herein may further comprise additional nucleotides to form a crRNA, e.g., following the guide sequence at its 3' end with the following exemplary nucleotide sequences: GUUUUAGAGCUAUGCUGUUUUG in a 5 'to 3' orientation (SEQ ID NO: 170). In the case of sgrnas, the above guide sequences may further comprise additional nucleotides (scaffold sequences) to form the sgrnas, e.g., having the following exemplary nucleotide sequences after the 3' end of the guide sequences: GUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCACCGAGUCGGUGCUUUU (SEQ ID NO: 171) or GUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCACCGAGUCGGUGC (SEQ ID NO: 172), which is SEQ ID NO:171 without four terminal U, in a 5 'to 3' orientation. In some embodiments, the four terminal U of SEQ ID NO. 171 are absent. In some embodiments, only 1, 2 or 3 of the four ends U of SEQ ID NO. 171 are present.
In some embodiments, the sgrnas comprise any one of the guide sequences of SEQ ID NOs 1-117 and additional nucleotides to form a crRNA, e.g., having the following exemplary nucleotide sequences after the guide sequence at its 3' end: GUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUGGCACCGAGUCGGUGC in a 5 'to 3' orientation (SEQ ID NO: 173). SEQ ID NO. 173 reference wild type guide RNA: GUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCACCGAGUCGGUGC (SEQ ID NO: 172) lacks 8 nucleotides. Other exemplary scaffold nucleotide sequences are provided in table 4. In some embodiments, the sgRNA comprises any of the guide sequences and additional guide scaffold sequences of SEQ ID NOS: 1-117 in a 5 'to 3' orientation in Table 4, including modified versions of the scaffold sequences as shown.
In some embodiments, the guide RNA is an sgRNA comprising any of the sequences shown in Table 2 (SEQ ID NOS: 218-334 and 335-426). In some embodiments, the guide RNA is a chemically modified guide RNA. In some embodiments, the guide RNA is a chemically modified single guide RNA. The chemically modified guide RNAs may comprise one or more of the modifications as shown in table 2. The chemically modified guide RNA may comprise one or more of the modified nucleotides of any of SEQ ID NOS 1006, 1010-1012 and 1014-1017.
In some embodiments, the guide RNA is a sgRNA comprising any of SEQ ID NOS: 218-334 with at least one chemical modification as disclosed herein. In some embodiments, the guide RNA is a sgRNA comprising a sequence having at least 99%, 98%, 97%, 96%, 95%, 94%, 93%, 92%, 91% or 90% identity to any one of SEQ ID NOs 218-334 having at least one chemical modification disclosed herein.
In some embodiments, the guide RNA is a sgRNA comprising the modification pattern shown in SEQ ID NO 1016 or 1017. In some embodiments, the guide RNA is a sgRNA comprising a sequence having at least 99%, 98%, 97%, 96%, 95%, 94%, 93%, 92%, 91% or 90% identity to any one of the nucleic acids of SEQ ID NOS 335-426.
In some embodiments, the guide RNA comprises a sgRNA comprising the modification pattern shown in SEQ ID NO 1006. In some embodiments, the guide RNA comprises a sgRNA comprising a modified nucleotide of SEQ ID NO. 1006, including a guide sequence comprising a sequence selected from the group consisting of SEQ ID NO. 1-117. In some embodiments, the guide RNA is a sgRNA comprising the sequence SEQ ID NO. 1008 or a sequence having at least 99%, 98%, 97%, 96%, 95%, 94%, 93%, 92%, 91% or 90% identity to SEQ ID NO. 1008.
In some embodiments, the guide RNA is a single guide RNA comprising any of the sequences SEQ ID NOs 335-426 and 1008 or a sequence having at least 99%, 98%, 97%, 96%, 95%, 94%, 93%, 92%, 91% or 90% identity to any of the sequences SEQ ID NOs 335-426 and 1008. In some embodiments, the guide RNA is a single guide RNA comprising any of the sequences SEQ ID NOs 32, 64, 67, 68, 74, 76, 84, 86, 90, 91, and 115. In some embodiments, the guide RNA is a single guide RNA comprising any of the sequences SEQ ID NOs 341, 373, 376, 377, 383, 385, 393, 395, 399, 400, and 424 or a sequence having at least 99%, 98%, 97%, 96%, 95%, 94%, 93%, 92%, 91% or 90% identity to any of the sequences SEQ ID NOs 341, 373, 376, 377, 383, 385, 393, 395, 399, 400, and 424.
The guide RNA may further comprise trRNA. In each of the compositions and method embodiments described herein, crRNA and trRNA can be associated as a single RNA (sgRNA) or can be on separate RNAs (dgrnas). In the case of sgrnas, the crRNA and trRNA components may be covalently linked, for example, via phosphodiester bonds or other covalent bonds. In some embodiments, crRNA and/or trRNA sequences can be referred to as "scaffolds" or "conserved portions" of guide RNAs.
In each of the compositions, uses, and method embodiments described herein, the guide RNA can comprise two RNA molecules in the form of a "double guide RNA" or a "dgRNA. The dgRNA comprises a first RNA molecule comprising a crRNA comprising a guide sequence such as shown in table 2; and a second RNA molecule comprising trRNA. The first RNA molecule and the second RNA molecule may not be covalently linked, but may form an RNA duplex via base pairing between the crRNA and the portion of the trRNA.
In each of the compositions, uses, and method embodiments described herein, the guide RNA can comprise a single RNA molecule in the form of a "single guide RNA" or "sgRNA. The sgrnas may comprise crrnas (or portions thereof) covalently linked to trrnas, which comprise the guide sequences shown in table 2. The sgrnas may comprise 17, 18, 19 or 20 consecutive nucleotides of the guide sequences shown in table 2. In some embodiments, the crRNA and trRNA are covalently linked via a linker. In some embodiments, the sgrnas form a stem-loop structure via base pairing between the crrnas and portions of the trrnas. In some embodiments, the crRNA and trRNA are covalently linked via one or more linkages that are not phosphodiester linkages.
In some embodiments, the trRNA can comprise all or a portion of the trRNA sequence derived from a naturally occurring CRISPR/Cas system. In some embodiments, the trRNA comprises a truncated or modified wild-type trRNA. the length of the trRNA depends on the CRISPR/Cas system used. In some embodiments, the trRNA comprises or consists of: 5. 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 40, 50, 60, 70, 80, 90, 100, or more than 100 nucleotides. In some embodiments, the trRNA may comprise certain secondary structures, such as one or more hairpin structures or stem-loop structures or one or more carina structures.
In some embodiments, a composition is provided comprising one or more guide RNAs comprising any of the guide sequences in table 2. In some embodiments, a composition is provided comprising one or more guide RNAs comprising any of the guide sequences in table 2, wherein the nucleotide of SEQ ID NO 170, 171, 172 or 173 follows the guide sequence at its 3' end. In some embodiments, one or more guide RNAs comprising any of the guide sequences in Table 2 are modified according to the modification pattern of any of SEQ ID NOS 1006, 1010-1012 and 1014-1017, wherein the nucleotide of SEQ ID NOS 170, 171, 172 or 173 follows the guide sequence at its 3' end.
In some embodiments, a composition is provided comprising one or more guide RNAs comprising any of the guide sequences in table 2. In one aspect, a composition comprising one or more gRNAs is provided that comprises a guide sequence that is at least 99%, 98%, 97%, 96%, 95%, 94%, 93%, 92%, 91% or 90% identical to any of the nucleic acids of SEQ ID NOS.1-117.
In other embodiments, a composition is provided that comprises at least one, e.g., at least two, grnas comprising a guide sequence selected from any two or more of the guide sequences shown in table 2. In some embodiments, the composition comprises at least two grnas, each comprising a guide sequence having at least 99%, 98%, 97%, 96%, 95%, 94%, 93%, 92%, 91%, or 90% identity to any of the guide sequences shown in table 2.
In some embodiments, the guide RNA compositions of the invention are designed to recognize (e.g., hybridize to) a target sequence in CIITA. For example, the CIITA target sequence can be recognized and cleaved by a provided Cas lyase comprising guide RNAs. In some embodiments, the RNA-guided DNA binding agent, such as Cas lyase, can be directed to a target sequence in CIITA by a guide RNA, wherein the guide sequence of the guide RNA hybridizes to the target sequence, and the RNA-guided DNA binding agent, such as Cas lyase, cleaves the target sequence.
In some embodiments, the selection of one or more guide RNAs is determined based on a target sequence within CIITA. In some embodiments, the composition comprising one or more guide sequences comprises a guide sequence that is complementary to a corresponding genomic region shown in table 2 according to coordinates from a reference genome hg 38. The leader sequence of other embodiments may be complementary to any of the sequences listed in table 2 immediately adjacent to the genomic coordinates within CIITA. For example, the leader sequence of other embodiments may be complementary to a sequence of 10 contiguous nucleotides ± 10 nucleotides comprising the genomic coordinates listed in table 2.
Without being bound by any particular theory, modifications in certain regions of the target gene (e.g., frame shift mutations resulting from insertion/deletion due to nuclease-mediated DSBs) may be more difficult to tolerate than mutations in other regions, and thus the location of the DSBs is an important factor that may cause a reduction in the amount or type of protein. In some embodiments, grnas that are complementary or have complementarity to target sequences within a target gene are used to direct RNA-guided DNA binding agents to specific locations in the target gene.
In some embodiments, the guide sequence has at least 99%, 98%, 97%, 96%, 95%, 94%, 93%, 92%, 91%, 90%, 85% or 80% identity to a target sequence present in a target gene. In some embodiments, the targeting sequence has at least 99%, 98%, 97%, 96%, 95%, 94%, 93%, 92%, 91%, 90%, 85% or 80% identity to a target sequence present in a human CIITA gene.
In some embodiments, the target sequence may be complementary to a guide sequence of a guide RNA. In some embodiments, the degree of complementarity or identity between the guide sequence of the guide RNA and its corresponding target sequence may be at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100%. In some embodiments, the target sequence and the guide sequence of the gRNA may be 100% complementary or identical. In other embodiments, the target sequence and the guide sequence of the gRNA may contain at least one mismatch. For example, the target sequence and the guide sequence of a gRNA may contain 1, 2, 3, or 4 mismatches, wherein the total length of the guide sequence is 20. In some embodiments, the target sequence and the guide sequence of the gRNA may contain 1-4 mismatches, wherein the guide sequence is 20 nucleotides.
In some embodiments, the compositions or formulations disclosed herein comprise mRNA comprising an Open Reading Frame (ORF) encoding an RNA-guided DNA binding agent, such as a Cas nuclease as described herein. In some embodiments, mRNA is provided, used, or administered comprising an ORF encoding an RNA-guided DNA binding agent such as a Cas nuclease.
Modification of gRNA
In some embodiments, the gRNA (e.g., sgRNA, short sgRNA, dgRNA, or crRNA) is modified. In the context of the grnas described herein, the term "modified" or "modification" includes modifications described above, including, for example, (a) terminal modifications, such as 5 'terminal modifications or 3' terminal modifications, including 5 'or 3' protected terminal modifications; (b) Nucleobase (or "base") modification, including base substitution or removal; (c) Sugar modifications, including modifications at the 2', 3' and/or 4' positions; (d) internucleoside linkage modification; and (e) backbone modifications, which may include phosphodiester linkages and/or ribose modifications or substitutions. Modifications of the nucleotide at a given position include modifications or substitutions of phosphodiester linkages immediately 3' of the sugar of the nucleotide. Thus, for example, a nucleic acid comprising a phosphorothioate between a first sugar and a second sugar at the 5' end is considered to comprise a modification at position 1. The term "modified gRNA" generally refers to a gRNA that has modifications to the chemical structure of one or more of the base, sugar, and phosphodiester linkages, or backbone moieties (including nucleotide phosphates), all as detailed and exemplified herein.
Other descriptions and exemplary modification modes are provided in table 1 of WO2019/237069, published 12 months 12 in 2019, the entire contents of which are incorporated herein by reference.
In some embodiments, the gRNA comprises modifications at 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16 or more YA sites. In some embodiments, the pyrimidine of the YA site comprises a modification (including modifications that alter internucleoside linkages of the sugar immediately following the pyrimidine). In some embodiments, the adenine of the YA site comprises a modification (including a modification that alters internucleoside linkages of the sugar immediately following the adenine). In some embodiments, the pyrimidine and adenine of the YA site comprise modifications, such as sugar, base, or internucleoside linkage modifications. The YA modification may be any type of modification set forth herein. In some embodiments, the YA modification comprises one or more of phosphorothioate, 2'-OMe, or 2' -fluoro. In some embodiments, the YA modification comprises a pyrimidine modification comprising one or more of phosphorothioate, 2' -OMe, 2' -H, inosine, or 2' -fluoro. In some embodiments, the YA modification comprises a bicyclic ribose analog (e.g., LNA, BNA, or ENA) within the RNA duplex region containing one or more YA sites. In some embodiments, the YA modification comprises a bicyclic ribose analog (e.g., LNA, BNA, or ENA) within the RNA duplex region containing the YA site, wherein the YA modification is distal to the YA site.
In some embodiments, the guide sequence (or guide region) of the gRNA comprises 1, 2, 3, 4, 5, or more YA sites that can comprise a YA modification ("guide region YA sites"). In some embodiments, one or more YA sites located 5, 6, 7, 8, 9, or 10 of the 5 'end relative to the 5' end (where "5" etc. refers to position 5 relative to the 3 'end of the guide region, i.e., the nucleotide closest to 3' in the guide region) comprise a YA modification. The modified leader YA site comprises a YA modification.
In some embodiments, the modified leader YA site is located within 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, or 9 nucleotides of the 3' terminal nucleotide of the leader. For example, if the modified leader YA site is located within 10 nucleotides of the 3' terminal nucleotide of the leader and the leader has a length of 20 nucleotides, then the modified nucleotide of the modified leader YA site is located at any of positions 11 to 20. In some embodiments, the modified leader YA site is located at or after nucleotide 4, 5, 6, 7, 8, 9, 10, or 11 of the 5 'end relative to the 5' end.
In some embodiments, the modified leader YA site is different from the 5' modification. For example, the sgrnas can comprise a 5' modification as described herein and further comprise a modified guide region YA site. Alternatively, the sgRNA may comprise an unmodified 5' end and a modified leader YA site. Alternatively, the short sgrnas may comprise a modified 5' end and an unmodified leader YA site.
In some embodiments, the modified leader YA site comprises a modification that is not comprised by at least one nucleotide 5' to the leader YA site. For example, if nucleotides 1 to 3 comprise phosphorothioates, nucleotide 4 comprises only a 2'-OMe modification, and nucleotide 5 is a pyrimido comprising phosphorothioate of the YA site, the modified guide region YA site comprises a modification (phosphorothioate) not comprised by at least one nucleotide (nucleotide 4) located 5' to the guide region YA site. In another example, if nucleotides 1 to 3 comprise phosphorothioates and nucleotide 4 is a pyrimidine of the YA site and comprises a 2' -OMe, the modified guide region YA site comprises a modification (2 ' -OMe) that is not comprised by at least one nucleotide (any of nucleotides 1 to 3) located 5' to the guide region YA site. This condition is also always met if the unmodified nucleotide is located 5' to the modified leader YA site.
In some embodiments, the modified leader YA site comprises a modification as described above for the YA site. The guide region of the gRNA can be modified according to any embodiment, including modified guide regions set forth herein. Any of the embodiments set forth elsewhere in this disclosure may be combined with any of the preceding embodiments, where applicable.
In some embodiments, the 5 'and/or 3' end regions of the gRNA are modified.
In some embodiments, the terminal (i.e., last) 1, 2, 3, 4, 5, 6, or 7 nucleotides in the 3' terminal region are modified. Throughout, this modification may be referred to as a "3' end modification". In some embodiments, the terminal (i.e., last) 1, 2, 3, 4, 5, 6, or 7 nucleotides in the 3' terminal region comprise more than one modification. In some embodiments, the 3' end modification comprises or further comprises any one or more of the following: a modified nucleotide selected from the group consisting of a 2 '-O-methyl (2' -O-Me) modified nucleotide, a 2'-O- (2-methoxyethyl) (2' -O-moe) modified nucleotide, a 2 '-fluoro (2' -F) modified nucleotide, a Phosphorothioate (PS) linkage between nucleotides, an inverted abasic modified nucleotide, or a combination thereof. In some embodiments, the 3 'modification comprises or further comprises a modification of 1, 2, 3, 4, 5, 6, or 7 nucleotides of the 3' end of the gRNA. In some embodiments, the 3' modification comprises or further comprises a PS linkage, wherein the linkage is between the last nucleotide and the penultimate nucleotide. In some embodiments, the 3' modification comprises or further comprises two PS linkages between the last three nucleotides. In some embodiments, the 3' modification includes or further includes four PS linkages between the last four nucleotides. In some embodiments, the 3' terminal modification comprises or further comprises a PS linkage between any one or more of the last 2, 3, 4, 5, 6, or 7 nucleotides. In some embodiments, a gRNA comprising a 3 'end modification comprises or further comprises a 3' tail, wherein the 3 'tail comprises modifications of any one or more nucleotides present in the 3' tail. In some embodiments, the 3' tail is fully modified. In some embodiments, the 3' tail comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 1-2, 1-3, 1-4, 1-5, 1-6, 1-7, 1-8, 1-9, or 1-10 nucleotides, optionally wherein any one or more of these nucleotides is modified. In some embodiments, grnas comprising a 3' protection end modification are provided. In some embodiments, the 3' tail comprises 1 to about 20 nucleotides, 1 to about 15 nucleotides, 1 to about 10 nucleotides, 1 to about 5 nucleotides, 1 to about 4 nucleotides, 1 to about 3 nucleotides, and 1 to about 2 nucleotides. In some embodiments, the gRNA does not comprise a 3' tail.
In some embodiments, the 5' terminal region is modified, e.g., the first 1, 2, 3, 4, 5, 6, or 7 nucleotides in the gRNA are modified. Throughout, this modification may be referred to as a "5' modification". In some embodiments, the first 1, 2, 3, 4, 5, 6, or 7 nucleotides in the 5' terminal region comprise more than one modification. In some embodiments, at least one of the terminal (i.e., pre) 1, 2, 3, 4, 5, 6, or 7 nucleotides of the 5' end is modified. In some embodiments, both the 5 'and 3' end regions (e.g., ends) of the gRNA are modified. In some embodiments, only the 5' end region of the gRNA is modified. In some embodiments, only the 3 'terminal region (plus or minus the 3' tail) of the conserved portion of the gRNA is modified. In some embodiments, the gRNA comprises a modification at 1, 2, 3, 4, 5, 6, or 7 of the first 7 nucleotides of the 5' end region of the gRNA. In some embodiments, the gRNA comprises modifications at 1, 2, 3, 4, 5, 6, or 7 of the 7 terminal nucleotides of the 3' terminal region. In some embodiments, 2, 3, or 4 of the first 4 nucleotides of the 5 'terminal region, and/or 2, 3, or 4 of the terminal 4 nucleotides of the 3' terminal region are modified. In some embodiments, 2, 3, or 4 of the first 4 nucleotides of the 5' terminal region are linked via Phosphorothioate (PS) linkages. In some embodiments, the modification to the 5 'end and/or the 3' end comprises a 2 '-O-methyl (2' -O-Me) or 2'-O- (2-methoxyethyl) (2' -O-moe) modification. In some embodiments, the modification comprises a 2 '-fluoro (2' -F) modification of the nucleotide. In some embodiments, the modification comprises Phosphorothioate (PS) linkage between nucleotides. In some embodiments, the modification comprises inverting the abasic nucleotide. In some embodiments, the modification comprises a protecting end modification. In some embodiments, the modification comprises more than one modification selected from the group consisting of a protecting terminal modification, 2'-O-Me, 2' -O-moe, 2 '-fluoro (2' -F), phosphorothioate (PS) linkage between nucleotides, and inverted abasic nucleotides. In some embodiments, equivalent modifications are contemplated.
In some embodiments, grnas comprising a 5 'end modification and a 3' end modification are provided. In some embodiments, the gRNA comprises modified nucleotides that are not at the 5 'or 3' end.
In some embodiments, there is provided an sgRNA comprising a stem modification, wherein the stem modification comprises a modification to any one or more of US1 to US12 in the stem region. In some embodiments, there is provided an sgRNA comprising a stem modification, wherein the stem modification comprises a modification to at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or all 12 nucleotides in a stem region. In some embodiments, there is provided an sgRNA comprising a stem-up modification, wherein the stem-up modification comprises 1, 2, 3, 4, or 5 YA modifications in the YA site. In some embodiments, the upper stem modification comprises a 2' -OMe modified nucleotide, a 2' -O-moe modified nucleotide, a 2' -F modified nucleotide, and/or combinations thereof. Other modifications described herein, such as 5 'terminal modifications and/or 3' terminal modifications, may be combined with the upper stem modifications.
In some embodiments, the sgrnas comprise modifications in the hairpin region. In some embodiments, the hairpin region modification comprises at least one modified nucleotide selected from the group consisting of a 2 '-O-methyl (2' -OMe) modified nucleotide, a 2 '-fluoro (2' -F) modified nucleotide, and/or a combination thereof. In some embodiments, the hairpin region modification is in the hairpin 1 region. In some embodiments, the hairpin region modification is in the hairpin 2 region. In some embodiments, the hairpin modification comprises 1, 2, or 3 YA modifications in the YA site. In some embodiments, the hairpin modification comprises at least 1, 2, 3, 4, 5, or 6 YA modifications. Other modifications described herein, such as upper stem modifications, 5 'terminal modifications, and/or 3' terminal modifications, may be combined with modifications in the hairpin region.
In some embodiments, the gRNA comprises a substituted and optionally shortened hairpin 1 region, wherein at least one of the following nucleotide pairs is substituted in the substituted and optionally shortened hairpin 1 with a Watson-Crick pairing (Watson-Crick pairing) nucleotide: h1-1 and H1-12, H1-2 and H1-11, H1-3 and H1-10 and/or H1-4 and H1-9. "Watson-Crick paired nucleotides" include any pair capable of forming Watson-Crick base pairs, including A-T, A-U, T-A, U-A, C-G and G-C pairs, and pairs including modified versions of any of the foregoing nucleotides having the same base pairing preference. In some embodiments, hairpin 1 region lacks any one or both of H1-5 to H1-8. In some embodiments, the hairpin 1 region lacks one, two, or three of the following nucleotide pairs: h1-1 and H1-12, H1-2 and H1-11, H1-3 and H1-10 and/or H1-4 and H1-9. In some embodiments, the hairpin 1 region lacks 1 to 8 nucleotides of the hairpin 1 region. In any of the foregoing embodiments, the lack of nucleotides can allow one or more nucleotide pairs substituted with Watson-Crick paired nucleotides (H1-1 and H1-12, H1-2 and H1-11, H1-3 and H1-10, and/or H1-4 and H1-9) to form base pairs in the gRNA.
In some embodiments, the gRNA further comprises a reduced upper stem region that lacks at least 1 nucleotide, such as any of the reduced upper stem regions indicated in table 7 of U.S. application No. 62/946,905, the contents of which are incorporated herein by reference in their entirety or elsewhere described herein, which upper stem region can be combined with any of the reduced or substituted hairpin 1 regions described herein.
In some embodiments, the sgrnas provided herein are short single guide RNAs (short sgrnas), e.g., comprise a conserved portion of the sgrnas that comprise a hairpin region, wherein the hairpin region lacks at least 5 to 10 nucleotides or 6 to 10 nucleotides. In some embodiments, 5 to 10 nucleotides or 6 to 10 nucleotides are contiguous.
In some embodiments, the short sgrnas lack at least nucleotides 54 to 58 (AAAAA) of the conserved portion of spyCas9 sgrnas. In some embodiments, the short sgrnas are non-spyCas 9 sgrnas lacking nucleotides corresponding to nucleotides 54 to 58 (AAAAA) of the conserved portion of spyCas9, as determined by, for example, pairwise or structural alignment.
In some embodiments, the short sgrnas described herein comprise a conserved portion comprising a hairpin region, wherein the hairpin region lacks 5, 6, 7, 8, 9, 10, 11, or 12 nucleotides. In some embodiments, the absent nucleotides are 5 to 10 absent nucleotides or 6 to 10 absent nucleotides. In some embodiments, the absent nucleotides are contiguous. In some embodiments, the lack of nucleotides spans at least a portion of hairpin 1 and a portion of hairpin 2. In some embodiments, 5 to 10 of the lacking nucleotides comprise or consist of nucleotides 54 to 58, 54 to 61 or 53 to 60 of SEQ ID NO. 172.
In some embodiments, the short sgrnas described herein further comprise a junction region, wherein the junction region lacks at least one nucleotide (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 nucleotides in the junction region). In some embodiments, the short sgrnas lack each nucleotide in the junction region.
In some embodiments, the SpyCas9 short sgRNA described herein comprises the sequence NNNNNNNNNNNNNNNNNNNNGUUUUAGAGCUAGAAAUAGCAAGUUAA AAUAAGGCUAGUCCGUUAUCACGAAAGGGCACCGAGUCGGUGCU (SEQ ID NO: 1005).
In some embodiments, the short sgrnas described herein comprise a modification pattern as shown in mN NNNNN NNNNNNNNNNNNGUUUUAGAmGmCmUmAmGmAmAmAmUmAmGmCAA GUUAAAAUAAGGCUAGUCCGUUAUCACGAAAGGGCACCGAGUCGGmUm GmC mU (SEQ ID NO: 1006), wherein A, C, G, U and N are adenine, cytosine, guanine, uracil, and any ribonucleotide, respectively, unless otherwise specified. m indicates a 2' o-methyl modification, and indicates phosphorothioate linkage between nucleotides.
In certain embodiments, using SEQ ID No. 172 ("exemplary SpyCas9 sgRNA-1") as an example, exemplary SpyCas9 sgRNA-1 further comprises one or more of:
A. A shortened hairpin 1 region or a substituted and optionally shortened hairpin 1 region, wherein
1. At least one of the following nucleotide pairs was Watson-Crick paired nucleotide substitutions in hairpin 1: h1-1 and H1-12, H1-2 and H1-11, H1-3 and H1-10, and/or H1-4 and H1-9, and hairpin 1 region is optionally absent
any one or both of H1-5 to H1-8,
b. one, two or three of the following nucleotide pairs: h1-1 and H1-12, H1-2 and H1-11, H1-3 and H1-10, and H1-4 and H1-9, or
c. 1 to 8 nucleotides of hairpin 1 region; or (b)
2. The shortened hairpin 1 region lacks 6 to 8 nucleotides, preferably 6 nucleotides; and
a. one or more of positions H1-1, H1-2 or H1-3 are deleted or substituted with respect to the exemplary SpyCas9sgRNA-1 (SEQ ID NO: 172), or
b. One or more of positions H1-6 to H1-10 are substituted relative to the exemplary SpyCas9sgRNA-1 (SEQ ID NO: 172); or (b)
3. The shortened hairpin 1 region lacks 5 to 10 nucleotides, preferably 5 to 6 nucleotides, and one or more of positions N18, H1-12 or N is substituted relative to exemplary SpyCas9sgRNA-1 (SEQ ID NO: 172); or (b)
B. A shortened upper stem region, wherein the shortened upper stem region lacks 1 to 6 nucleotides and wherein 6, 7, 8, 9, 10, or 11 nucleotides of the shortened upper stem region comprise less than or equal to 4 substitutions relative to exemplary SpyCas9sgRNA-1 (SEQ ID NO: 172); or (b)
C. Substitution at any one or more of LS6, LS7, US3, US10, B3, N7, N15, N17, H2-2, and H2-14 relative to exemplary SpyCas9 sgRNA-1 (SEQ ID NO: 172), wherein the substituent nucleotide is neither pyrimidine followed by adenine, nor adenine preceding the pyrimidine; or (b)
D. Exemplary SpyCas9 sgRNA-1 (SEQ ID NO: 172) having an upper stem region, wherein the upper stem modification comprises modification of any one or more of US1 to US12 in the upper stem region, wherein
1. The modified nucleotide is optionally selected from a 2 '-O-methyl (2' -O-Me) modified nucleotide, a 2'-O- (2-methoxyethyl) (2' -O-moe) modified nucleotide, a 2 '-fluoro (2' -F) modified nucleotide, phosphorothioate (PS) linkages between nucleotides, inverted abasic modified nucleotides, or a combination thereof; or (b)
2. The modified nucleotides optionally include 2' -OMe modified nucleotides.
In certain embodiments, the exemplary SpyCas9 sgRNA-1 or sgrnas (such as the sgrnas comprising the exemplary SpyCas9 sgrnas-1) further comprise a 3 'tail, e.g., a 3' tail of 1, 2, 3, 4, or more nucleotides. In certain embodiments, the tail comprises one or more modified nucleotides. In certain embodiments, the modified nucleotide is selected from the group consisting of a 2 '-O-methyl (2' -O-Me) modified nucleotide, a 2'-O- (2-methoxyethyl) (2' -O-moe) modified nucleotide, a 2 '-fluoro (2' -F) modified nucleotide, phosphorothioate (PS) linkages between nucleotides, inverted abasic modified nucleotides, or a combination thereof. In certain embodiments, the modified nucleotide comprises a 2' -OMe modified nucleotide. In certain embodiments, the modified nucleotides comprise PS linkages between nucleotides. In certain embodiments, the modified nucleotide comprises a 2' -OMe modified nucleotide and a PS linkage between nucleotides.
In some embodiments, the grnas described herein further comprise a junction region (nexus region), wherein the junction region lacks at least one nucleotide.
In some embodiments, the gRNA is chemically modified. A gRNA comprising one or more modified nucleosides or nucleotides is referred to as a "modified" gRNA or a "chemically modified" gRNA, used to describe the presence of one or more non-natural and/or naturally occurring components or configurations used in place of or in addition to the typical A, G, C and U residues. The modified nucleosides and nucleotides can include one or more of the following: (i) Alterations, such as substitutions (exemplary backbone modifications), of one or both of the unbound oxygens in the phosphodiester backbone linkages and/or one or more of the bound oxygens; (ii) Modification, e.g., substitution (exemplary sugar modification), of the ribose component, e.g., the 2' hydroxyl group on ribose; (iii) Batch displacement of the phosphate moiety with a "dephosphorylation" linker (exemplary backbone modification); (iv) Modification or substitution of naturally occurring nucleobases, including the use of atypical nucleobases (exemplary base modifications); (v) Substitution or modification of the ribose-phosphate backbone (exemplary backbone modifications); (vi) Modification of the 3 'or 5' end of the oligonucleotide, such as removal, modification or substitution of a terminal phosphate group, or binding of a moiety, cap or linker (such 3 'or 5' cap modification may comprise sugar and/or backbone modification); and (vii) modification or substitution of sugar (exemplary sugar modifications).
Chemical modifications such as those listed above can be combined to yield modified grnas comprising nucleosides and nucleotides (collectively "residues") that can have two, three, four, or more modifications. For example, modified residues may have modified sugars and modified nucleobases. In some embodiments, each base of the gRNA is modified, e.g., all bases have a modified phosphate group such as a phosphorothioate group. In certain embodiments, all or substantially all of the phosphate groups of the gRNA molecule are replaced with phosphorothioate groups. In some embodiments, the modified gRNA comprises at least one modified residue at or near the 5' end of the RNA. In some embodiments, the modified gRNA comprises at least one modified residue at or near the 3' end of the RNA.
In some embodiments, the gRNA comprises one, two, three, or more modified residues. In some embodiments, at least 5% (e.g., at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or 100%) of the positions in the modified gRNA are modified nucleosides or nucleotides.
In some embodiments of backbone modification, the phosphate group of the modified residue may be modified by replacing one or more oxygens with different substituents. Furthermore, modified residues, such as those present in modified nucleic acids, may include bulk substitution of the unmodified phosphate moiety with a modified phosphate group as described herein. In some embodiments, backbone modification of the phosphate backbone may include creating a change in uncharged linkers or charged linkers with asymmetric charge distribution.
Examples of modified phosphate groups include phosphorothioates, phosphoroselenos, boranophosphates (borono phosphates), boranophosphates (borano phosphate ester), hydrogen phosphonates, phosphoramidates, alkyl or aryl phosphonates and phosphotriesters.
Scaffolds that can mimic nucleic acids can also be constructed in which the phosphate linker and ribo are replaced with nuclease resistant nucleosides or nucleotide substitutes. Such modifications may include backbone modifications and sugar modifications. In some embodiments, nucleobases can be tethered by a surrogate backbone. Examples may include, but are not limited to, morpholino, cyclobutyl, pyrrolidine, and Peptide Nucleic Acid (PNA) nucleoside substitutes.
Modified nucleosides and modified nucleotides can include one or more modifications to the sugar group, i.e., at the sugar modification. For example, the 2' hydroxyl (OH) group may be modified, e.g., replaced by a plurality of different "oxy" or "deoxy" substituents. In some embodiments, modification of the 2 'hydroxyl group may enhance the stability of the nucleic acid, as the hydroxyl group may no longer be deprotonated to form a 2' -alkoxide. Examples of 2' hydroxyl modifications may include alkoxy OR aryloxy (OR, where "R" may be, for example, alkyl, cycloalkyl, aryl, aralkyl, heteroaryl, OR sugar); polyethylene glycol (PEG), O (CH) 2 CH 2 O) n CH 2 CH 2 OR, wherein R may be, for example, H OR optionally substituted alkyl, and n may be an integer from 0 to 20. In some embodiments, the 2 'hydroxyl modification may be 2' -O-Me. In some embodiments, the 2' hydroxyl modification may be a 2' -fluoro modification that replaces the 2' hydroxyl with fluoro. In some embodiments, the 2 'hydroxyl modification may include a "locked" nucleic acid (LNA), where the 2' hydroxyl may be modified, for example, by C 1-6 Alkylene or C 1-6 The heteroalkylene bridge is attached to the 4' carbon of the same ribose, where an exemplary bridge may include methyleneA radical, propylene, diethyl ether or an amino bridge. In some embodiments, the 2' hydroxyl modification may include "unlocking" the nucleic acid (UNA), wherein the ribose ring lacks a C2' -C3' bond. In some embodiments, the 2' hydroxyl modification may include Methoxyethyl (MOE) (OCH) 2 CH 2 OCH 3 For example PEG derivatives).
"deoxy" 2' modifications may include hydrogen (i.e., deoxyribose, e.g., at a protruding portion of a partial dsRNA); halo (e.g., bromo, chloro, fluoro, or iodo); amino (where amino may be, for example, NH 2 The method comprises the steps of carrying out a first treatment on the surface of the Alkylamino, dialkylamino, heterocyclyl, arylamino, diarylamino, heteroarylamino, diheteroarylamino, or amino acid); NH (CH) 2 CH 2 NH) n CH2CH 2 -amino (wherein amino may be, for example, as described herein), -NHC (O) R (wherein R may be, for example, alkyl, cycloalkyl, aryl, aralkyl, heteroaryl or sugar), cyano; a mercapto group; alkyl-thio-alkyl; thioalkoxy; and alkyl, cycloalkyl, aryl, alkenyl, and alkynyl groups optionally substituted with amino groups, e.g., as described herein.
The sugar modification may comprise a sugar group that may also contain one or more carbons having a stereochemical configuration opposite the corresponding carbon in ribose. Thus, the modified nucleic acid may comprise a nucleotide containing, for example, arabinose as a sugar. The modified nucleic acid may also comprise an abasic sugar. These abasic sugars may also be further modified at one or more of the constituent sugar atoms. The modified nucleic acid may also comprise one or more sugars in the L form, such as L-nucleosides.
The modified nucleosides and modified nucleotides described herein that can be incorporated into a modified nucleic acid can include modified bases, also referred to as nucleobases. Examples of nucleobases include, but are not limited to, adenine (A), guanine (G), cytosine (C), and uracil (U)). These nucleobases can be modified or fully substituted to give modified residues that can be incorporated into modified nucleic acids. The nucleobases of the nucleotides may be independently selected from purines, pyrimidines, purine analogues or pyrimidine analogues. In some embodiments, nucleobases can include naturally occurring derivatives and synthetic derivatives of bases, for example.
In embodiments employing dual guide RNAs, each of the crRNA and tracr RNA may contain modifications. Such modifications may be at one or both ends of the crRNA and/or tracr RNA. In embodiments comprising the sgrnas, one or more residues at one or both ends of the sgrnas may be chemically modified, or the entire sgrnas may be chemically modified. Certain embodiments comprise a 5' modification. Certain embodiments comprise a 3' modification. In certain embodiments, one or more or all of the nucleotides in the single stranded overhang of the gRNA molecule are deoxynucleotides.
In some embodiments, the gRNA disclosed herein comprises one of the modification modes disclosed in WO2018/107028A1 published at 6-14 of 2018, the contents of which are hereby incorporated by reference in their entirety.
The terms "mA", "mC", "mU" or "mgs" may be used to indicate 2' -O-Me modified nucleotides. The terms "fA", "fC", "fU" or "fG" may be used to indicate a 2' -F substituted nucleotide. ". X" can be used to describe PS modifications. The terms a, C, U, or G may be used to indicate a nucleotide linked to the next (e.g., 3') nucleotide by PS bonds. The terms "mA", "mC", "mU" or "mgx" may be used to indicate a nucleotide that has been substituted with 2'-O-Me and linked to the next (e.g., 3') nucleotide by PS linkages.
C. Ribonucleoprotein complexes
In some embodiments, the present disclosure provides compositions comprising one or more grnas comprising one or more guide sequences from table 2 and an RNA-guided DNA binding agent, e.g., a nuclease, such as a Cas nuclease, such as Cas9. In some embodiments, the RNA-guided DNA binding agent has a lyase activity, which may also be referred to as double-stranded endonuclease activity. In some embodiments, the RNA-guided DNA binding agent comprises a Cas nuclease. Examples of Cas9 nucleases include those of type II CRISPR systems and modified (e.g., engineered or mutated) versions thereof of streptococcus pyogenes, staphylococcus aureus (s. Aureus), and other prokaryotes (see the list in the following paragraph). See, for example, US2016/0312198A1; US2016/0312199 A1. Other examples of Cas nucleases include Csm or Cmr complexes of type III CRISPR systems, or Cas10, csm1 or Cmr2 subunits thereof; and a cascade complex of a type I CRISPR system, or a Cas3 subunit thereof. In some embodiments, the Cas nuclease can be from a type IIA, type IIB, or type IIC system. For a discussion of various CRISPR systems and Cas nucleases, see, e.g., makarova et al, nat. Rev. Microbiol.9:467-477 (2011); makarova et al, nat. Rev. Micwall, 13:722-36 (2015); shmakov et al, MOLECULAR CELL,60:385-397 (2015). In some embodiments, the RNA-guided DNA binding agent comprises Cas nickase. In some embodiments, the RNA-guided nickase is modified or derived from a Cas protein, such as a class 2 Cas nuclease (which may be, for example, a type II, type V, or type VI Cas nuclease). Class 2 Cas nucleases include, for example, cas9, cpf1, C2 and C2C3 proteins and modifications thereof.
Non-limiting exemplary species from which Cas nuclease or Cas nickase may be derived include streptococcus pyogenes (Streptococcus pyogenes), streptococcus thermophilus (Streptococcus thermophilus), streptococcus, staphylococcus aureus (Staphylococcus aureus), listeria innocuous (Listeria innocua), lactobacillus gasseri (Lactobacillus gasseri), franciscensis novinae (3835), waldens succinogenes (Wolinella succinogenes), gardnerella (Sutterella wadsworthensis), amoebacteria gamma (gammophila), neisseria (Neisseria meningitidis), campylobacter jejuni (Campylobacter jejuni), pasteurella multocida (Pasteurella multocida), fibric acid producing bacteria (Fibrobacter succinogene), rhodospirillus (Rhodospirillum rubrum), rhodobacter darunae (Nocardiopsis dassonvillei), streptomyces roseus (Streptomyces pristi naespiralis), streptomyces viridis (Streptomyces viridochromogenes), streptomyces viridis produced, rhodosporidium (Streptosporangium roseum), rhodosporum, bacillus stearothermophilus (Ali cyclobacillus acidocaldarius), bacillus pseudomycosis (Bacillus pseudomycoides), bacillus pumilus (Bacillus selenitireducens), lactobacillus salivarius (Bacillus selenitireducens) and lactobacillus (Bacillus selenitireducens) are expressed by the bacteria, the species of genus rhodococcus (Polaromonas sp.)), alligator (Crocosphaera watsonii), alligator, microcystis aeruginosa (Microcystis aerugi nosa), synechococcus (Synechococcus sp.)), acetobacter arabicum (Acetohalobium arabaticu m), methanococcus halophilus (Ammonifex degensii), klebsiella pyrolysitum (Caldicelulosiruptor becscii), candida desulphus (Candidatus Desulforudis), clostridium botulinum (Clostridium botulin um), clostridium difficile (Clostridium difficile), gordonia macrogoldens (Finegoldia) magnna, thermophilic anaerobe (Natranaerobius thermophilus), anaerobic enterobacter thermophilus (Pelotomaculum thermopropionicum), thermophilic temperature thiobacillus (Acidithiobacillus caldus), sulfolobus acidophilus (Acidithiobacillus ferrooxidans), heterochromous (Allochromatium vinosum), haemophilus (marinobabacter sp.), nitrococcus halophilus (Nitrosococcus halophilus), rhodococcus vachelli (Nitrosococcus watsoni), alteromonas plankton (Pseudoalteromonas halo planktis), candida racemosa (Methanohal obium evestigatum), methanotrophic (Methanohal obium evestigatum), foamy bacteria (Anabaena variabilis), rhodococcus macrogoldens (Arthrospira platensis), spirulina (35, rhodobacter sphaeromonas (Arthrospira platensis), spirulina (35, rhodochrous sp (Arthrospira platensis), spirulina (35) and other species of genus rhodochrous (35 Streptococcus (Streptococcus pasteurian us), neisseria gray (Neisseria cinerea), campylobacter erythropolis (Campylobacter lari), corynebacterium parvum (Parvibaculum lavamentivorans) for detergent, corynebacterium diphtheriae (Corynebacteri um diphtheria), streptococcus (acidococcus sp.), strain Mao Luoke (Lachnospiracea e bacterium) ND2006 and marine chlorine-free bacteria (Acaryochloris marina).
In some embodiments, the Cas nuclease is a Cas9 nuclease from streptococcus pyogenes. In some embodiments, the Cas nuclease is a Cas9 nuclease from streptococcus thermophilus. In some embodiments, the Cas nuclease is a Cas9 nuclease from neisseria meningitidis. In some embodiments, the Cas nuclease is a Cas9 nuclease from staphylococcus aureus. In some embodiments, the Cas nuclease is a Cpf1 nuclease from franciscensis novica. In some embodiments, the Cas nuclease is a Cpf1 nuclease from the genus amino acid coccus. In some embodiments, the Cas nuclease is a Cpf1 nuclease from Mao Luoke bacteria ND 2006. In other specific examples, the Cas nuclease is a Cpf1 nuclease from: francisella tularensis (Francisella tularensis), mao Luoke, vibrio ruminalis (Butyrivibrio proteoclasticus), pachyrhizus (Peregrinibacteria bacterium), pachyrhizus (Parcubacteria bacterium), smith's bacteria (Smithlla), amino acid coccus, mycoplasma methanolica candidate species (Candidatus Methanoplasma termitum), eubacterium parvulum (Eubacterium eligens), moraxella bovis (Moraxella bovoculi), leptospira paddy (Leptospira inadai), porphyromonas canis (Porphyromonas crevioricanis), prevotella catarrhalis (Prevotella disiens), or Porphyromonas kii (Porphyromonas macacae). In certain embodiments, the Cas nuclease is a Cpf1 nuclease from the genus amino acid coccus or Mao Luoke bacteria.
In some embodiments, the Cas nickase is derived from a Cas9 nuclease from streptococcus pyogenes. In some embodiments, the Cas nickase is derived from a Cas9 nuclease from streptococcus thermophilus. In some embodiments, the Cas nickase is a nickase form of a Cas9 nuclease from neisseria meningitidis. See, e.g., WO/2020081568, which describes Nme2Cas 9D 16A nickase fusion proteins. In some embodiments, the Cas nickase is derived from a Cas9 nuclease from staphylococcus aureus. In some embodiments, the Cas nickase is derived from a Cpf1 nuclease from francissamia newfashioned. In some embodiments, the Cas nickase is derived from a Cpf1 nuclease from the genus amino acid coccus. In some embodiments, the Cas nickase is derived from a Cpf1 nuclease from Mao Luoke bacteria ND 2006. In other embodiments, the Cas nickase is derived from a Cpf1 nuclease from: francisella tularensis, mao Luoke, vibrio ruminalis, pachyrhizus, smith's, amino acid coccus, termite methane mycoplasma candidate species, bacillus bifidus, moraxella bovis, leptospira paddy, porphyromonas canis, prevotella descense or Porphyromonas kii. In certain embodiments, the Cas nickase is derived from a Cpf1 nuclease from the amino acid coccus or the chaetoviridae family. As discussed elsewhere, the nickase may be derived from a nuclease by inactivating one of the two catalytic domains, for example by mutating the active site residues necessary for nucleolysis (such as D10, H840 of N863 in Spy Cas 9). Those of skill in the art will be familiar with techniques that readily identify corresponding residues in other Cas proteins, such as sequence alignment and structural alignment, which will be discussed in detail below.
In some embodiments, the gRNA together with the RNA-guided DNA binding agent is referred to as a ribonucleoprotein complex (RNP). In some embodiments, the RNA-guided DNA binding agent is a Cas nuclease. In some embodiments, the gRNA together with the Cas nuclease is referred to as a Cas RNP. In some embodiments, the RNP comprises a type I, type II, or type III component. In some embodiments, the Cas nuclease is a Cas9 protein from a type II CRISPR/Cas system. In some embodiments, the gRNA together with Cas9 is referred to as Cas9 RNP.
Wild-type Cas9 has two nuclease domains: ruvC and HNH. RuvC domains cleave non-target DNA strands, and HNH domains cleave target DNA strands. In some embodiments, the Cas9 protein comprises more than one RuvC domain and/or more than one HNH domain. In some embodiments, the Cas9 protein is a wild-type Cas9. In each of the composition, use, and method embodiments, the Cas induces a double strand break in the target DNA.
In some embodiments, a chimeric Cas nuclease is used, wherein one domain or region of the protein is replaced with a portion of a different protein. In some embodiments, the Cas nuclease domain can be replaced with a domain from a different nuclease, such as Fok 1. In some embodiments, the Cas nuclease can be a modified nuclease.
In other embodiments, the Cas nuclease or Cas nickase can be from a type I CRISPR/Cas system. In some embodiments, the Cas nuclease can be a component of a cascade complex of a type I CRISPR/Cas system. In some embodiments, the Cas nuclease can be a Cas3 protein. In some embodiments, the Cas nuclease can be from a type III CRISPR/Cas system. In some embodiments, the Cas nuclease may have RNA cleavage activity.
In some embodiments, the RNA-guided DNA binding agent has single-strand nicking enzyme activity, i.e., one DNA strand can be cleaved to create a single-strand break, also known as a "nick". In some embodiments, the RNA-guided DNA binding agent comprises Cas nickase. Nicking enzymes are enzymes that create a nick in dsDNA, i.e., cleave one strand of a DNA duplex but not the other strand. In some embodiments, the Cas nickase is a version of a Cas nuclease (e.g., a Cas nuclease discussed above) in which the endonuclease active site is inactivated, e.g., by one or more changes in the catalytic domain (e.g., a point mutation). For a discussion of Cas nickases and exemplary catalytic domain changes, see, e.g., U.S. patent No. 8,889,356. In some embodiments, the Cas nickase, such as Cas9 nickase, has an inactive RuvC or HNH domain.
In some embodiments, the RNA-guided DNA binding agent is modified to contain only one functional nuclease domain. For example, the agent protein may be modified such that one of the nuclease domains is mutated or deleted entirely or partially to reduce its nucleic acid cleavage activity. In some embodiments, a nicking enzyme with a RuvC domain having reduced activity is used. In some embodiments, a nicking enzyme with an inactive RuvC domain is used. In some embodiments, a nicking enzyme having a reduced activity HNH domain is used. In some embodiments, a nicking enzyme having an inactive HNH domain is used.
In some embodiments, conserved amino acids within the Cas protein nuclease domain are substituted to reduce or alter nuclease activity. In some embodiments, the Cas nuclease may comprise amino acid substitutions in the RuvC or RuvC-like nuclease domain. Exemplary amino acid substitutions in RuvC or RuvC-like nuclease domains include D10A (based on streptococcus pyogenes Cas9 protein). See, e.g., zetsche et al (2015) Cell 10 month 22:163 (3): 759-771. In some embodiments, the Cas nuclease may comprise amino acid substitutions in the HNH or HNH-like nuclease domain. Exemplary amino acid substitutions in HNH or HNH-like nuclease domains include E762A, H840A, N863A, H983A and D986A (based on streptococcus pyogenes Cas9 protein). See, e.g., zetsche et al (2015). Other exemplary amino acid substitutions include D917A, E1006A and D1255A (based on the New inland Francisella U112 Cpf1 (FNCpf 1) sequence (UniProtKB-A0Q 7Q2 (CPF1_FRATN)).
In some embodiments, the mRNA encoding the nicking enzyme will be provided in combination with a pair of guide RNAs that are complementary to the sense and antisense strands, respectively, of the target sequence. In this embodiment, the guide RNA directs the nicking enzyme to the target sequence and introduces the DSB by creating a nick on opposite strands of the target sequence (i.e., double nicking). In some embodiments, the use of double nicks may improve specificity and reduce off-target effects. In some embodiments, a nicking enzyme is used in conjunction with two individual guide RNAs targeting opposite strands of the DNA to create a double nick in the target DNA. In some embodiments, a nicking enzyme is used in conjunction with two individual guide RNAs selected in close proximity to create a double nick in the target DNA.
In some embodiments, the RNA-guided DNA binding agent lacks lyase and nicking enzyme activity. In some embodiments, the RNA-guided DNA binding agent comprises a dCas DNA binding polypeptide. dCas polypeptides have DNA binding activity and are substantially devoid of catalytic (lyase/nickase) activity. In some embodiments, the dCas polypeptide is a dCas9 polypeptide. In some embodiments, the RNA-guided DNA binding agent or dCas DNA binding polypeptide lacking lyase and nickase activity is a version of a Cas nuclease in which the endonuclease active site is inactivated, e.g., by one or more changes in the catalytic domain (e.g., point mutations) (e.g., cas nucleases discussed above). See, for example, US2014/0186958 A1; US 2015/0166980A1.
In some embodiments, the RNA-guided DNA binding agent comprises one or more heterologous functional domains (e.g., is or comprises a fusion polypeptide).
In some embodiments, the RNA-guided DNA binding agent comprises apodec 3 deaminase. In some embodiments, the apodec 3 deaminase is apodec 3A (a 3A). In some embodiments, A3A is human A3A. In some embodiments, A3A is wild-type A3A.
In some embodiments, the RNA-guided DNA binding agent comprises a deaminase and an RNA-guided nicking enzyme. In some embodiments, the mRNA further comprises a linker that links the sequencing encoded A3A to the sequencing encoded RNA guided nicking enzyme sequence. In some embodiments, the linker is an organic molecule, group, polymer, or chemical moiety. In some embodiments, the linker is a peptide linker. In some embodiments, the peptide linker is any amino acid stretch having at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 15, at least 20, at least 25, at least 30, at least 40, at least 50, or more amino acids. In some embodiments, the peptide linker is a 16 residue "XTEN" linker or variant thereof (see, e.g., examples; and Schellenberger et al, A recombinant polypeptide extends the in vivo half-life of peptides and proteins in a tunable manger. Nat. Biotechnol.27,1186-1190 (2009)). In some embodiments, the XTEN linker comprises the sequence SGSETPGTSES ATPES (SEQ ID NO: 900), SGSETPGTSESA (SEQ ID NO: 901) or SGSETP GTSESATPEGGSGGS (SEQ ID NO: 902). In some embodiments, the peptide linker comprises one or more sequences selected from SEQ ID NOS 903-913.
In some embodiments, the heterologous functional domain may facilitate delivery of the RNA-guided DNA binding agent into the nucleus. For example, the heterologous functional domain may be a Nuclear Localization Signal (NLS). In some embodiments, RNA-guided DNA binding agents can be fused to 1-10 NLS. In some embodiments, RNA-guided DNA binding agents can be fused to 1-5 NLS. In some embodiments, the RNA-guided DNA binding agent can be fused to one NLS. In the case of one NLS, the NLS may be fused at the N-terminus or C-terminus of the RNA-guided DNA binding agent sequence. It may also be inserted into an RNA-guided DNA binding agent sequence. In other embodiments, the RNA-guided DNA binding agent may be fused to more than one NLS. In some embodiments, the RNA-guided DNA binding agent can be fused to 2, 3, 4, or 5 NLSs. In some embodiments, the RNA-guided DNA binding agent can be fused to two NLS. In some cases, the two NLSs may be the same (e.g., two SV40 NLSs) or different. In some embodiments, the RNA-guided DNA binding agent is fused to two NLS sequences (e.g., SV 40) at the carboxy terminus. In some embodiments, the RNA-guided DNA binding agent can be fused to two NLSs, one linked at the N-terminus and one linked at the C-terminus. In some embodiments, RNA-guided DNA binding agents can be fused to 3 NLS. In some embodiments, the RNA-guided DNA binding agent may not be fused to the NLS. In some embodiments, the NLS may be a mono-part sequence, such as SV40 NLS, PKKKRKV (SEQ ID NO: 600) or PKKKKRRV (SEQ ID NO: 601). In some embodiments, the NLS may be a duplex sequence such as NLS, KRPAATKKAGQAKKKK (SEQ ID NO: 602) of a nucleoplasmin. In a particular embodiment, a single PKKKRKV (SEQ ID NO: 600) NLS may be fused at the C-terminus of an RNA directed DNA binding agent. One or more linkers are optionally included at the fusion site.
In some embodiments, the RNA-guided DNA binding agent comprises an editing agent. An exemplary editing agent is BC22n, which comprises homo sapiens apodec 3A fused to streptococcus pyogenes-D10A Cas9 nickase by an XTEN linker, and mRNA encoding BC22 n. mRNA encoding BC22n is provided (SEQ ID NO: 804).
In some embodiments, the heterologous functional domain may be capable of modifying the intracellular half-life of the RNA-guided DNA binding agent. In some embodiments, the half-life of the RNA-guided DNA binding agent may be increased. In some embodiments, the half-life of the RNA-guided DNA binding agent may be reduced. In some embodiments, the heterologous functional domain may be capable of increasing the stability of the RNA-guided DNA binding agent. In some embodiments, the heterologous functional domain may be capable of reducing the stability of the RNA-guided DNA binding agent. In some embodiments, the heterologous functional domain can serve as a signal peptide for protein degradation. In some embodiments, protein degradation may be mediated by proteolytic enzymes, such as proteasome, lysosomal proteases, or calpain (calpain). In some embodiments, the heterologous functional domain may comprise a PEST sequence. In some embodiments, the RNA-guided DNA binding agent can be modified by the addition of ubiquitin or polyubiquitin chains. In some embodiments, the ubiquitin can be ubiquitin-like protein (UBL). Non-limiting examples of ubiquitin-like proteins include small ubiquitin-like modifier (SUMO), ubiquitin cross-reactive protein (UCRP, also known as interferon stimulatory gene-15 (ISG 15)), ubiquitin-related modifier-1 (URM 1), neuronal-progenitor-cell expressed dysplasia protein-8 (NEDD 8, known as Rub1 in saccharomyces cerevisiae), human leukocyte antigen F-related (FAT 10), autophagy-8 (ATG 8) and autophagy-12 (ATG 12), fau ubiquitin-like protein (FUB 1), membrane anchored UBL (MUB), ubiquitin folding modifier-1 (UFM 1) and ubiquitin-like protein-5 (UBL 5).
In some embodiments, the heterologous functional domain may be a marker domain. Non-limiting examples of marker domains include fluorescent proteins, purification tags, epitope tags, and reporter sequences. In some embodiments, the marker domain may be a fluorescent protein. Non-limiting examples of suitable fluorescent proteins include Green fluorescent proteins (e.g., GFP-2, tagGFP, turboGFP, sfGFP, EGFP, emerald (Emerald), azami Green, monomeric Azami Green, copGFP, aceGFP, zsGreen 1), yellow fluorescent proteins (e.g., YFP, EYFP, citrine, venus, YPet, phiYFP, zsYellow 1), blue fluorescent proteins (e.g., EBFP2, azurite, mKalamal, GFPuv, sapphire (Sapphire), T-Sapphire (T-Sapphire)), cyan fluorescent proteins (e.g., ECFP, cerulean, cyPet, amCyan, midorishi-Cyan), red fluorescent proteins (e.g., mKate2, mPlum, dsRed monochrome, mCherry, mRFP1, dsRed-Express, dsRed2, dsRed monochrome, hcRed string, hcRed1, asRed2, FP611, mRasberry, mStrawberry, jred), and Orange fluorescent proteins (mOrange, mKO, kusabira-Orange, monomeric Kusabira-Orange, mTangerine, tdTomato) or any other suitable fluorescent protein. In other embodiments, the marker domain may be a purification tag and/or an epitope tag. Non-limiting exemplary tags include glutathione-S-transferase (GST), chitin Binding Protein (CBP), maltose Binding Protein (MBP), thioredoxin (TRX), poly (NANP), tandem Affinity Purification (TAP) tag, myc, acV5, AU1, AU5, E, ECS, E2, FLAG, HA, nus, softag1, softag 3, strep, SBP, glu-Glu, HSV, KT3, S, S1, T7, V5, VSV-G, 6 XHis, 8 XHis, biotin Carboxyl Carrier Protein (BCCP), polyHis, and calmodulin. Non-limiting exemplary reporter genes include glutathione-S-transferase (GST), horseradish peroxidase (HRP), chloramphenicol Acetyl Transferase (CAT), beta-galactosidase, beta-glucuronidase, luciferase, or fluorescent proteins.
In other embodiments, the heterologous functional domain may target an RNA-guided DNA binding agent to a particular organelle, cell type, tissue, or organ. In some embodiments, the heterologous functional domain can target an RNA-guided DNA binding agent to the granulosa.
In other embodiments, the heterologous functional domain may be an effector domain, such as an editing domain. When the RNA-guided DNA binding agent is directed to its target sequence, for example, when the Cas nuclease is directed to the target sequence by the gRNA, the effector domain (such as an editing domain) can modify or affect the target sequence. In some embodiments, the effector domain (such as an editing domain) may be selected from a nucleic acid binding domain, a nuclease domain (e.g., a non-Cas nuclease domain), an epigenetic modification domain, a transcriptional activation domain, or a transcriptional repressor domain. In some embodiments, the heterologous functional domain is a nuclease, such as a fokl nuclease. See, for example, U.S. patent No. 9,023,649. In some embodiments, the heterologous functional domain is a transcriptional activator or repressor. See, e.g., qi et al, "Repurposing CRISPR as an RNA-guid ed platform for sequence-specific control of gene expression", cell 152:1173-83 (2013); perez-Pinera et al, "RNA-guided gene activation by CRISPR-Cas9-ba sed transcription factors", nat. Methods 10:973-6 (2013); mali et al, "CAS9 tra nscriptional activators for target specificity screening and paired nickases for c ooperative genome engineering", nat. Biotechnol.31:833-8 (2013); gilbert et al, "CRISPR-mediated modular RNA-guided regulation of transcription in eukaryo tes", cell 154:442-51 (2013). Thus, RNA-guided DNA binding agents essentially become transcription factors that can be guided to bind to a desired target sequence using guide RNA.
D. Determination of guide RNA efficacy
In some embodiments, the efficacy of the guide RNA is determined when delivered or expressed with other components that form the RNP (e.g., RNA-guided DNA binding agents). In some embodiments, the guide RNA is expressed with an RNA-guided DNA binding agent, such as a Cas protein, e.g., cas 9. In some embodiments, the guide RNA is delivered to or expressed in a cell line that has stably expressed an RNA-guided DNA nuclease (such as a Cas nuclease or a nickase, e.g., a Cas9 nuclease or nickase). In some embodiments, the guide RNA is delivered to the cell as part of the RNP. In some embodiments, the guide RNA is delivered into the cell with mRNA encoding an RNA-guided DNA nuclease (such as a Cas nuclease or nickase, e.g., cas9 nuclease or nickase).
As described herein, use of the RNA-guided DNA nucleases and guide RNAs disclosed herein can cause DSB, SSB, and/or site-specific binding that results in nucleic acid modifications in DNA or pre-mRNA that, when repaired by cellular mechanisms, can produce errors in the form of insertion/deletion (indel) mutations. A variety of mutations due to insertions/deletions can alter the reading frame, introduce premature stop codons or induce exon skipping, and thus produce a nonfunctional protein.
In some embodiments, the efficacy of a particular guide RNA is determined based on an in vitro model. In some embodiments, the in vitro model is a T cell line. In some embodiments, the in vitro model is HEK 293T cells. In some embodiments, the in vitro model is a HEK293 cell stably expressing Cas9 (hek293_cas 9). In some embodiments, the in vitro model is a lymphoblastoid cell line. In some embodiments, the in vitro model is primary human T cells. In some embodiments, the in vitro model is primary human B cells. In some embodiments, the in vitro model is primary human peripheral blood lymphocytes. In some embodiments, the in vitro model is primary human peripheral blood mononuclear cells.
In some embodiments, the number of off-target sites in the in vitro model where a deletion or insertion occurs is determined, for example, by analyzing genomic DNA from cells transfected in vitro with Cas9 mRNA and guide RNA. In some embodiments, such assays comprise analyzing genomic DNA from cells transfected in vitro with Cas9 mRNA, guide RNA, and donor oligonucleotides. Exemplary procedures for such assays are provided in the working examples below.
In some embodiments, the efficacy of a particular gRNA is determined on a plurality of in vitro cell models used to guide the RNA selection process. In some embodiments, the data is compared to data for the selected guide RNA against the cell line. In some embodiments, multiple cell models are used for cross-screening.
In some embodiments, the efficacy of a particular guide RNA is determined based on an in vivo model. In some embodiments, the in vivo model is a rodent model. In some embodiments, the rodent model is a mouse that expresses a target gene. In some embodiments, the rodent model is a mouse that expresses the CIITA gene. In some embodiments, the rodent model is a mouse that expresses a human CIITA gene. In some embodiments, the rodent model is a mouse that expresses the B2M gene. In some embodiments, the rodent model is a mouse that expresses a human B2M gene. In some embodiments, the in vivo model is a non-human primate, such as a cynomolgus monkey.
In some embodiments, the efficacy of the guide RNA is assessed by mid-target cleavage efficiency. In some embodiments, the efficacy of the guide RNA is measured by the percentage of editing at the target location, e.g., CIITA or B2M. In some embodiments, depth sequencing can be used to identify the presence of modifications (e.g., insertions, deletions) introduced by gene editing. The percent insertion/deletion can be calculated from the next generation sequencing "NGS".
In some embodiments, the efficacy of the guide RNA is measured by the number and/or frequency of insertions/deletions at off-target sequences within the genome of the target cell type. In some embodiments, effective guide RNAs are provided that produce insertions/deletions at off-target sites at very low frequencies (e.g., < 5%) in a cell population and/or relative to the frequency of insertion/deletion production at target sites. Thus, the present disclosure provides guide RNAs that do not exhibit off-target insertion/deletion formation in a target cell type (e.g., T cells or B cells), or that produce an off-target insertion/deletion formation frequency of <5% in a cell population and/or relative to the insertion/deletion production frequency at a target site. In some embodiments, the present disclosure provides guide RNAs that do not exhibit any off-target insertion/deletion formation in a target cell type (e.g., T cells or B cells). In some embodiments, guide RNAs are provided that produce insertions/deletions at fewer than 5 off-target sites, e.g., as assessed by one or more of the methods described herein. In some embodiments, for example, as assessed by one or more of the methods described herein, guide RNAs are provided that produce insertions/deletions at less than or equal to 4, 3, 2, or 1 off-target sites. In some embodiments, the off-target site is not present in a protein coding region in the genome of the target cell (e.g., T cell or B cell).
In some embodiments, linear amplification is used to detect gene editing events, such as the formation of insertion/deletion ("indel") mutations, translocations, and Homology Directed Repair (HDR) events in target DNA. For example, linear amplification of primers with unique sequence tags can be used and the tagged amplification products isolated (hereinafter referred to herein as "uit", or "unique identifier tagging" methods).
In some embodiments, the efficacy of the guide RNA is measured by the number of chromosomal rearrangements within the target cell type. The Kromatid dGH assay can be used to detect chromosomal rearrangements including, for example, translocation, reciprocal translocation, translocation to off-target chromosomes, deletions (i.e., chromosomal rearrangements in which fragments are lost in the cell replication cycle due to editing events). In some embodiments, the target cell type has less than 10, less than 8, less than 5, less than 4, less than 3, less than 2, or less than 1 chromosomal rearrangements. In some embodiments, the target cell type does not have a chromosomal rearrangement.
Delivery of gRNA compositions
Lipid nanoparticles (LNP compositions) are well known means for delivering nucleotide and protein cargo (cargo) and can be used to deliver guide RNAs, compositions or pharmaceutical formulations disclosed herein. In some embodiments, the LNP composition delivers the nucleic acid, protein, or nucleic acid along with the protein.
In some embodiments, the invention includes a method of delivering any of the grnas disclosed herein to a subject, wherein the grnas are formulated as LNPs. In some embodiments, the LNP comprises a gRNA and Cas9 or mRNA encoding Cas 9.
In some embodiments, the invention includes a composition comprising any of the disclosed grnas and LNPs. In some embodiments, the composition further comprises Cas9 or mRNA encoding Cas 9.
In some embodiments, the LNP composition comprises a cationic lipid. In some embodiments, the LNP composition comprises octadecyl-9, 12-dienoic acid (9 z,12 z) -3- ((4, 4-bis (octyloxy) butanoyl) oxy) -2- (((((3- (diethylamino) propoxy) carbonyl) oxy) methyl) propyl ester (also known as (9 z,12 z) -octadecyl-9, 12-dienoic acid 3- ((4, 4-bis (octyloxy) butanoyl) oxy) -2- (((((3- (diethylamino) propoxy) carbonyl) oxy) methyl) propyl ester) or another ionizable lipid. See, for example, WO/2017/173054 and references described therein. In some embodiments, the LNP composition comprises a molar ratio of cationic lipid amine to RNA phosphate (N: P) of about 4.5, 5.0, 5.5, 6.0, or 6.5. In some embodiments, in the case of LNP lipids, the terms cationic and ionizable are interchangeable, e.g., wherein the ionizable lipid is cationic according to pH.
In some embodiments, the grnas disclosed herein are formulated as LNP compositions for use in the preparation of a medicament for treating a disease or disorder.
Electroporation is a well known means of delivering cargo, and any electroporation method may be used to deliver any of the grnas disclosed herein. In some embodiments, electroporation can be used to deliver any of the grnas disclosed herein and Cas9 or mRNA encoding Cas9.
In some embodiments, the invention includes a method of delivering any of the grnas disclosed herein to an ex vivo cell, wherein the gRNA is formulated as an LNP or not formulated as an LNP. In some embodiments, the LNP comprises a gRNA and Cas9 or mRNA encoding Cas9.
In some embodiments, the guide RNA compositions described herein, alone or encoded on one or more carriers, are formulated in or administered via a lipid nanoparticle; see, for example, WO/2017/173054 and WO 2019/067992, the contents of which are incorporated herein by reference in their entirety.
In certain embodiments, the invention includes DNA or RNA vectors encoding any one guide RNA comprising any one or more of the guide sequences described herein. In some embodiments, the vector comprises nucleic acid that does not encode a guide RNA in addition to the guide RNA sequence. Nucleic acids that do not encode guide RNAs include, but are not limited to, promoters, enhancers, regulatory sequences, and nucleic acids that encode an RNA-guided DNA nuclease (which may be a nuclease such as Cas 9). In some embodiments, the vector comprises one or more nucleotide sequences encoding crrnas, trrnas, or both crrnas and trrnas. In some embodiments, the vector comprises one or more nucleotide sequences encoding sgrnas and mRNA encoding an RNA-guided DNA nuclease (which may be a Cas nuclease, such as Cas9 or Cpf 1). In some embodiments, the vector comprises one or more nucleotide sequences encoding crrnas, trrnas, and mrnas encoding RNA-guided DNA nucleases, which can be Cas proteins, such as Cas9. In one embodiment, cas9 is from streptococcus pyogenes (i.e., spy Cas 9). In some embodiments, the nucleotide sequence encoding crRNA, trRNA, or crRNA and trRNA (which may be sgRNA) comprises or consists of: a guide sequence flanked by all or a portion of a repeat sequence from a naturally occurring CRISPR/Cas system. The nucleic acid comprising or consisting of crRNA, trRNA or crRNA and trRNA may further comprise a vector sequence, wherein the vector sequence comprises or consists of: nucleic acids that are not found naturally with crrnas, trrnas, or both crrnas and trrnas.
Therapeutic methods and uses
Any of the engineered cells and compositions described herein can be used in methods of treating a variety of diseases and conditions as described herein. In some embodiments, genetically modified cells (engineered cells) and/or genetically modified cell (engineered cells) populations and compositions are useful in methods of treating a variety of diseases and disorders. In some embodiments, methods of treating any of the diseases or conditions described herein are contemplated, comprising administering any one or more of the compositions described herein.
In some embodiments, the methods and compositions described herein are useful for treating diseases or conditions in which delivery of a therapeutic agent is desired. In some embodiments, the invention provides a method of providing immunotherapy in a subject, the method comprising administering to the subject an effective amount of an engineered cell (or population of engineered cells) as described herein, e.g., a cell of any of the foregoing cellular aspects and embodiments.
In some embodiments, the methods comprise administering to the subject a composition comprising an engineered cell described herein as adoptive cell transfer therapy. In some embodiments, the engineered cell is an allogeneic cell.
In some embodiments, the methods comprise administering to a subject a composition comprising an engineered cell described herein, wherein the cell produces, secretes, and/or expresses a polypeptide suitable for treating a disease or disorder in the subject (e.g., targets a subjectA body). In some embodiments, the cells act as a cell factory to produce soluble polypeptides. In some embodiments, the cells act as a cell factory to produce antibodies. In some embodiments, the cell continuously secretes the polypeptide in vivo. In some embodiments, the cells continue to secrete the polypeptide for at least 1, 2, 3, 4, 5, or 6 weeks after in vivo transplantation. In some embodiments, the cells continue to secrete the polypeptide for more than 6 weeks after in vivo transplantation. In some embodiments, the soluble polypeptide (e.g., antibody) is produced by the cell at least 10 per day 2 、10 3 、10 4 、10 5 、10 6 、10 7 Or 10 8 The concentration of each copy was generated. In some embodiments, the polypeptide is an antibody and is produced by the cell at least 10 per day 8 The concentration of each copy was generated.
In some embodiments of the methods, the methods comprise administering a lymphocyte depleting agent or immunosuppressant prior to administering to the subject an effective amount of an engineered cell as described herein, e.g., a cell of any of the foregoing cellular aspects and embodiments. In another aspect, the invention provides a method of preparing an engineered cell (e.g., an engineered cell population).
Immunotherapy is the treatment of diseases by activating or suppressing immune responses. Immunotherapy designed to elicit or amplify an immune response is classified as activated immunotherapy. Cell-based immunotherapy has been shown to be effective in treating some cancers. Immune effector cells, such as lymphocytes, macrophages, dendritic cells, natural killer cells, cytotoxic T Lymphocytes (CTLs), T helper cells, B cells, or progenitor cells thereof, such as Hematopoietic Stem Cells (HSCs) or induced pluripotent stem cells (ipscs), may be programmed to function in response to abnormal antigens expressed on the surface of tumor cells. Cancer immunotherapy thus allows components of the immune system to destroy tumors or other cancer cells. Cell-based immunotherapy has also been demonstrated to be effective in treating autoimmune diseases or graft rejection. Immune effector cells, such as regulatory T cells (tregs) or mesenchymal stem cells, may be programmed to function in response to autoantigens or transplantation antigens expressed on the surface of normal tissue.
In some embodiments, the invention provides a method of preparing an engineered cell (e.g., an engineered cell population). The engineered cell populations can be used in immunotherapy.
In some embodiments, the invention provides a method of treating a subject in need thereof, comprising administering an engineered cell prepared by a method of preparing a cell described herein, e.g., a method of any of the foregoing aspects and embodiments of the cell preparation methods.
In some embodiments, the engineered cells can be used to treat cancer, infectious disease, inflammatory disease, autoimmune disease, cardiovascular disease, neurological disease, ophthalmic disease, kidney disease, liver disease, musculoskeletal disease, red blood cell disease, or transplant rejection.
In some embodiments, the engineered cells may be used as cell therapies including allogeneic stem cell therapies. In some embodiments, the cell therapy comprises induced pluripotent stem cells (ipscs). ipscs can be induced to differentiate into other cell types including, for example, beta islet cells, neurons, and blood cells. In some embodiments, the cell therapy comprises hematopoietic stem cells. In some embodiments, the stem cells include mesenchymal stem cells that can develop into bone, cartilage, muscle, and fat cells. In some embodiments, the stem cells comprise ocular stem cells. In some embodiments, allogeneic stem cell transplantation includes allogeneic bone marrow transplantation. In some embodiments, the stem cells comprise Pluripotent Stem Cells (PSCs). In some embodiments, the stem cells comprise induced Embryonic Stem Cells (ESCs).
The engineered cells of the invention are suitable for further engineering, for example by introducing other edited or modified genes or alleles. In some embodiments, the polypeptide is a wild-type or variant TCR. The cells of the invention may also be suitable for further engineering by introducing exogenous nucleic acids encoding, for example, a targeted receptor (e.g., TCR, CAR, uniCAR). CARs are also known as chimeric immune receptors, chimeric T cell receptors, or artificial T cell receptors.
In some embodiments, the cell therapy is a transgenic T cell therapy. In some embodiments, the cell therapy comprises transgenic T cells that target wilms' tumor 1 (WT 1). In some embodiments, the cell therapy comprises a commercially available T cell therapy, such as a targeted recipient of a CAR T cell therapy or a donor nucleic acid encoding a targeted recipient. There are currently a number of targeted receptors approved for cell therapies. The cells and methods provided herein can be used with these known constructs. Commercially approved cell products including targeted receptor constructs for use as cell therapies include, for example(tisamgenlecleicel); />(alemtujopsis (axicabtagene ciloleucel)); tecartus TM (blatty (brexucabtagene autoleucel)); peptide Bei Lusai (Tabelecteicosel)Viralym-M (ALVR 105); and Viralym-C.
In some embodiments, the method provides for administering the engineered cells to the subject, wherein the administering is injection. In some embodiments, the methods provide for administering the engineered cells to the subject, wherein the administering is intravascular injection or infusion. In some embodiments, the methods provide for administering the engineered cells to the subject, wherein the administration is a single dose.
In some embodiments, the methods provide for alleviating a sign or symptom associated with a disease in a subject treated with a composition disclosed herein. In some embodiments, the subject has a response to treatment with a composition disclosed herein for more than one week. In some embodiments, the subject has a response to treatment with a composition disclosed herein for more than two weeks. In some embodiments, the subject has a response to treatment with a composition disclosed herein for more than three weeks. In some embodiments, the subject has a response to treatment with a composition disclosed herein for more than one month.
In some embodiments, methods provide for administering an engineered cell to a subject, and wherein the subject is responsive to the administered cell, including alleviating a sign or symptom associated with a disease treated by cell therapy. In some embodiments, the subject has a response lasting more than one week. In some embodiments, the subject has a response lasting more than one month. In some embodiments, the subject has a response lasting at least 1-6 weeks.
TABLE 4 additional sequences
/>
/>
/>
/>
/>
/>
/>
/>
/>
/>
/>
/>
/>
/>
/>
/>
/>
/>
/>
/>
/>
/>
/>
/>
/>
/>
/>
/>
/>
/>
/>
/>
/>
/>
/>
/>
/>
/>
/>
/>
/>
/>
(in each of the sequences described in the above tables or herein, the modified sequences may be unmodified or alternatively modified.)
Examples
The following examples are provided to illustrate certain disclosed embodiments and should not be construed as limiting the scope of the disclosure in any way.
Example 1 general procedure
1.1. Preparation of lipid nanoparticles
In general, the lipid component is dissolved in 100% ethanol in various molar ratios. RNA cargo (e.g., cas9 mRNA and sgRNA) was dissolved in 25mM citrate buffer, 100mM NaCl (pH 5.0), resulting in an RNA cargo concentration of approximately 0.45 mg/mL.
The lipid nucleic acid assemblies contain the ionizable lipid (octadeca-9, 12-dienoic acid (9Z, 12Z) -3- ((4, 4-bis (octyloxy) butanoyl) oxy) -2- ((((3- (diethylamino) propoxy) carbonyl) oxy) methyl) propyl ester, also known as (9Z, 12Z) -octadeca-9, 12-dienoic acid 3- ((4, 4-bis (octyloxy) butanoyl) oxy) -2- ((((3- (diethylamino) propoxy) carbonyl) oxy) methyl) propyl ester), cholesterol, DSPC, and PEG2k-DMG in a molar ratio of 50:38:9:3, respectively. The lipid nucleic acid assemblies were formulated at a lipid amine to RNA phosphate (N: P) molar ratio of about 6 and a gRNA to mRNA ratio of 1:1 or 1:2 by weight.
LNP compositions were prepared using cross-flow techniques using lipid-containing ethanol mixed with two volumes of RNA solution and an impinging jet of one volume of water. Lipid-containing ethanol was mixed with two volumes of RNA solution via mixing crossover. The fourth water flow is mixed with the outlet flow of the cross via an in-line tee (see WO2016010840 fig. 2). The LNP composition was kept at room temperature for 1 hour and further diluted with water (approximately 1:1 v/v). LNP compositions were concentrated using tangential flow filtration on a flat plate cartridge (Sartorius, 100kD MWCO) and their buffer was exchanged into 50mM Tris, 45mM NaCl, 5% (w/v) sucrose, pH 7.5 (TSS) using a PD-10 desalting column (GE). Alternatively, LNP is optionally concentrated using a 100kDa Amicon spin filter and its buffer is exchanged into the TSS using a PD-10 desalting column (GE). The resulting mixture was then filtered using a 0.2 μm sterile filter. The final LNP was stored at 4℃or-80℃until further use.
In vitro transcription of mRNA ("IVT")
Capping and polyadenylation mRNAs containing N1-methyl pseudo-U were produced by in vitro transcription using linearized plasmid DNA templates and T7 RNA polymerase. Plasmid DNA containing T7 promoter, transcribed sequence and polyadenylation sequence was linearized by incubation with XbaI for 2 hours at 37 ℃ under the following conditions: 200 ng/. Mu.L plasmid, 2U/. Mu.L XbaI (NEB) and 1 Xreaction buffer. XbaI was deactivated by heating the reaction at 65℃for 20 minutes. Linearized plasmids were purified from enzymes and buffer salts. The IVT reaction for producing modified mRNA was performed by incubation at 37 ℃ for 1.5-4 hours under the following conditions: 50 ng/. Mu.L of linearized plasmid; GTP, ATP, CTP and N1-methyl pseudo-UTP (Trilink), each 2-5 mM; 10-25mM ARCA (Trilink); 5U/. Mu. L T7 RNA polymerase (NEB); 1U/. Mu.L of murine ribonuclease inhibitor (NEB); 0.004U/. Mu.L of inorganic E.coli pyrophosphatase (NEB); and 1 x reaction buffer. TURBO deoxyribonuclease (ThermoFisher) was added to a final concentration of 0.01U/. Mu.L, and the reaction was incubated for an additional 30 minutes to remove the DNA template. mRNA was purified using MegaClear Transcription Clean-up kit (ThermoFisher) or RNeasy Maxi kit (Qiagen) according to the manufacturer's protocol. Alternatively, mRNA is purified via a precipitation scheme (in some cases, followed by HPLC-based purification). Briefly, after deoxyribonuclease digestion, mRNA was purified using LiCl precipitation, ammonium acetate precipitation, and sodium acetate precipitation. For HPLC purified mRNA, after LiCl precipitation and reconstitution, the mRNA is purified by RP-IP HPLC (see, e.g., kariko et al, nucleic Acids Research,2011, volume 39, stage 21 e 142). Fractions selected for pooling were pooled and desalted by sodium acetate/ethanol precipitation as described above. In another alternative, the mRNA is purified by LiCl precipitation followed by further purification by tangential flow filtration. RNA concentration was determined by measuring absorbance at 260nm (Nanodrop) and transcripts were analyzed by capillary electrophoresis with Bioanlayzer (Agilent).
Streptococcus pyogenes ("Spy") Cas9 mRNA was generated from plasmid DNA encoding the open reading frame according to SEQ ID NOS 801-803 (see the sequences in Table 4). BC22n mRNA is produced from plasmid DNA encoding the open reading frames according to SEQ ID NOS 804-805. BC22 mRNA is produced from plasmid DNA encoding an open reading frame according to SEQ ID NO. 806. UGI mRNA was produced from plasmid DNA encoding the open reading frames according to SEQ ID NOS 807-808. When reference is made below to SEQ ID NOS 801-808 with respect to RNA, it is understood that T should be replaced by U (which is N1-methyl pseudouridine as described above). Messenger RNAs used in the examples include 5 'caps and 3' polyadenylation regions, e.g., up to 100nt, and are identified in Table 4 by SEQ ID NOS: 801-808.
1.3. Next generation sequencing ("NGS") and analysis for mid-target editing efficiency
According to the manufacturer's scheme, quickExract is used TM Genomic DNA was extracted from the DNA extraction solution (Lucigen, catalog number QE 09050).
In order to quantitatively determine editing efficiency at a target location in a genome, deep sequencing was used to identify the presence of insertions and deletions introduced by gene editing. PCR primers are designed around target sites within the gene of interest (e.g., TRAC), and genomic regions of interest are amplified. Primer sequence design was performed according to the standards in the art.
Additional PCR was performed according to the manufacturer's protocol (Illumina) to add chemistry to sequencing. Amplicons were sequenced on an Illumina MiSeq instrument. After eliminating reads with low quality scores, the reads were compared to a human reference genome (e.g., hg 38). Reads overlapping the target region of interest are realigned with local genomic sequences to improve the alignment. The number of wild type reads was then calculated relative to the number of reads containing mutations with C to T, mutations with C to A/G, or insertions/deletions. Insertions and deletions were scored in a 20bp region centered on the predicted Cas9 cleavage site. Percent insertions/deletions are defined as the total number of sequencing reads that insert or delete one or more bases within a 20bp scoring region divided by the total number of sequencing reads (including wild-type). The mutation of C to T or the mutation of C to A/G was scored in a 40bp region comprising 10bp upstream and 10bp downstream of the 20bp sgRNA target sequence. The percent editing of C to T is defined as the total number of sequencing reads with one or more mutations of C to T within the 40bp region divided by the total number of sequencing reads (including wild type). The percentage of mutation of C to A/G was similarly calculated.
Example 2 screening 1 of CIITA guide RNA
CIITA guide RNAs were screened for efficacy in T cells by assessing loss of MHC class II cell surface expression. The percentage of T cells negative for MHC class II proteins ("MHC II negative%") was determined after CIITA editing.
2.1. Editing T cells with ribonucleoprotein
Cas9 editing activity was assessed using electroporation of Cas9 Ribonucleoprotein (RNP). After thawing, pan CD3+ T cells were plated at 0.5X10 6 The density of individual cells per milliliter was plated in T cell RPMI medium consisting of RPMI 1640 (Invitrogen, catalog No. 22400-089) containing 5% (v/v) fetal bovine serum, 1 XGlutamax (Gibco, catalog No. 35050-061), 50. Mu.M 2-mercaptoethanol, 100. Mu.M nonessential amino acids (Invitrogen, catalog No. 11140-050), 1mM sodium pyruvate, 10mM HEPES buffer, 1% penicillin-streptomycin, and 100U/mL recombinant human interleukin-2 (Peprotech, catalog No. 200-02). By Dynabeads TM Human T-Expander CD3/CD28 (3:1, invitrogen) activated T cells. Cells were expanded in T cell RPMI medium for 72 hours prior to RNP transfection.
RNPs were generated by pre-annealing the crRNA and trRNA (SEQ ID NO: 215) individually targeted to CIITA by mixing equal amounts of reagents and incubating at 95℃for 2 minutes and cooling to room temperature. The double guide (dgRNA) consisting of pre-annealed crRNA and trRNA was incubated with recombinant Spy Cas9 protein (SEQ ID NO: 800) to form Ribonucleoprotein (RNP) complexes. An RNP mixture of 50 μm dgRNA and 50 μm Cas9-NLS protein was prepared and incubated for 10 min at 25 ℃. mu.L of RNP mix was combined with 100,000 cells in 20. Mu. L P3 electroporation buffer (Lonza). 22. Mu.L of RNP/cell mixture was transferred to corresponding wells of a Lonza short 96-well electroporation plate. Cells were electroporated in triplicate with the manufacturer's pulse code. T cell RPMI medium was added to the cells immediately after electroporation. The electroporated T cells are then cultured. Two days after editing, a portion of the electroporated T cells were collected for NGS sequencing.
2.2. Flow cytometry
On day 7 post-editing, T cells were phenotyped by flow cytometry to determine MHC class II protein expression. Briefly, T cells were targeted to HLA-DR antibodiesCatalog number 307622) and isotype control AF647Catalog number 400234). The cells were then washed, processed on a Cytoflex flow cytometer (Beckman Coulter) and analyzed using FlowJo suite software. T cells are gated based on size, shape, viability and MHC class II expression. PCR and subsequent NGS analysis were performed on the DNA samples. Table 5 and FIG. 1A show the results in CD3 + Results of percent editing after CIITA editing with various guides in T cells. Table 5 and FIG. 1A show the results of CIITA editing with various guides in T cells using HLA-DR as a percentage of labeled MHC-II negative cells.
TABLE 5 percentage of editing after CIITA editing and percentage of HLA-DR-cells
EXAMPLE 3 sgRNA dose response editing
3.1T cell preparation
Healthy human donor blood cell apheresis is commercially available (Hemacare) and cells are washed on a LOVO device and resuspendedPBS/EDTA buffer (Miltenyi Biotec catalog No. 130-070-525). UsingPlus and->LS Disposable kit T cells were isolated via positive selection using CD4 and CD8 magnetic beads (Miltenyi Biotec catalog No. 130-030-401/130-030-801). T cells were aliquoted into vials and stored frozen +. >CS10 (StemCell Technologies catalog number 07930) and Plasmalyte a (Baxter catalog number 2B 2522X) for future use.
After thawing, T cells were grown at 1.5X10 6 The individual cells/mL were plated in Optmizer based medium containing CTS Optmizer T cell expansion SFM (Gibco, catalog No. A3705001), 5% human AB serum (Gemini, catalog No. 100-512), 1% penicillin-streptomycin, 1 XGlutamax, 10mM HEPES, 200U/mL recombinant human interleukin-2 (Peprotech, catalog No. 200-02), 5ng/mL recombinant human interleukin 7 (Peprotech, catalog No. 200-07) and 5ng/mL recombinant human interleukin 15 (Peprotech, catalog No. 200-15). T cells in this Medium with TransAct TM (1:100 dilution, miltenyi Biotec) for 48 hours.
3.2T cell editing
LNP compositions containing mRNA encoding Cas9 (SEQ ID NO: 802) and sgRNA targeting CIITA were formulated as described in example 1. Each LNP formulation was incubated at 37℃for 5 minutes in OpTmizer-based medium with cytokines as described above supplemented with 10. Mu.g/ml recombinant human ApoE3 (Peprotech, cat. No. 350-02). 48 hours after activation, T cells were washed and suspended in OpTmizer medium with cytokines as described and without human serum. Pre-incubated LNP mixtures were added to each well, resulting in final concentrations as described in table 6. Also included is a control group that included non-edited T cells (without LNP). After 24 hours, T cells were collected, washed and cultured in OpTmizer-based medium for 7 days, then collected for evaluation by NGS and flow cytometry. All groups were performed in duplicate wells (n=2). Expanded T cells were cryopreserved for functional assays. NGS analysis was performed as described for a single set of duplicate samples in example 1. Table 6 and fig. 2A show the results of percentage editing after CIITA editing with various guides in T cells.
TABLE 6 percent insertion/deletion after CIITA editing in total T cells (n=1)
3.3 flow cytometry
On day 7 post-editing, T cells were phenotyped by flow cytometry to determine MHC class II protein expression. Briefly, T cells were incubated with an antibody (Biolegend, cat. No. 361706) targeting HLA-DR DP-DQ, followed by washing and analysis on a Cytoflex flow cytometer (Beckman Coulter). Data analysis was performed using FlowJo suite software. T cells are gated based on size, shape, viability, and MHC class II (HLA-DRDP-DQ) expression. Table 7 and FIG. 2B show the results of percentage of MHC-II negative cells (HLA-DR-DP-DQ-) after CIITA editing with various primers in CD4+, CD8+ or total T cells.
TABLE 7 average percentage of MHC class II negative cells after CIITA editing
EXAMPLE 4 CIITA guide RNA
4.1T cell preparation
Healthy human donor blood cell line (Hemacare) was obtained commercially, and cells were washed and resuspended in 2% pbs/EDTA buffer. UsingCD4/CD8 microbead kit (Miltenyi Biotec catalog No. 130-122-352), T cells were isolated via positive selection on MultiMACS (Miltenyi Biotec catalog No. 130-098-637). T cells were aliquoted into vials and stored frozen +. >CS10 (StemCell Technologies catalog number 07930).
After thawing, T cells were grown at 1.0X10 6 The density of individual cells/ml was plated on X-VIVO 15 TM Serum-free hematopoietic cell culture medium (Lonza Bioscience) containing 5% (v/v) fetal bovine serum, 55. Mu.M 2-mercaptoethanol, 10mM N-acetyl-L- (+) -cysteine, 10U/mL penicillin-streptomycin, plus 1 Xcytokines (200U/mL recombinant human interleukin-2, 5ng/mL recombinant human interleukin-7, and 5ng/mL recombinant human interleukin-15) in a T cell basal medium. By TransAct TM (1:100 dilution, miltenyi Biotec) activated T cells. Cells were subjected to electroporation in the presence of TransAct TM Is expanded in T cell basal medium for 48 hours.
4.2 editing T cells with ribonucleoprotein
RNP is produced by pre-annealing the individual crrnas and trrnas by mixing equal amounts of reagents and incubating for 2min at 95 ℃ and rapidly cooling. Double-guide consisting of preannealed crRNA and trRNA (dgRNA) was combined with Spy Cas9 protein (SEQ ID NO: 800) at 2:1dgRNA +.The protein molar ratios are incubated together to form Ribonucleoprotein (RNP) complexes. 96 well Nucleofector Using P3 Primary cells TM Kit (Lonza, catalog No. V4 SP-3960) and manufacturer pulse code were used to transfect CD3 with RNP in duplicate at the concentrations indicated in table 8 + T cells. T cell media was added to cells immediately after nuclear transfection and cultured for 2 or more days.
Four days after nuclear transfection, genomic DNA was prepared and NGS analysis was performed as described in example 1. Table 8 and FIG. 3A show the results in CD3 + Results of percent editing after CIITA editing with various guides in T cells.
4.3. Flow cytometry
On day 10 post-editing, T cells were phenotyped by flow cytometry to determine MHC class II protein expression. Briefly, T cells were incubated in a mixture of antibodies targeting HLA-DR-DP-DQ (Biolegend, cat. No. 361704) and CD3 (Biolegend, cat. No. 300322). The cells were then washed, processed on a Cytoflex flow cytometer (Beckman Coulter) and analyzed using FlowJo suite software. T cells are gated based on size, shape, viability and MHC class II expression. Table 8 and FIG. 3B show the results in CD3 + Results of percentage of MHC-II negative cells after CIITA editing with various primers in T cells. \
TABLE 8 percentage of editing after CIITA editing and percentage of MHC-II negative cells
/>
Example 5-T cell editing, CIITA guide RNA with Cas9 and BC22
5.1T cell preparation
T cells were edited with trans UGI and BC22 or Cas9 at CIITA locus to assess the effect of the editing type on MHC class II antigens.
Following manufacturer's prescriptionT cells were prepared from the leukocyte removal product using EasySep human T cell isolation kit (Stem Cell Technology, catalog No. 17951). T cells were cryopreserved in a cryo-store of cryo-store CS10 medium (catalog number 07930) for future use. After thawing, T cells were plated at 1.0X10 6 Individual cells/mL were plated in T cell R10 medium consisting of RPMI 1640 (Corning, catalog number 10-040-CV) containing 10% (v/v) fetal bovine serum, 2mM Glutamax (Gibco, catalog number 35050-061), 22. Mu.M of 2-mercaptoethanol, 100. Mu.M of nonessential amino acids (Corning, catalog number 25-025-Cl), 1mM sodium pyruvate, 10mM HEPES buffer, 1% penicillin-streptomycin plus 100U/mL recombinant human interleukin-2 (Peprotech, catalog number 200-02). By usingHuman T-activator CD3/CD28 (Gibco, catalog number 11141D) activates T cells. Cells were expanded in T cell medium for 72 hours prior to mRNA transfection.
5.2T cell editing by RNA electroporation
Solutions containing mRNA encoding Cas9 protein (SEQ ID NO: 801), BC22 (SEQ ID NO: 806) or UGI (SEQ ID NO 807) were prepared in sterile water. The CIITA of 50 μm targeting sgrnas was removed from its storage plate and denatured at 95 ℃ for 2 min, followed by cooling on ice. 72 hours after activation, T cells were harvested, centrifuged, and at 12.5X10 6 The concentration of individual T cells per milliliter was resuspended in P3 electroporation buffer (Lonza). For each well to be electroporated, 1X 10 will be used as described in Table 9 5 The individual T cells were mixed with 200ng of editing mRNA, 200ng of UGI mRNA and 20pmol of sgRNA in P3 electroporation buffer, with a final volume of 20. Mu.L. This mixture was transferred in duplicate to a 96 well nucleoactor TM The plates were electroporated with the manufacturer's pulse code. The electroporated T cells were allowed to stand in 180. Mu. l R10 medium plus 100U/mL recombinant human interleukin-2, and then transferred to a new flat bottom 96 well plate. The resulting plates were incubated at 37℃for 4 days. On day 10 post-editing, cells were collected for flow cytometry analysis and NGS sequencing.
5.3 flow cytometry and NGS sequencing
On day 10 post-editing, T cells were phenotyped by flow cytometry to target HLA-DR, DQ, DP-PE as described in example 4Catalog number 361704) and isotype control-PE (>Catalog number 400234) determines MHC class II protein expression. The DNA samples were subjected to PCR and subsequent NGS analysis as described in example 1. Table 9 shows CIITA gene editing and MHC class II negative results for cells edited with BC 22. Table 10 shows CIITA gene editing and MHC class II negative results for cells edited with Cas 9.
TABLE 9 percentage of editing and percentage of MHC-II negative cells after CIITA editing with BC22
/>
/>
TABLE 10 percentage of editing and percentage of MHC-II negative cells after CIITA editing with Cas9
/>
/>
* The G016111 target sequence is subject to naturally occurring C/T single nucleotide polymorphism.
Example 6 dose response and multiple editing
The three guides G016086, G016092 and G016067 from table 9 were further characterized for their editing efficacy as the amount of guide increased and combined with guides targeting TRAC (G013009, G016016 or G016017) and B2M (G015991, G015995 or G015996). In general, unless otherwise specified, guide RNAs identified as "gxxxxx" as used throughout the examples refer to 100nt modified versions of sgrnas, unless otherwise specified, such as those shown in the tables provided herein.
Cell preparation, activation and electroporation were performed as described in example 5 with the following deviations. Editing was performed using two mRNA species encoding BC22 (SEQ ID NO: 806) and UGI (SEQ ID NO: 807), respectively. Edits were assessed at various concentrations of sgrnas, as indicated in tables 11 and 12. When multiple guides are used in a single reaction, each guide represents a quarter of the total guide concentration.
On day 10 post-editing, T cells were phenotyped by flow cytometry to determine MHC class II protein expression as described in example 6. In addition, B2M detection was performed by B2M-FITC antibody (BioLegend, catalog No. 316304) and CD3 expression was determined using CD3-BV605 antibody (BioLegend, catalog No. 317322). The DNA samples were subjected to PCR and subsequent NGS analysis as described in example 1. Table 11 provides MHC class II negative flow cytometry results and NGS edits of cells edited with BC22 and individual guides targeting CIITA, wherein fig. 4A plots the percent conversion of C to T and fig. 4B plots the percent MHC class II negative. Table 12 shows MHC class II negative results for cells simultaneously edited with CIITA, B2M, TRAC and TRBC primers.
Table 11-percentage of MHC-II negative cells after CIITA editing and NGS results (n=2)
Tables 12-CIITA, TRAC, TRBC and percentage of antigen negative cells after B2M editing
EXAMPLE 7 comparison of sgRNA in T cells
T cells were edited at CIITA locus Cas9 to assess the effect of the editing type on MHC class II antigens.
7.1T cell preparation
A healthy human donor blood cell line (Hemacare) was obtained and cells were washed on a LOVO device and resuspended PBS/EDTA buffer (Miltenyi Biotec catalog No. 130-070-525). UsingPlus and->LS Disposable kit T cells were isolated via positive selection using CD4 and CD8 magnetic beads (Miltenyi Biotec catalog No. 130-030-401/130-030-801). T cells were aliquoted into vials and stored frozen +.>CS10 (StemCell Technologies catalog number 07930) and Plasmalyte a (Baxter catalog number 2B 2522X) for future use. After thawing, T cells were grown at 1.0X10 6 The density of individual cells/ml was plated on X-VIVO 15 TM A T cell basal medium comprising a serum-free hematopoietic cell culture medium (Lonza Bioscience) containing 5% (v/v) fetal bovine serum, 50. Mu.M 2-mercaptoethanol, 10mM N-acetyl-L- (+) -cysteine, 10U/mL penicillin-streptomycin, plus 1 Xcytokines (200U/mL recombinant human interleukin-2, 5ng/mL recombinant humanInterleukin-7 and 5. Mu.g/mL recombinant human interleukin-15). By TransAct TM (1:100 dilution, miltenyi Biotec) activated T cells. Cells were subjected to electroporation in the presence of TransAct TM Is expanded in T cell basal medium for 72 hours.
7.2T cell editing with RNA electroporation
Solutions containing mRNA encoding Cas9 (SEQ ID NO: 802) and mRNA encoding UGI (SEQ ID NO: 807) were prepared in sterile water. The guide RNA was denatured at 95℃for 2 min, followed by cooling on ice. T cells were harvested 72 hours after activation and at 12.5×10 6 The concentration of individual T cells per milliliter was resuspended in P3 electroporation buffer (Lonza). For each well to be electroporated, 1X 10 will be described in Table 13 5 The individual T cells were mixed with 200ng of editing mRNA, 200ng of UGI mRNA and 40pmol of sgRNA in P3 electroporation buffer, with a final volume of 20. Mu.L. This mixture was transferred in duplicate to a 96 well nucleoactor TM The plates were electroporated with the manufacturer's pulse code. The electroporated T cells were immediately allowed to stand in an Optmizer-based medium without cytokines. Cells were incubated in Optmizer-based medium with cytokines for 4 days at 37 ℃. After 96 hours, some cells were harvested for NGS analysis and the remaining T cells were 1:3 diluted into fresh OpTmizer-based medium with cytokines. The electroporated T cells were then cultured for an additional 11 days and collected for flow cytometry analysis.
7.3 flow cytometry
On day 11 post-editing, T cells were phenotyped by flow cytometry to target HLA-DR, DQ, DP-FITC [ ] as described in example 4Catalog number 361706) determines MHC class II protein expression. Table 13 shows MHC class II protein expression after electroporation with UGI mRNA combined with Cas 9.
TABLE 13 percentage of MHC-II negative cells after CIITA editing
/>
/>
* The concentration may have technical problems
EXAMPLE 8 CIITA insertion
8.1T cell preparation
Healthy human donor blood cell line (Hemacare) was obtained commercially, and cells were washed and resuspended in 2% pbs/EDTA buffer. UsingCD4/CD8 microbead kit (Miltenyi Biotec catalog No. 130-122-352), T cells were isolated via positive selection on MultiMACS (Miltenyi Biotec catalog No. 130-098-637). T cells were aliquoted into vials and stored frozen +.>CS10 (StemCell Technologies catalog number 07930).
After thawing, T cells were grown at 1.0X10 6 The density of individual cells/ml was plated on X-VIVO 15 TM Serum-free hematopoietic cell culture medium (Lonza Bioscience) containing 5% (v/v) fetal bovine serum, 55. Mu.M 2-mercaptoethanol, 10mM N-acetyl-L- (+) -cysteine, 10U/mL penicillin-streptomycin, plus 1 Xcytokines (200U/mL recombinant human interleukin-2, 5ng/mL recombinant human interleukin-7, and 5ng/mL recombinant human interleukin-15) in a T cell basal medium. The following day, T cells were treated with TransAct TM Activation (1:100 dilution, miltenyi Biotec). Cells were subjected to electroporation in the presence of TransAct TM Is expanded in T cell basal medium for 48 hours.
8.2T cell editing with ribonucleoprotein and AAV
The sgRNA was selected for incubation with recombinant Sp.Cas9-NLS protein (SEQ ID NO: 800) to form Ribonucleoprotein (RNP) complexes. The CIITA-targeted sgrnas were denatured at 95 ℃ for 2 min, followed by cooling at room temperature. An RNP mixture of 40uM sgRNA and 20uM Cas9-NLS protein was prepared and incubated at 25℃for 10 min. 2.5. Mu.L of RNP mix was combined with 1,000,000 CD3+ T cells in 20. Mu. L P3 electroporation buffer (Lonza). 25. Mu.L of RNP/cell mixture was transferred to corresponding wells of a Lonza short 96-well electroporation plate. Cells were electroporated in duplicate with the manufacturer's pulse code. Immediately after nuclear transfection, T cell basal medium was added to the cells and the cells were transferred to 24-well plates containing T cell medium containing cytokines. AAV constructs were designed to encode the mCherry reporter gene flanked by homology arms immediately 5 'and 3' of the cleavage site (SEQ ID nos. 1001-1003) of each guide. At 3X 10 5 AAV was added to the corresponding wells at the multiplicity of infection (MOI). The next day cells were transferred to 24 well Grex plates (Wilson Wolf, catalog No. 80192) and expanded for 10 days by changing the medium according to the manufacturer's protocol.
8.3 flow cytometry
On day 10 post-editing, T cells were phenotyped by flow cytometry to determine MHC class II protein expression and mCherry reporter expression. Briefly, T cells were isolated from CD4-BV 605%Directory number 317438), CD8-AF700 (/ -for)>Directory number 344724) and HLA-DR, DQ, DP-FITC (+.>Catalog number 361706) was incubated in an antibody cocktail. The cells were then washed, processed on a Cytoflex flow cytometer (Beckman Coulter) and analyzed using FlowJo suite software. Gating T cells based on size, shape, followed by CD4 and CD8 gatingAnd (5) controlling. The inserts were then quantified using mCherry expression as shown in table 14 and fig. 5A. MHC class II expression was also determined to quantify the frequency of editing, as shown in table 15 and fig. 5B.
Table 14-average percentage of cells positive for mCherry after editing.
TABLE 15 average percentage of MHC class II negative cells after editing
EXAMPLE 9 LNP titration in fixed ratio UGI T cells of BC22n
LNP delivery to activated human T cells was used to assess the efficacy of single site editing with Cas9 or BC22 n.
T cell preparation.
Healthy human donor blood cell apheresis is commercially available (Hemacare) and cells are washed on a LOVO device and resuspended PBS/EDTA buffer (Miltenyi Biotec catalog No. 130-070-525). UsingPlus and->LS Disposable kit T cells were isolated via positive selection using CD4 and CD8 magnetic beads (Miltenyi Biotec catalog No. 130-030-401/130-030-801). T cells were aliquoted into vials and stored frozen +.>CS10 (StemCell Technologies catalog number 07930) and Plasmalyte a (Baxter catalog number 2B 2522X) for future use. SolutionAfter freezing, T cells were plated at 1.0X10 6 The density of individual cells/ml was plated on X-VIVO 15 TM Serum-free hematopoietic cell culture medium (Lonza Bioscience) containing 5% (v/v) fetal bovine serum, 50. Mu.M 2-mercaptoethanol, 10mM N-acetyl-L- (+) -cysteine, 10U/mL penicillin-streptomycin, plus 1 Xcytokines (200U/mL recombinant human interleukin-2, 5ng/mL recombinant human interleukin-7, and 5ng/mL recombinant human interleukin-15) in a T cell basal medium. By TransAct TM (1:100 dilution, miltenyi Biotec) activated T cells. Cells were expanded in T cell basal medium for 72 hours prior to LNP transfection. />
9.2T cell editing
Each RNA species, i.e., UGI mRNA, sgRNA, or edited mRNA, was formulated in LNP separately as described in example 1. The edited mRNA encodes BC22n (SEQ ID NO: 805) or Cas9 (SEQ ID NO: 803). The sgRNA (SEQ ID NO: 395) targeting CIITA (G016086) was used. UGI mRNA (SEQ ID NO: 807) was delivered in the experimental Cas9 and BC22n groups to normalize the amount of lipid. Previous experiments have established that UGI mRNA when used with Cas9 mRNA does not affect the overall editing or editing profile. LNP compositions were mixed to a fixed total mRNA weight ratio of 6:3:2 for editing mRNA, guide RNA, and UGI mRNA, respectively, as described in table 16. The LNP mixture was incubated at 37 ℃ for 5 minutes in T cell basal medium with 6% cynomolgus monkey serum (Bioreclamation IVT, cat-No. CYN 220760) instead of fetal bovine serum.
72 hours after activation, T cells were washed and suspended in basal T cell medium. Pre-incubation of LNP mixture at 1X 10 5 Individual T cells/well are added to each well. During the experiment, T cells were incubated with 5% CO at 37 °c 2 Incubation was performed. T cell media was changed on days 6 and 8 after activation and tenth after activation, cells were harvested for analysis by NGS and flow cytometry. NGS analysis was performed as described in example 1. Table 16 and fig. 6A describe T cell editing. In all guides tested, the total edits and edits with C as T show a direct, dose-responsive relationship with increasing amounts of BC22n mRNA, UGI mRNA, and guide. The conversion of insertions/deletions and C into A or G is inversely related to the dosageThe lower doses in (a) resulted in a higher percentage of these mutations. In Cas9 edited samples, total editing and insertion/deletion activity increased with total RNA dose.
Table 16-percentage compiled as total reading-single guide delivery (n=2)
On day 10 after activation, T cells were phenotyped by flow cytometry to measure cell surface protein loss using antibodies targeting HLA DR DQ DP-PE (BioLegend, catalog No. 361704) and DAPI (BioLegend, catalog No. 422801) as described in example 5. A subset of unedited cells were treated with isotype control-PE Catalog number 400234) were incubated together.
Table 17 and FIG. 6B report the phenotyping results as the percentage of cells negative for antibody binding. The percentage of antigen-negative cells for both BC22n and Cas9 samples increased in a dose-responsive manner as total RNA increased. For all tested guides, cells edited with BC22n displayed similar or higher protein knockouts compared to cells edited with Cas 9.
Table 17-flow cytometry data-percentage of MHC class II negative cells (n=2)
EXAMPLE 10 off target analysis
10.1 biochemical off-target analysis
Biochemical methods (see, e.g., cameron et al, nature methods.6,600-606; 2017) were used to determine potential off-target genomic sites cleaved by Cas9 using specific guides targeting CIITA. In this experiment, two human CIITA-targeted sgrnas were screened using genomic DNA purified from lymphoblastic cell line NA24385 (Coriell Institute) and three control primers with known off-target profiles. The number of potential off-target sites detected in biochemical assays using primer concentrations of 192nM and 64nM Cas9 protein is shown in table 18.
Table 18: biochemical off-target analysis
10.2 Targeted sequencing to verify potential off-target sites
Potential off-target sites predicted by detection assays, such as the biochemical methods used above, can be assessed using targeted sequencing of identified potential off-target sites to determine whether off-target cleavage at the site is detected.
In one method, cas9 and sgrnas of interest (e.g., the sgrnas with potential off-target sites for evaluation) are introduced into primary T cells. T cells are then lysed and primers flanking the potential off-target sites are used to generate amplicons for NGS analysis. The identification of a certain level of insertion/deletion may verify potential off-target sites, while the lack of insertion/deletion found at potential off-target sites may indicate false positives in the off-target prediction assay used.
Example 11 multiple editing of T cells by sequential LNP delivery
T cells are engineered by a series of gene disruptions and insertions. Healthy donor cells were sequentially treated with four LNP compositions, each LNP co-formulated with mRNA encoding Cas9 (SEQ ID No. 802) and sgrnas targeting TRAC (G013006), TRBC (G016239), CIITA (G013676) or HLA-A (G018995). The LNP composition was formulated with lipid A, cholesterol, DSPC and PEG2k-DMG in a molar ratio of 50:38.5:10:1.5, respectively. The lipid nucleic acid assemblies were formulated at a lipid amine to RNA phosphate (N: P) molar ratio of about 6 and a gRNA to mRNA ratio of 1:2 by weight. The transgenic T cell receptor (WT 1 TCR) (SEQ ID NO: 1000) targeting Wilms' tumor antigen was integrated into the TRAC cleavage site by using AAV delivery homology directed repair templates.
T cell preparation
T cells were isolated from leukopenia products (STEMCELL Technologies) of three healthy HLA-A2+ donors. T cells were isolated using EasySep human T cell isolation kit (STEMCELL Technologies, catalog No. 17951) following the manufacturer's protocol and cryopreserved using a Cryostor CS10 (STEMCELL Technologies, catalog No. 07930). One day before T cell editing was started, cells were thawed and plated in T Cell Activation Medium (TCAM): CTS Optmizer (Thermofiser, catalog No. A3705001) was left standing overnight, and the medium was supplemented with 2.5% human AB serum (Gemini, catalog No. 100-512), 1 XGlutaMAX (Thermofiser, catalog No. 35050061), 10mM HEPES (Thermofiser, catalog No. 15630080), 200U/mL IL-2 (Peprotech, catalog No. 200-02), IL-7 (Peprotech, catalog No. 200-07), IL-15 (Peprotech, catalog No. 200-15).
LNP treatment and expansion of T cells
LNP compositions were prepared daily in ApoE-containing medium and delivered to T cells as described in table 19 and below.
Table 19: -edit sequence of T cell engineering
| Group of | Day 1 | Day 2 | Day 3 | Day 4 |
| 1 | Unedited with | Unedited with | Unedited with | Unedited with |
| 2 | TRBC | CIITA | TRAC | HLA-A |
| 3 | TRBC | HLA-A | TRAC | CIITA |
| 4 | TRBC | TRAC |
On day 1, LNP compositions as indicated in Table 19 were incubated at a concentration of 5 μg/mL in TCAM containing 5 μg/mL rhApoE3 (Peprotection, catalog number 350-02). At the same time, T cells were harvested, washed, and at 2X 10 6 The individual cells/ml density was resuspended in TCAM with a 1:50 dilution of T cell TransAct human reagent (Miltenyi, catalog number 130-111-160). T cells and LNP-ApoE medium were mixed at a 1:1 ratio and plated in flasks overnight.
On day 2, LNP compositions as indicated in Table 19 were incubated at a concentration of 25. Mu.g/mL in TCAM containing 20. Mu.g/mL rhApoE3 (Peprotection, catalog No. 350-02). The LNP-ApoE solution was then added to the appropriate culture at a 1:10 ratio.
On day 3, TRAC-LNP compositions were incubated at a concentration of 5. Mu.g/mL in TCAM containing 10. Mu.g/mL of rhApoE3 (Peprotech, cat. No. 350-02). T cells were harvested, washed, and at 1X 10 6 The individual cells/ml density was resuspended in TCAM. T cells and LNP-ApoE medium were mixed at a 1:1 ratio and plated in flasks. WT1 AAV (SEQ ID NO: 1000) was then used in 3X 10 5 The MOI of each genome copy/cell was added to each group.
On day 4, LNP compositions as indicated in Table 19 were incubated at a concentration of 5 μg/mL in TCAM containing 5 μg/mL rhApoE3 (Peprotection, catalog number 350-02). The LNP-ApoE solution was then added to the appropriate culture at a 1:1 ratio.
On days 5-11: t cells were transferred to T Cell Expansion Medium (TCEM) of 24-well GREX plate (Wilson Wolf, catalog No. 80192): in CTS Optmizer (Thermofiser, catalog No. A3705001), the medium was supplemented with 5% CTS immune cell serum replacement (Thermofiser, catalog No. A2596101), 1 XGlutaMAX (Thermofiser, catalog No. 35050061), 10mM HEPES (Thermofiser, catalog No. 15630080), 200U/mL IL-2 (Peprotech, catalog No. 200-02), IL-7 (Peprotech, catalog No. 200-07) and IL-15 (Peprotech, catalog No. 200-15). Cells were expanded according to the manufacturer's protocol. T cells were expanded for 6 days with medium changed every other day. The CELLs were counted using a Vi-CELL counter (Beckman Coulter) and fold expansion was calculated by dividing CELL yield by starting material as shown in table 20.
TABLE 20 fold expansion after multiple editing T cell engineering
| Group of | Donor A | Donor B | Donor C | Average value of | SD |
| 1 | 331.40 | 362.24 | 533.18 | 408.94 | 108.69 |
| 2 | 61.82 | 72.15 | 116.13 | 83.37 | 28.84 |
| 3 | 64.08 | 76.29 | 157.75 | 99.37 | 50.92 |
| 4 | No data | 146.78 | 331.67 | 239.22 | 130.74 |
11.3. Quantitative T cell editing by flow cytometry and NGS
After expansion, the edited T cells were analyzed by flow cytometry to determine HLA-A2 expression (HLA-A + ) HLA-DR-DP-DQ expression (MHC II) after CIITA knockdown + ) WT1-TCR expression (CD 3) + Vb8 + ) And residual endogenous TCR expression (CD 3) + Vb8 - ) Or mismatched TCR expression (CD 3) + Vb8 Low and low ). Incubating T cells with a mixture of antibodies targeting: CD4 (Biolegend, catalog No. 300524), CD8 (Biolegend, catalog No. 301045), vb8 (Biolegend, catalog No. 348106), CD3 (Biolegend, catalog No. 300327), HLA-A2 (Biolegend, catalog No. 343306), HLA-DRDPDQ (Biolegend, catalog No. 361706), CD62L (Biolegend, catalog No. 304844), CD45RO (Biolegend, catalog No. 304230). The cells were then washed and analyzed on a Cytoflex LX instrument (Beckman Coulter) using FlowJo suite software. T cells were gated according to size and CD4/CD8 status, followed by measurement of the expression of the edit and insert markers. The percentage of cells expressing the relevant cell surface proteins after sequential T cell engineering is shown in table 21 and figures 7A-7F (for CD8 + T cells) and table 22 and figures 8A-8F (for CD 4) + T cells). Complete editing of CD4 + Or CD8 + Percentage gating of T cells to CD3 + Vb8 + HLA-A - MHC II - Percent of the total weight of the composition. High levels of HLA-A and MHC II knockdown were observed in the edited samples, as well as WT1-TCR insertion and endogenous TCR KO. Genomic DNA was prepared and NGS analysis was performed as described in example 1, except for flow cytometry analysis, to determine the rate of editing at each target site. Table 23 and FIGS. 9A-9D show the results of percent editing at CIITA, HLA-A and TRBC1/2 loci, where the pattern across groups is consistent with that identified by flow cytometry. In all groups, the TRBC1/2 locus was edited to >90-95％。
/>
Table 23: percent insertion/deletion at CIITA, HLA-A, TRBC1 and TRBC2 after sequential T-cell editing
EXAMPLE 12 NK cell functional killing assay
T cells that were edited in various combinations to destroy CIITA, HLA-A or B2M or overexpress HLa-E were tested for their ability to resist Natural Killer (NK) cell mediated killing.
12.1. Engineered T cells and purification
After thawing, pan CD3+ T cells (StemCell, HLA-A 02.01/A03.01) were expanded at 0.5X10 ≡ 6 The density of individual cells per milliliter was plated in T cell RPMI medium consisting of RPMI 1640 (Invitrogen, catalog No. 22400-089) containing 5% (v/v) fetal bovine serum, 1 XGluatmax (Gibco, catalog No. 35050-061), 50. Mu.M 2-mercaptoethanol, 100. Mu.M nonessential amino acids (Invitrogen, catalog No. 11140-050), 1mM sodium pyruvate, 10mM HEPES buffer, 1% penicillin-streptomycin, and 100U/mL recombinant human interleukin-2 (Peprotech, catalog No. 200-02). By TransAct TM (1:100 dilution, miltenyi Biotec) activated T cells.
T cells were edited to disrupt the B2M gene one day after activation as described in table 24. Briefly, LNP compositions containing Cas9 mRNA and B2M-targeted sgRNA G000529 (SEQ ID NO: 216) were formulated as described in example 1. LNP compositions were incubated at 37℃for 15 min in RPMI-based medium with cytokines as described above supplemented with 1. Mu.g/ml recombinant human ApoE3 (Peprotech, cat. No. 350-02). LNP mixtures were added to two million activated T cells to give a final concentration of 2.5 μg total LNP/mL.
TABLE 24 sequence of sequential editing and Virus transduction
| Conditions (conditions) | Day 1 | Day 2 | Day 3 |
| Unedited with | |||
| B2M - | B2M LNP | ||
| B2M - +HLA-E | B2M LNP | HLA-E lentiviruses | |
| HLA-A - MHC II - | CIITA LNP | HLA-A LNP | |
| HLA-A - | HLA-A LNP |
Two days after activation, additional T cells were edited with LNP composition to disrupt CIITA genes. As described for B2M editing, this editing was performed using LNP compositions containing Cas9 mRNA and CIITA-targeted sgRNA G013675 (comprising the sgrnas of SEQ ID NO:27, as shown in table 2). The LNP composition used in this step was formulated with lipid A, cholesterol, DSPC and PEG2k-DMG in molar ratios of 50:38.5:10:1.5, respectively. The lipid nucleic acid assemblies were formulated at a lipid amine to RNA phosphate (N: P) molar ratio of about 6 and a gRNA to mRNA ratio of 1:2 by weight.
Three days after activation, all edited and unedited cells were resuspended in fresh medium without tranact. B2M edited T cell samples were transduced with lentivirus expressing HLA-E by EF1a promoter (SEQ ID No. 1004) by centrifugation at 1000g for 1 hour at 37℃with an MOI of 10. The CIITA-edited T cell sample was further edited with LNP composition to disrupt the HLA-A gene. The editing was performed as described above for B2M editing using LNP composition containing Cas9 mRNA and HLA-A targeted sgRNA G019000 formulated with lipid a, cholesterol, DSPC and PEG2k-DMG at a molar ratio of 50:38.5:10:1.5, respectively. The lipid nucleic acid assemblies were formulated at a lipid amine to RNA phosphate (N: P) molar ratio of about 6 and a gRNA to mRNA ratio of 1:2 by weight. Four days after activation, all cells were transferred to GREX plates (Wilson Wolf, catalog No. 80240M) for expansion.
Seven days after activation, HLA-E-infected T cells were selected for HLA-E expression using biotinylated anti-HLA-E antibodies (Biolegend) and anti-biotin microbeads (Miltenyi Biotec, catalog No. 130-090-485) and magnetic LS columns (Miltenyi Biotec, catalog No. 130-042-401) according to the manufacturer's protocol.
Similarly, nine days after activation, CIITA-edited T cells were negative selected for lack of MHC II expression using biotinylated anti-HLA class II antibodies (Miltenyi, catalog number 130-104-823), anti-biotin microbeads (Miltenyi Biotec, catalog number 130-090-485) and magnetic LS columns (Miltenyi Biotec, catalog number 130-042-401) according to the manufacturer's protocol.
12.2 flow cytometry
NK cell mediated cytotoxicity against engineered T cells was determined. For this, T cells were co-cultured with HLA-B/C matched CTV labeled NK cells at effector to target ratios (E: T) of 10:1, 5:1, 2.5:1, 1.25:1 and 0.625:1 for 21 hours. Cells were stained with 7AAD (Pharmingen, catalog No. 559925), treated on a Cytoflex flow cytometer (Beckman Coulter) and analyzed using FlowJo suite software. T cells are gated based on CTV negativity, size, and shape and viability. Table 25 and fig. 10 show the percentage of T cell lysis after NK cell challenge.
TABLE 25 percent T cell lysis after NK cell challenge of engineered T cells
Example 13: HLA-A and CIITA partial match in NK cell in vivo killing mouse model
Implantation of 1.5X10 to female NOG-hIL-15 mice 6 Primary NK cells were then injected 4 weeks later with engineered T cells containing luciferase +/-HLA-A, CIITA or HLA-A/CIITA KO to determine if 1) the implanted NK cells could readily lyse control T cells (B2M -/- ) And 2) adding a partial match edit (HLA-A or CIITA) to provide a protective effect for NK cells to lyse T cells in vivo.
13.1. Preparation of T cells containing luciferase +/-HLA-A, CIITA or HLA-A/CIITA KO
T cells were isolated from peripheral blood of healthy human donors with the following MHC I phenotypes: HLA-A.times.02:01:01G, 03:01:01G, HLA-B.times.07:02:01G, HLA-C.times.07:02:01G. Briefly, the leukocyte removal package (Stemcell Technologies) was treated in ammonium chloride RBC lysis buffer (Stemcell Technologies; catalog No. 07800) for 15 minutes to lyse the erythrocytes. Peripheral Blood Mononuclear Cell (PBMC) counts were determined after lysis and T cell isolation was performed using EasySep human T cell isolation kit (Stemc ell Technologies, catalog No. 17951) according to the manufacturer's protocol. Isolated cd3+ T cells were resuspended in a router CS10 medium (Stemcell Technologies, cat# 07930) and frozen in liquid nitrogen until further use.
Frozen T cells were grown at 1X 10 6 Cell concentration of individual cells/mL was thawed in T Cell Growth Medium (TCGM) composed of OpTmizer TCGM as described in example 3 and further supplemented with 100U/mL recombinant human interleukin-2 (Peprotech, cat# 200-02), 5ng/mL IL-7 (Peprotech, cat# 200-07), 5ng/mL IL-15 (Peprotech, cat# 200-15). T cell TransAct at 37℃with 1:100 dilution TM (Miltenyi Biotec, catalog No. 130-111-160) cells were activated for 24 hours.
24 hours after activation, 500. Mu.l of 1X 10 in fresh TCGM without cytokines were taken up 6 Individual T cells were transduced by centrifugation at 1000xG for 60 min at 37 ℃ with 150 μl luciferase lentivirus (Imanis Life Sciences, catalog No. LV 050L). Transduced cells were expanded in TCGM with cytokines in 24-well G-Rex plates (Wilson Wolf, catalog No. 80192M) at 37 ℃ for 24 hours.
48 hours after activation, luciferase LV-infected T cells were edited to disrupt the B2M or HLA-A genes. Briefly, LNP compositions containing mRNA encoding cas9 (SEQ ID NO: 802) and sgRNA G019000 targeting HLA-A (SEQ ID NO: 217) were formulated with lipid A, cholesterol, DSPC, and PEG2k-DMG at 50:38.5:10:1.5 molar ratios, respectively. The lipid nucleic acid assemblies were formulated at a lipid amine to RNA phosphate (N: P) molar ratio of about 6 and a gRNA to mRNA ratio of 1:2 by weight. LNP compositions containing Cas9 mRNA and B2M-targeted sgRNA G000529 (SEQ ID NO: 216) were formulated as described in example 1. LNP compositions were incubated at 37℃for 15 min in an Optmizer TCGM further supplemented with 1. Mu.g/ml recombinant human ApoE3 (Peprotech, catalog No. 350-02) in the absence of serum or cytokines. T cells were washed and suspended in TCGM with cytokines. The pre-incubated LNP and T cells were mixed to produce a final concentration of 0.5e 6T cells/mL and LNP of 2.5 μg total RNA/mL in TCGM with 5% human AB serum, 100U/mL recombinant human interleukin-2 (Peprotech, cat. No. 200-02), 5ng/mL IL-7 (Peprotech, cat. No. 200-07), 5ng/mL IL-15 (Peprotech, cat. No. 200-15). Another set of cells was mock-edited with medium containing ApoE3 but no LNP composition. All cells were incubated at 37℃for 24 hours.
72 hours after activation, cells were edited to destroy CIITA and LNP was administered on luciferase and HLA-A edited cells or luciferase cells alone. Briefly, cells were transduced with LNP compositions containing Cas9 mRNA and sgRNA G013675 (comprising the sgRNA of SEQ ID NO:27, as shown in Table 2) as described for HLA-A editing. 96 hours after activation, cells were washed and transferred to 24-well G-Rex. Media with fresh cytokines was changed every 2 days. Day 15 post-activation, GFP was used with BD FACS Aria Flow Sorter + The edited T cells were sorted on cells to enrich for cells expressing luciferase. For the B2M KO luciferase group, in GFP + And MHC-I - Cells were sorted up. The sorted cells were allowed to stand overnight in TCGM medium with cytokines in an incubator at 37 ℃. The following day, T cells were diluted 1:100 with T cell TransAct TM The stimulation was continued for a further 24 hours. 24 hours after restimulation, the TransAct was washed off and T cells were cultured and maintained in G-Rex plates for 15 days with periodic replacement of medium and cytokines.
15 days after restimulation, NK cell mediated cytotoxicity against engineered T cells was measured in vitro as in example 12, with the following exceptions. Analysis was performed using an Optmizer TCGM with 100. Mu.l/ml IL-2. T cells were co-cultured overnight with HLA-B/C matched CTV labeled NK cells at effector to target ratios (E: T) of 10:1, 5:1, 2.5:1, 1.25:1 and 0.625:1. Cells were incubated with BrightGlo luciferase reagent (plagmager, cat# E2620) and treated in ClarioStar on CellTiter Glo program to determine NK cell lysis of T cells based on luciferase signal. Table 26 shows the percentage of T cell lysis after NK cell challenge. In vitro, B2M editing cells displayed sensitivity to NK killing, while HLA-A editing, CIITA editing and HLA-A, CIITA dual editing cells displayed protection against NK-mediated lysis.
TABLE 26 percent lysis of luciferase-transduced T cells following NK cell challenge
13.2. Protection of HLA-A and CIITA double knockout T cells from NK killing
For in vivo studies, NK cells isolated from white blood cell harvest by methods known in the art were washed with HBSS (Gibco, catalog No. 14025-092) and washed at 10X 10 6 Individual cells/ml were resuspended for injection into 150 μl HBSS. 22 female NOG-hIL-15 mice (Taconic) were dosed by tail vein injection of 1.5e6 isolated NK cells. An additional 27 female NOG-hIL-15 served as a control for non-NK injections.
Mice were injected with unedited or engineered T cells as described in table 26 28 days after NK cell injection. Briefly, 16 days after the second activation, wash in PBS and at 6×10 6 After resuspension of individual cells/150 μl in HBSS solution, engineered T cells were injected.
IVIS imaging of live mice was performed to identify luciferase-positive T cells by IVIS spectroscopy. IVIS imaging was performed 6 hours, 24 hours, 48 hours, 8 days, 13 days, 18 days and 27 days after T cell injection. Mice were prepared for imaging by intraperitoneal injection of D-fluorescein at 10 μl/g body weight, about 150 μl per animal, according to manufacturer's recommendations. Animals were anesthetized and then placed in an IVIS imaging unit. Visualization is performed with the exposure time set to automatic, field D, medium binning and F/stop set to 1. Table 27 and FIG. 11A show the emissivity (photons/s/cm 2/sr) of luciferase-expressing T cells present at various time points after injection. FIG. 11B shows the emissivity (photons/s/cm 2/sr) of luciferase-expressing T cells present in different groups of mice after 27 days. In vivo, B2M editing cells displayed sensitivity to NK killing, while HLA-A editing, CIITA editing and HLA-A, CIITA dual editing cells displayed protection against NK mediated lysis. Unexpectedly, even after reduction of one of the three highly polymorphic MHC class I proteins (HLA-A), the cells are still protected from NK-mediated rejection.
Table 27-emissivity (photons/s/cm 2/sr) of luciferase expressing T cells from treated mice at intervals after T cell injection.
/>
Example 14: HLA-A and CIITA partial match in NK cell in vivo killing mouse model
Implantation of 1.5X10 to female NOG-hIL-15 mice 6 Primary NK cells were then injected 4 weeks later with engineered T cells containing luciferase +/-HLA-A, CIITA KO and HD1 TCR to determine if 1) the implanted NK cells could readily lyse control T cells (B2M -/- ) And 2) adding partial match editing (HLA-A&CIITA) provides a protective effect for NK cell lysis in vivo of T cells with exogenous HD1 TCR.
14.1. Preparation of T cells containing luciferase +/-HLA-A/CIITA KO and HD1 TCR
T cells were isolated from peripheral blood of healthy human donors with the following MHC I phenotypes: HLA-A.times.02:01:01G, 03:01:01G, HLA-B.times.07:02:01G, HLA-C.times.07:02:01G. Briefly, the leukocyte removal package (Stemcell Technologies) was treated in ammonium chloride erythrocyte lysis buffer (Stemcell Technologies; catalog No. 07800) for 15 minutes to lyse the erythrocytes. Peripheral Blood Mononuclear Cell (PBMC) counts were determined after lysis and T cell isolation was performed using EasySep human T cell isolation kit (Stemc ell Technologies, catalog No. 17951) according to the manufacturer's protocol. Isolated cd3+ T cells were resuspended in a router CS10 medium (Stemcell Technologies, cat# 07930) and frozen in liquid nitrogen until further use.
Frozen T cells were grown at 1.5X10 6 Cell concentration of individual cells/ml was thawed in T Cell Activation Medium (TCAM) consisting of OpTmizer TCGM, described in example 3Is described in (2) and is further supplemented with 100U/mL recombinant human interleukin-2 (Peprotech, catalog number 200-02), 5ng/mL IL-7 (Peprotech, catalog number 200-07), 5ng/mL IL-15 (Peprotech, catalog number 200-15). The cells were allowed to stand at 37℃for 24 hours.
T cells were counted and at 2 x 10 at 24 hours after thawing 6 Each cell/ml was resuspended in TCAM medium and 1:50 Transact was added. The cells were mixed and incubated at 37℃for 20 to 30 minutes. LNP compositions containing mRNA encoding Cas9 (SEQ ID NO: 802) and CIITA-targeted sgRNA G013675 (comprising the sgRNA of SEQ ID NO:27, as shown in Table 2) were formulated with lipid A, cholesterol, DSPC, and PEG2k-DMG at 50:38.5:10:1.5 molar ratios, respectively. The lipid nucleic acid assemblies were formulated at a lipid amine to RNA phosphate (N: P) molar ratio of about 6 and a gRNA to mRNA ratio of 1:2 by weight. 5 μg/ml of LNP composition was incubated for 15 min at 37℃in an Optmizer TCAM further supplemented with 5 μg/ml recombinant human ApoE3 (Peprotech, catalog number 350-02). Pre-incubated LNP compositions and T cells were mixed with Transact to produce 1X 10 in TCAM medium with 2.5% human AB serum, 100U/mL recombinant human interleukin-2 (Peprotech, cat. No. 200-02), 5ng/mL IL-7 (Peprotech, cat. No. 200-07) and 5ng/mL IL-15 (Peprotech, cat. No. 200-15) 6 Final concentrations of LNP of individual T cells/ml and 2.5 μg total RNA/ml. Another set of cells was mock-edited with medium containing ApoE3 but no LNP composition. All cells were incubated at 37℃for 24 hours.
48 hours after activation, all groups were transduced with the EF1α -GFP-Luc lentivirus. Lentiviruses were removed from-80 ℃ and thawed on ice. Cells were collected by group and centrifuged at 500×g for 5min to wash out LNP composition and medium. Cells were individually grouped in 2X 10 groups 6 Individual cells/ml were resuspended in TCAM medium. Subsequently, 500. Mu.l of the cell suspension was transferred to a sterile eppendorf tube (total 1X 10) 6 Individual cells) and 100 μl of lentivirus was added. The cells were centrifuged at 1000 XG for 60 minutes at 37 ℃. After centrifugation, cells were combined according to their groups and at 1×10 6 Individual cells/ml TCAM medium was resuspended, containing human AB serum at a final concentration of 2.5%,100U/mL recombinant human interleukin-2 (Peprotech, catalog number 200-02), 5ng/mL IL-7 (Peprotech, catalog number 200-07) and 5ng/mL IL-15 (Peprotech, catalog number 200-15) were incubated at 37℃for 24 hours.
72 hours after activation, luciferase-transduced T cells were treated with LNP compositions to disrupt the TRAC gene and further treated with HD1 AAV to insert HD1 TCR at the TRAC locus. Cells were collected by group and centrifuged at 500 Xg for 5min to wash out lentiviruses and medium. The cells were then incubated at 1X 10 6 Individual cells/ml were resuspended in TCAM medium. LNP compositions containing mRNA encoding Cas9 (SEQ ID NO: 802) and TRAC-targeted sgRNA G013006 (SEQ ID NO: 203) were formulated with lipid A, cholesterol, DSPC and PEG2k-DMG at a molar ratio of 50:38.5:10:1.5, respectively. The lipid nucleic acid assemblies were formulated at a lipid amine to RNA phosphate (N: P) molar ratio of about 6 and a gRNA to mRNA ratio of 1:2 by weight. 5 μg/ml LNP composition was incubated for 15 min at 37℃in an Optmizer TCAM further supplemented with 5 μg/ml recombinant human ApoE3 (Peprotection, catalog number 350-02). Pre-incubated LNP compositions and T cells were mixed with Transact to generate 1X 10 in TCAM with 2.5% human AB serum, 100U/mL recombinant human interleukin-2 (Peprotech, cat. No. 200-02), 5ng/mL IL-7 (Peprotech, cat. No. 200-07) and 5ng/mL IL-15 (Peprotech, cat. No. 200-15) 6 Final concentrations of LNP of individual T cells/ml and 2.5 μg total RNA/ml. Vials of EF1 alpha-HD 1AAV were thawed on a laboratory bench and at 3 x 10 5 GC/cells were added to TRAC LNP treated cells. The cells were then incubated at 37℃for 24 hours.
Cells were then treated 96 hours after activation and the final round of editing was performed with TRBC LNP alone or in combination with HLA-A LNP. The B2M KO group was treated with the B2M LNP. Cells were collected by group and centrifuged at 500×g for 5min to wash out LNP composition and medium. The cells were then incubated at 1X 10 6 Individual cells/ml were resuspended in TCAM medium. Briefly, LNP compositions containing mRNA encoding Cas9 (SEQ ID NO: 802) and HLA-A targeted sgRNA G018995 (SEQ ID NO: 214) were formulated as described in example 1. LNP compositions containing Cas9 mRNA and B2M-targeting sgRNA G000529 (SEQ ID NO: 216) and methods of using Cas9 mRNA and TRBC-targeting LNP compositionsLNP compositions of sgRNA G016239 (SEQ ID NO: 211) were formulated with lipid A, cholesterol, DSPC and PEG2k-DMG in a 50:38.5:10:1.5 molar ratio, respectively. The lipid nucleic acid assemblies were formulated at a lipid amine to RNA phosphate (N: P) molar ratio of about 6 and a gRNA to mRNA ratio of 1:2 by weight. 5 μg/ml of LNP composition was incubated for 15 min at 37℃in an Optmizer TCAM further supplemented with 5 μg/ml recombinant human ApoE3 (Peprotech, catalog number 350-02). Pre-incubated LNP compositions and T cells were mixed with Transact to generate 1X 10 in TCAM with 2.5% human AB serum, 100U/mL recombinant human interleukin-2 (Peprotech, cat. No. 200-02), 5ng/mL IL-7 (Peprotech, cat. No. 200-07) and 5ng/mL IL-15 (Peprotech, cat. No. 200-15) 6 Final concentrations of LNP of individual T cells/ml and 2.5 μg total RNA/ml. For simultaneous TRBC and HLA-A editing, LNP and ApoE3 were formulated at 4 x final concentration, followed by first adding TRBC LNP to T cells and incubating for 15 min at 37 ℃. After incubation, the cells were incubated for 24 hours with the addition of the pre-formulated HLA-A LNP composition.
After the last round of editing, cells were washed by spinning at 500 XG for 5min and resuspended in TCGM medium containing 5% human AB serum, 100U/mL recombinant human interleukin-2 (Peprotech, cat. No. 200-02), 5ng/mL IL-7 (Peprotech, cat. No. 200-07) and 5ng/mL IL-15 (Peprotech, cat. No. 200-15).
Day 5 post-activation, GFP using BD FACS Aria Flow Sorter + T cells were sorted and edited on cells to enrich for cells expressing luciferase. The sorted cells were allowed to stand overnight in TCGM medium with cytokines in an incubator at 37 ℃. The following day, T cells were diluted 1:100 with T cell TransAct TM The stimulation was continued for a further 24 hours. 24 hours after restimulation, transAct was washed away TM T cells were cultured and maintained in G-Rex plates for 15 days with periodic replacement of medium and cytokines.
The level of editing was confirmed via flow cytometry 15 days after the first re-stimulation, and the cells were washed and resuspended in HBSS buffer for injection.
HLA-A and CIITA double knockout T cells demonstrate protective effects on NK killing
For in vivo studies, NK cells isolated from white blood cell harvest by methods known in the art were washed with HBSS (Gibco, catalog No. 14025-092) and washed at 10X 10 6 Individual cells/ml were resuspended for injection into 150 μl HBSS. 30 female NOG-hIL-15 mice (Tacouc) were injected by tail vein 1.5X10 6 Individual isolated NK cells were administered. Another 25 female NOG-hIL-15 served as a control for non-NK injections.
Mice were injected with unedited or engineered T cells as described in table 28 day after NK cell injection. Briefly, 16 days after the second activation, wash in PBS and at 6.0×10 6 After resuspension of individual cells/150. Mu.L in HBSS solution, 0.2X10 were injected 6 Engineering T cells.
IVIS imaging of live mice was performed to identify luciferase-positive T cells by IVIS spectroscopy. IVIS imaging was performed 24 hours, 48 hours, 72 hours, 6 days, 10 days, 13 days, 17 days, 20 days, 24 days, 27 days, 31 days, 34 days, 38 days, 42 days, 44 days, 48 days, 55 days, 63 days, 72 days, 77 days, 85 days, and 91 days after T cell injection. Mice were prepared for imaging by intraperitoneal injection of D-fluorescein at about 150 μl per animal at 10 μl/g body weight, according to manufacturer's recommendations. Animals were anesthetized and then placed in an IVIS imaging unit. Visualization is performed with the exposure time set to automatic, field D, medium binning, and F/stop set to 1. Table 29 and FIG. 12A show the emissivity (photons/s/cm 2/sr) of luciferase-expressing T cells present at various time points to 91 days after injection. FIG. 12B shows the emissivity (photons/s/cm 2/sr) of luciferase-expressing T cells present in different groups of mice after 31 days. In vivo, B2M-edited cells displayed sensitivity to NK killing, while HLA-A, CIITA double-edited cells displayed protection against NK-mediated lysis.
TABLE 28 engineering of T cells
Table 29-total flux (photons/s) of T cells expressing luciferase from treated mice at intervals after T cell injection.
/>
Example 15: MHC I and MHC II KO in vivo efficacy of HD 1T cells
Implantation of 0.2X10 into female NOG-hIL-15 mice 6 Personal acute lymphoblastic leukemia cell line 697-Luc2 followed by injection of 10X 10 6 Engineered T cells with various edits are used to determine whether the edits provide a specific anti-tumor effect. The T cell group studied included: non-edited T cell control group (697 only); TRAC and TRBC have edited T cells (TCR KO); TRAC and TRBC have T cells edited and inserted into HD1 (TCR KO/WT1 insertion); TRAC and TRBC have T cells (HLA-AKO) that edit, insert HD1, and destroy HLA-A; TRAC and TRBC have edited, insert HD1 and HLA-A and CIITA have edited T cells (alloWT 1); and TRAC and TRBC have edited and inserted HD1 in the presence of DNA PKi compound and HLA-A and CIITA have edited T cells (alloWT 1+PKi compound 1).
T cell preparation
HLA-A2 + T cells of the donor (110046967) were isolated from the leukopenia product (STEMCELL Technologies) of healthy donors. T cells were isolated using EasySep human T cell isolation kit (STEMCELL Technologies, catalog No. 17951) following the manufacturer's protocol and cryopreserved using a Cryostor CS10 (STEMCELL Technologies, catalog No. 07930). One day before T cell editing was started, cells were thawed and plated in T Cell Activation Medium (TCAM): CTS Optmizer (thermosipher, # A3705001) was left standing overnight, and the medium was supplemented with 2.5% human AB serum (Gemini, # 100-512), 1 XGlutaMAX (thermosipher, # 35050061), 10mM HEPES (thermosipher, # 15630080), 200U/mL IL-2 (Peprotech, # 200-02), IL-7 (Peprotech, # 200-07), IL-15 (Pep) rotech，#200-15)。
15.2. Multiple editing of T cells by sequential LNP delivery
T cells were prepared by sequentially treating healthy donor cells with four LNP compositions, each of which was co-formulated with Cas9 mRNA and sgrnas targeting TRAC, TRBC, CIITA and HLA-A. The lipid fraction of the LNP composition includes lipid A, cholesterol, DSPC, and PEG2k-DMG in a molar ratio of 50:38.5:10:1.5, respectively. The lipid nucleic acid assemblies were formulated at a lipid amine to RNA phosphate (N: P) molar ratio of about 6 and a gRNA to mRNA ratio of 1:2 by weight. By using AAV delivery homology directed repair templates shown in table 30, small molecule inhibitors that bind DNA dependent protein kinases, WT1 targeted transgenic TCRs were site-specifically integrated into the TRAC cleavage site to increase tgTCR insertion rate. The inhibitor hereinafter referred to as "DNAPKI compound 1" is 9- (4, 4-difluorocyclohexyl) -7-methyl-2- ((7-methyl- [1,2,4] triazolo [1,5-a ] pyridin-6-yl) amino) -7, 9-dihydro-8H-purin-8-one, also depicted as:
DNAPKI compound 1 was prepared as follows:
general information
All reagents and solvents were purchased from commercial suppliers and used as received or synthesized according to the procedures cited. All intermediates and final compounds were purified using flash column chromatography on silica gel. NMR spectra were recorded on a Bruker or Varian 400MHz spectrometer and NMR data in CDCl3 at ambient temperature was collected. Chemical shifts are reported in parts per million (ppm) relative to CDCl3 (7.26). The data for 1H NMR are reported below: chemical shift, multiplicity (br=broad, s=singlet, d=doublet, t=triplet, q=quartet, dd=doublet of doublet, dt=doublet of triplet, m=multiplet), coupling constant and integral. MS data were recorded on a Waters SQD2 mass spectrometer with electrospray ionization (ESI) source. The purity of the final compound was determined by UPLC-MS-ELS using a Waters Acquity H-Class liquid chromatograph equipped with a SQD2 mass spectrometer with a photodiode array (PDA) and an Evaporative Light Scattering (ELS) detector.
Example 1 Compound 1
Intermediate 1a: (E) -N, N-dimethyl-N' - (4-methyl-5-nitropyridin-2-yl) formamidine
To a solution of 4-methyl-5-nitro-pyridin-2-amine (5 g,1.0 eq) in toluene (0.3M) was added DMF-DMA (3.0 eq). The mixture was stirred at 110℃for 2h. The reaction mixture was concentrated under reduced pressure to give a residue and purified by column chromatography to give the product as a yellow solid (59%). 1 H NMR(400MHz,(CD 3 ) 2 SO)δ8.82(s,1H),8.63(s,1H),6.74(s,1H),3.21(m,6H)。
Intermediate 1b: (E) -N-hydroxy-N' - (4-methyl-5-nitropyridin-2-yl) carboxamidine
To a solution of intermediate 1a (4 g,1.0 eq.) in MeOH (0.2M) was added NH 2 OH HCl (2.0 eq). The reaction mixture was stirred at 80℃for 1 hour. The reaction mixture was filtered, and the filtrate was concentrated under reduced pressure to give a residue. The residue was partitioned between H 2 Between O and EtOAc, then extracted 2 times with EtOAc. The organic phase was concentrated under reduced pressure to give a residue and purified by column chromatography to give the product as a white solid (66%). 1H NMR (400 MHz, (CD) 3 ) 2 SO)δ10.52(d,J＝3.8Hz,1H),10.08(dd,J＝9.9,3.7Hz,1H),8.84(d,J＝3.8Hz,1H),7.85(dd,J＝9.7,3.8Hz,1H),7.01(d,J＝3.9Hz,1H),3.36(s,3H)。
Intermediate 1c: 7-methyl-6-nitro- [1,2,4] triazolo [1,5-a ] pyridine
To a solution of intermediate 1b (2.5 g,1.0 eq.) in THF (0.4M) was added trifluoroacetic anhydride (1.0 eq.) at 0 ℃. The mixture was stirred at 25℃for 18 hours. The reaction mixture was filtered, and the filtrate was concentrated under reduced pressure to give a residue. The residue was purified by column chromatography to give the product as a white solid (44%). 1 H NMR(400MHz,CDCl 3 )δ9.53(s,1H),8.49(s,1H),7.69(s,1H),2.78(d,J＝1.0Hz,3H)。
Intermediate 1d: 7-methyl- [1,2,4] triazolo [1,5-a ] pyridin-6-amine
To a mixture of Pd/C (10% w/w,0.2 eq.) in EtOH (0.1M) was added intermediate 1C (1.0 eq.) and ammonium formate (5.0 eq.). The mixture was heated at 105℃for 2 hours. The reaction mixture was filtered, and the filtrate was concentrated under reduced pressure to give a residue. The residue was purified by column chromatography to give the product as a pale brown solid. 1 H NMR(400MHz,(CD 3 ) 2 SO)δ8.41(s,2H),8.07(d,J＝9.0Hz,2H),7.43(s,1H),2.22(s,3H)。
Intermediate 1e: 8-methylene-1, 4-dioxaspiro [4.5] decane
/>
To a solution of methyl (triphenyl) phosphonium bromide (1.15 eq) in THF (0.6M) at-78 ℃ n-BuLi (1.1 eq) was added dropwise and the mixture was stirred at 0 ℃ for 1 hour. Subsequently, 1, 4-dioxaspiro [4.5]]Decan-8-one (50 g,1.0 eq) was added to the reaction mixture. The mixture was stirred at 25℃for 12 hours. Pouring the reaction mixture into NH at 0 DEG C 4 In aqueous Cl solution, H is used 2 O was diluted and extracted 3 times with EtOAc. The combined organic layers were concentrated under reduced pressure to give a residue, which was purified by column chromatography to give the product as a colorless oil (51%). 1 H NMR(400MHz,CDCl 3 )δ4.67(s,1H),3.96(s,4H),2.82(t,J＝6.4Hz,4H),1.70(t,J＝6.4Hz,4H)。
Intermediate 1f:7, 10-dioxadispiro [2.2.4 6 .2 3 ]Dodecane (dodecane)
To a solution of intermediate 4a (5 g,1.0 eq.) in toluene (3M) was added ZnEt dropwise at-40 deg.c 2 (2.57 eq.) and the mixture was stirred at-40℃for 1 hour. Followed by N at-40 DEG C 2 Diiodomethane (6.0 eq) was added drop wise to the mixture. Then at-20 ℃ under N 2 The mixture was stirred under atmosphere for 17 hours. Pouring the reaction mixture into NH at 0 DEG C 4 Aqueous Cl and extracted 2 times with EtOAc. The combined organic phases were washed with brine (20 mL), dried over anhydrous Na 2 SO 4 Dried, filtered, and the filtrate concentrated in vacuo. The residue was purified by column chromatography to give the product as a pale yellow oil (73%).
Intermediate 1g: spiro [2.5] octan-6-ones
Intermediate 4b (4 g,1.0 eq.) in 1:1THF/H 2 TFA (3.0 eq) was added to a solution in O (1.0M). At 20℃under N 2 The mixture was stirred for 2 hours under an atmosphere. The reaction mixture was concentrated under reduced pressure to remove THF, and the residue was adjusted to pH 7 with 2M NaOH (aqueous). The mixture was poured into water and extracted 3 times with EtOAc. The combined organic phases were washed with brine, dried over anhydrous Na 2 SO 4 Dried, filtered, and the filtrate concentrated in vacuo. The residue was purified by column chromatography to give the product as a pale yellow oil (68%). 1 H NMR(400MHz,CDCl 3 )δ2.35(t,J＝6.6Hz,4H),1.62(t,J＝6.6Hz,4H),0.42(s,4H)。
Intermediate 1h: n- (4-methoxybenzyl) spiro [2.5] oct-6-amine
To a mixture of intermediate 4c (2 g,1.0 eq) and (4-methoxyphenyl) methylamine (1.1 eq) in DCM (0.3M) was added AcOH (1.3 eq). At 20℃under N 2 The mixture was stirred under atmosphere for 1 hour. Next, naBH (OAc) was added to the mixture at 0deg.C 3 (3.3 eq.) and at 20℃under N 2 The mixture was stirred under atmosphere for 17 hours. The reaction mixture was concentrated under reduced pressure to remove DCM and the resulting residue was taken up with H 2 O was diluted and extracted 3 times with EtOAc. The combined organic layers were washed with brine, dried over Na 2 SO 4 Drying, filtration and concentration of the filtrate under reduced pressure gave a residue. The residue was purified by column chromatography to give the product as a grey solid (51%). 1 H NMR(400MHz,(CD 3 ) 2 SO)δ7.15–7.07(m,2H),6.77–6.68(m,2H),3.58(s,3H),3.54(s,2H),2.30(ddt,J＝10.1,7.3,3.7Hz,1H),1.69–1.62(m,2H),1.37(td,J＝12.6,3.5Hz,2H),1.12–1.02(m,2H),0.87–0.78(m,2H),0.13–0.04(m,2H)。
Intermediate 1i: spiro [2.5] oct-6-amines
To a suspension of Pd/C (10% w/w,1.0 eq.) in MeOH (0.25M) was added intermediate 4d (2 g,1.0 eq.) and at 80℃at 50Psi at H 2 The mixture was stirred in an atmosphere for 24 hours. The reaction mixture was filtered and the filtrate was concentrated under reduced pressure to give a residue which was purified by column chromatography to give the product as a white solid. 1 H NMR(400MHz,(CD 3 ) 2 SO)δ2.61(tt,J＝10.8,3.9Hz,1H),1.63(ddd,J＝9.6,5.1,2.2Hz,2H),1.47(td,J＝12.8,3.5Hz,2H),1.21–1.06(m,2H),0.82–0.72(m,2H),0.14–0.05(m,2H)。
Intermediate 1j: 2-chloro-4- (spiro [2.5] oct-6-ylamino) pyrimidine-5-carboxylic acid ethyl ester
At N 2 To a mixture of ethyl 2, 4-dichloropyrimidine-5-carboxylate (2.7 g,1.0 eq) and intermediate 1i (1.0 eq) in ACN (0.5-0.6M) was added K in one portion 2 CO 3 (2.5 equivalents). The mixture was stirred at 20℃for 12 hours. The reaction mixture was filtered, and the filtrate was concentrated under reduced pressure to give a residue. The residue was purified by column chromatography to give the product as a white solid (54%). 1 H NMR(400MHz,(CD 3 ) 2 SO)δ8.64(s,1H),8.41(d,J＝7.9Hz,1H),4.33(q,J＝7.1Hz,2H),4.08(d,J＝9.8Hz,1H),1.90(dd,J＝12.7,4.8Hz,2H),1.64(t,J＝12.3Hz,2H),1.52(q,J＝10.7,9.1Hz,2H),1.33(t,J＝7.1Hz,3H),1.12(d,J＝13.0Hz,2H),0.40–0.21(m,4H)。
Intermediate 1k: 2-chloro-4- (spiro [2.5] oct-6-ylamino) pyrimidine-5-carboxylic acid
To intermediate 1j (2 g,1.0 eq.) in 1:1THF/H 2 To a solution in O (0.3M) was added LiOH (2.0 eq). The mixture was stirred at 20℃for 12 hours. The reaction mixture was filtered, and the filtrate was concentrated under reduced pressure to give a residue. The residue was adjusted to pH 2 by 2M HCl and the precipitate was collected by filtration, washed with water, and dried in vacuo. The product was used directly in the next step without additional purification (82%). 1 H NMR(400MHz,(CD 3 ) 2 SO)δ13.54(s,1H),8.38(d,J＝8.0Hz,1H),8.35(s,1H),3.82(qt,J＝8.2,3.7Hz,1H),1.66(dq,J＝12.8,4.1Hz,2H),1.47–1.34(m,2H),1.33–1.20(m,2H),0.86(dt,J＝13.6,4.2Hz,2H),0.08(dd,J＝8.3,4.8Hz,4H)。
Intermediate 1l: 2-chloro-9- (spiro [2.5] oct-6-yl) -7, 9-dihydro-8H-purin-8-one
To intermediate 1k (1.5 g,1.0 eq) and Et 3 N (1.0 eq.) DPPA (1.0 eq.) was added to a mixture in DMF (0.3M). At N 2 The mixture was stirred under an atmosphere at 120 ℃ for 8 hours. The reaction mixture was poured into water. The precipitate was collected by filtration, washed with water, and dried under vacuum to give a residue which was used directly in the next step (67%) without additional purification. 1 H NMR(400MHz,(CD 3 ) 2 SO)δ11.68(s,1H),8.18(s,1H),4.26(ddt,J＝12.3,7.5,3.7Hz,1H),2.42(qd,J＝12.6,3.7Hz,2H),1.95(td,J＝13.3,3.5Hz,2H),1.82–1.69(m,2H),1.08–0.95(m,2H),0.39(tdq,J＝11.6,8.7,4.2,3.5Hz,4H)。
Intermediate 1m: 2-chloro-7-methyl-9- (spiro [2.5] oct-6-yl) -7, 9-dihydro-8H-purin-8-one
Intermediate 1l (1.0 g,1.0 eq.) and NaOH (5.0 eq.) at 1:1THF/H 2 MeI (2.0 equivalents) was added to the mixture in O (0.3-0.5M). At N 2 The mixture was stirred under an atmosphere at 20 ℃ for 12 hours. The reaction mixture was concentrated under reduced pressure to give a residue, which was purified by column chromatography to give the product as a pale yellow solid (67%). 1 H NMR(400MHz,CDCl 3 )δ7.57(s,1H),4.03(tt,J＝12.5,3.9Hz,1H),3.03(s,3H),2.17(qd,J＝12.6,3.8Hz,2H),1.60(td,J＝13.4,3.6Hz,2H),1.47–1.34(m,2H),1.07(s,1H),0.63(dp,J＝14.0,2.5Hz,2H),-0.05(s,4H)。
Compound 1: 7-methyl-2- ((7-methyl- [1,2,4] triazolo [1,5-a ] pyridin-6-yl) amino) -9- (spiro [2.5] oct-6-yl) -7, 9-dihydro-8H-purin-8-one
Intermediate 1m (1.0 eq.) and intermediate 1d (1.0 eq.), pd (dppf) Cl 2 (0.2 equivalent)Xantphos (0.4 eq.) and Cs 2 CO 3 (2.0 eq.) A mixture in DMF (0.2-0.3M) was degassed and N 2 Purge 3 times and at 130 ℃ under N 2 The mixture was stirred under atmosphere for 12 hours. The mixture was then poured into water and extracted 3 times with DCM. The combined organic phases were washed with brine, dried over Na 2 SO 4 Dried, filtered, and the filtrate concentrated in vacuo. The residue was purified by column chromatography to give the product as an off-white solid. 1 H NMR(400MHz,(CD 3 ) 2 SO)δ9.09(s,1H),8.73(s,1H),8.44(s,1H),8.16(s,1H),7.78(s,1H),4.21(t,J＝12.5Hz,1H),3.36(s,3H),2.43(s,3H),2.34(dt,J＝13.0,6.5Hz,2H),1.93–1.77(m,2H),1.77–1.62(m,2H),0.91(d,J＝13.2Hz,2H),0.31(t,J＝7.1Hz,2H).MS：405.5m/z[M+H]。
As illustrated in table 30, each group is edited in turn.
TABLE 30T cell engineering
LNP treatment and expansion of T cells
LNP compositions were formulated in ApoE-containing medium and delivered to T cells as follows: on day 1, LNP compositions as indicated in Table 30 were incubated at a concentration of 5 μg/mL in TCAM containing 5 μg/mL rhApoE3 (Peprotection 350-02). At the same time, T cells were harvested, washed, and at 2X 10 6 The individual cells/ml density was resuspended in TCAM with a 1:50 dilution of T cell TransAct human reagent (Miltenyi, 130-111-160). T cells and LNP-ApoE medium were mixed at a 1:1 ratio and plated in flasks overnight.
On day 2, LNP compositions as indicated in Table 30 were incubated at a concentration of 25. Mu.g/mL in TCAM containing 20. Mu.g/mL rhApoE3 (Peprotection, 350-02). The LNP-ApoE solution was then added to the appropriate culture at a 1:10 ratio.
On day 3, TRAC-LNP compositions (Table 30) were incubated at a concentration of 5ug/mL in TCAM containing 10 ug/mL rhApoE3 (Peprotection 350-02). At the same time, thin TCell harvesting, washing, and washing at 1X 10 6 The individual cells/ml density was resuspended in TCAM. T cells and LNP-ApoE medium were mixed at a 1:1 ratio and plated in flasks. WT1 AAV was then run at 3×10 5 The MOI of each GC/cell was added to the relevant group. Compound 1 was added to the relevant group at a final concentration of 0.25uM.
On day 4, LNP compositions as indicated in Table 30 were incubated at a concentration of 5ug/mL in TCAM containing 5ug/mL rhApoE3 (Peprotection 350-02). T cells were washed by centrifugation and washed at 1X 10 6 The density of individual cells per ml of LNP-ApoE solution was resuspended and then added to the appropriate culture at a 1:1 ratio.
On days 5 to 11, T CELLs were transferred to and expanded in GREX plate (Wilson Wolf) T CELL expansion medium (TCEM: CTS OptmIzer (Thermofilter #A 3705001) supplemented with 5% CTS immune CELL serum replacement (Thermofilter #A 2596101), 1 XGlutamax (Thermofilter # 35050061), 10mM HEPES (Thermofilter # 15630080), 200U/mL IL-2 (Peprotech # 200-02), IL-7 (Peprotech # 200-07), IL-15 (Peprotech # 200-15). Briefly, T CELLs were expanded for 6 days, fresh cytokines were supplemented every other day CELLs were counted using a Vi-CELL counter (Beckman Coulter) and expansion was calculated by dividing CELL yield by starting material.
15.4. Quantitative T cell editing by flow cytometry and NGS
After expansion, the edited T cells were stained in an antibody cocktail to determine HLA-A2 knockout (HLA-A 2 - ) HLA-DR-DP-DQ knockdown via CIITA knockdown (HLA-DRDPDQ) - ) WT1-TCR insertion (CD 3) + Vb8 + ) And expression of residual endogenous (CD 3) + Vb8 - ) Is a percentage of cells of the cell line. The cells were then washed and analyzed on a Cytoflex LX instrument (Beckman Coulter) using FlowJo suite software. T cells were gated according to size and cd8+ status, followed by determination of editing and insertion rate. Editing and insertion rates can be seen in table 31 and fig. 14A-14F. Percentage of fully edited alloWT1-T cells expressing WT1-TCR and knocked out HLA-A and CIITA was gated as CD3 + Vb8 + HLA-A-HLA-DRDPDQ-%. At the time of editing the sampleHigh levels of HLA-A and CIITA knockouts were observed, as well as WT1-TCR insertion and endogenous TCR KO. Notably, T cells receiving DNA PK inhibitor compound 1 showed improved editing efficiency.
IVIS imaging of live mice was performed to identify luciferase-positive tumor cells by IVIS spectroscopy. IVIS imaging was performed 2, 6, 9, 13, 16 and 18 days after T cell injection. Mice were prepared for imaging by intraperitoneal injection of D-fluorescein at 10 μl/g body weight, about 150 μl per animal, according to manufacturer's recommendations. Animals were anesthetized and then placed in an IVIS imaging unit. Visualization is performed with the exposure time set to automatic, field D, medium binning, and F/stop set to 1. Table 32 and FIG. 15 show the emissivity (photons/s/cm 2/sr) of luciferase-expressing T cells present from different time points to 18 days after injection.
TABLE 31T cell editing efficiency
Table 32-total flux (photons/s) of target cells expressing luciferase from treated mice at intervals after T cell injection.
/>
15.5. Engineered T cell cytokine release
Cytokine release profiles of engineered T cells prepared as described in examples 10.1 and 10.2 were determined. In vitro OCI-AML3 tumor cell killing assays were performed using engineered T cells, respectively (data not shown). The supernatant from the tumor cell killing assay was used to evaluate the cytokine release profile of each engineered T cell.
Briefly, TCR KO T cells, autologous WT 1T cells (TCR KO+WT1 TCR insert) and allogeneic WT 1T cells (as indicated in Table 33) were thawed and allowed to stand overnight in TCGM supplemented with IL-2, IL-7 and IL-15. The following day, a co-culture assay was established in which each group of engineered T cells was co-cultured with the OCI-AML3 target tumor. First, OCI-AML3 target tumor cells were pulsed with different concentrations (500, 50, 5, 0.5, 0.05 and 0.005 nM) of VLD peptide for 1 hour. T cells from each group were then counted and resuspended in TCGM medium without cytokines and co-cultured with pulsed OCI-AML3 at a 1:1e:t ratio. The number of T cells in the co-cultures was normalized to the insertion rate to maintain the ET identity between the different groups. After 24 hours of co-culture, supernatants from each co-culture sample were diluted 5-fold in diluent 2 from U-PLEX immunooncology group 1 (hu) assay kit (MSD, catalog number K151 AEL-2). 50 μl of diluted samples from each group were loaded onto Meso Scalediscovery (MSD) plates and incubated for 1 hour.
TABLE 33 engineering of T cells.
For each cytokine measured, biotinylated capture antibody from U-PLEX immunooncology group 1 (hu) assay (MSD, catalog number K151 AEL-2) was added to the indicated linker according to the kit protocol. The antibody-linker mixture was vortexed and incubated for 30 minutes at room temperature. After incubation, the plates were washed, sealed and stored overnight.
The next day, the calibrator containing the standard for each cytokine to be assayed (IL-2 and IFN-gamma) was reconstituted according to the manufacturer's instructions and diluted to generate a 4-fold standard curve.
The plates were washed and 50 μl of detection antibody solution (prepared according to kit instructions) was added to each well of the MSD plate. Plates were incubated for 1 hour.
After incubation, the plates were washed and immediately read on an MSD instrument. Cytokine release is shown in tables 34-35 and FIGS. 16A-16B.
Table 34: IFN-gamma
Table 35: IL-2
Example 16: HLA-A+CIITA DKO T cells do not elicit proliferation of host CD4 or CD8 in mixed lymphocyte reaction assays
T cells were isolated from peripheral blood of healthy human donors with the following MHC I phenotypes: HLA-A.times.02:01:01G, 03:01:01G, HLA-B.times.07:02:01G, HLA-C.times.07:02:01G. Briefly, the leukocyte removal package (Stemcell Technologies) was treated in ammonium chloride RBC lysis buffer (Stemcell Technologies; catalog No. 07800) for 15 minutes to lyse the erythrocytes. Peripheral Blood Mononuclear Cell (PBMC) counts were determined after lysis and T cell isolation was performed using EasySep human T cell isolation kit (Stemc ell Technologies, catalog No. 17951) according to the manufacturer's protocol. Isolated cd3+ T cells were resuspended in a router CS10 medium (Stemcell Technologies, cat# 07930) and frozen in liquid nitrogen until further use.
Frozen T cells were grown at 1.5X10 6 Cell concentration of individual cells/mL was thawed in T Cell Activation Medium (TCAM) composed of OpTmizer TCGM as described in example 3 and further supplemented with 100U/mL recombinant human interleukin-2 (Peprotech, cat# 200-02), 5ng/mL IL-7 (Peprotech, cat# 200-07), 5ng/mL IL-15 (Peprotech, cat# 200-15). The cells were allowed to stand at 37℃for 24 hours.
T cells were counted and at 2 x 10 at 24 hours after thawing 6 Each cell/ml was resuspended in TCAM medium and 1:50v/v TransAct (Miltenyi Biotec catalog No. 30-111-160) was added. Will be 1X 10 6 Individual cells were added to each well of a 24-well tissue culture plate, each group maintained 2 wells for engineering and 2 wells as unedited controls (engineered group: unedited)Editing or WT, B2M KO (also indicated as HLA-I or HLA-I class), CIITA (also indicated as HLA-II class or HLA-II) KO, B2M+CIITA DKO, HLA-A KO, HLA-A+CIITA DKO). The plates were transferred to a 37 ℃ incubator. LNP compositions containing mRNA encoding cas9 (SEQ ID NO: 802) and CIITA-targeted sgRNA G013675 (SEQ ID NO: 27) were formulated with lipid A, cholesterol, DSPC and PEG2k-DMG at a molar ratio of 50:38.5:10:1.5, respectively. The lipid nucleic acid assemblies were formulated at a lipid amine to RNA phosphate (N: P) molar ratio of about 6 and a gRNA to mRNA ratio of 1:2 by weight. 5 μg/mL of the LNP composition was incubated for 15 minutes at 37℃in an Optmizer TCAM further supplemented with 5 μg/mL recombinant human ApoE3 (Peprotech, catalog number 350-02). Pre-incubated LNP and T cells were mixed with Transact in 6 of 12 wells to produce 1X 10 in TCAM medium with 2.5% human AB serum, 100U/mL recombinant human interleukin-2 (Peprotech, cat. No. 200-02), 5ng/mL IL-7 (Peprotech, cat. No. 200-07), 5ng/mL IL-15 (Peprotech, cat. No. 200-15) 6 Final concentrations of LNP (2 wells in CIITA KO group, 2 wells in HLA-A+ciita DKO group and 2 wells in b2m+ciita DKO group) of individual T cells/ml and 2.5 μg total RNA/ml. All additional wells were mock-edited with medium containing ApoE3 but no LNP composition. All cells were incubated at 37℃for 24 hours.
24 hours after activation, 2 pre-untreated wells and 2 wells containing CIITA LNP were treated with the B2M LNP composition (for the B2M KO and b2m+ciita DKO groups); and 2 pre-untreated wells and 2 wells containing CIITA LNP were treated with the LNP composition of HLA-A (for HLA-AKO and HLA-A+ciita DKO groups). LNP compositions containing Cas9 mRNA and B2M-targeted sgRNA G000529 (SEQ ID NO: 216) and LNP compositions containing Cas 9-encoding mRNA (SEQ ID NO: 802) and HLA-A-targeted sgRNA G018995 (comprising the sgrnas of SEQ ID NO:214, as shown in table 4) were formulated with lipid a, cholesterol l, DSPC, and PEG2k-DMG in molar ratios of 50:38.5:10:1.5, respectively. The lipid nucleic acid assemblies were formulated at a lipid amine to RNA phosphate (N: P) molar ratio of about 6 and a gRNA to mRNA ratio of 1:2 by weight. 25ug/mL of the LNP composition was incubated at 37℃for 15 minutes in an Optmizer TCAM further supplemented with 20ug/mL recombinant human ApoE3 (Peprotech, catalog number 350-02). As described above, B2M and HLA-A LNP compositions were added to appropriate wells of 24-well plates to give final concentrations of LNP of 2.5 μg total RNA per mL in TCAM medium with 2.5% human AB serum, 100U/mL recombinant human interleukin-2 (Peprotech, catalog number 200-02), 5ng/mL IL-7 (Peprotech, catalog number 200-07), 5ng/mL IL-15 (Peprotech, catalog number 200-15). Another set of cells was mock-edited with medium containing ApoE3 but no LNP composition to act as an unedited or WT control. All cells were incubated at 37℃for 24 hours.
24 hours after the second round of editing, cells were washed by spinning at 500 XG for 5min and resuspended in a solution containing 5% CTS TM Immune cells SR (Gibco catalog number A2596101), 100U/mL recombinant human interleukin-2 (Peprotech, catalog number 200-02), 5ng/mL IL-7 (Peprotech, catalog number 200-07) and 5ng/mL IL-15 (Peprotech, catalog number 200-15) in TCEM medium. Cells were cultured and maintained for 7 days in G-Rex plates with periodic replacement of media and cytokines, after which they were resuspended in a resistor CS10 medium (Stemcell Technologies, cat# 07930) and frozen in liquid nitrogen until further use.
For the MLR assay, six groups of donor T cells (wild-type unedited, B2M KO, HLA-A KO, CIITA KO, HLA-A+CIITA DKO, B2M+CIITA DKO) were thawed and expressed as 1X 10 6 Re-suspension of individual/mL in TCGM+100U/mL IL2, 0.5ng/mL IL-7&IL-15 (donor and host HLA genotypes are shown in Table 36 below). Thawing Peripheral Blood Mononuclear Cells (PBMC) from 3 hosts (autologous, allogeneic (HLA-B and C matched) and positive control (HLA-A, HLA-B and HLA-C mismatched) to 1X 10 6 Re-suspension of individual/mL in TCGM+100U/mL IL2, 0.5ng/mL IL-7&IL-15. The donor cells and host cells were allowed to stand overnight in an incubator at 37 ℃. The next day, donor cell flasks were irradiated at 4000rad and briefly centrifuged, and each group was irradiated at 1X 10 6 The individual/mL was resuspended in TCGM without cytokines. Host PBMC from two hosts were depleted of CD56 using CD56 microbeads (Miltenyi Biotec, catalog No. 130-050-401) + And (3) cells. Will be about 1X 10 6 Cells from each host were stored in 15mL tubes for unlabeled flow control. 18X 10 for labeling each host 6 One bottle Cell Trace Violet (Thermo Fisher, cat.No.C 34571) was brought to room temperature and reconstituted with 20. Mu.L DMSO to yield a 5mM CTV stock solution. The host cells were grown at about 1X 10 6 The samples/mL were resuspended in phosphate buffered saline (Corning, catalog number 21-040-CV) and transferred to another 50mL conical tube. After adding 18 μl CTV to the tube to stain the host cells, the tube was transferred to a 37 ℃ incubator for 15 minutes. Thereafter, the tube was filled to 40mL with TCGM without cytokine to absorb any unbound dye. The labeled host cells were then briefly centrifuged at 500 Xg for 5 min at 1X 10 6 The individual/mL was resuspended in TCGM without cytokines. Each well was plated with 50,000 cells/50. Mu.L/Kong Suzhu PBMC from the appropriate host. 200,000 host cells per 200. Mu.L/Kong Tupu in wells requiring 4-fold host cells (control samples to normalize data). In the host cells labeled "host+TransAct" (proliferation positive control), 50,000 cells/50. Mu.L/Kong Suzhu PBMC were inoculated followed by 1. Mu.L of T cell TransAct TM Human (Miltenyi Biotec, catalog No. 130-111-160) and the volumes of these wells were complemented to 200. Mu.L with cytokine-free TCGM. The irradiated donor cells were plated at 150,000 cells/150. Mu.L/Kong Tupu. For the flow control, 50,000 cells from one donor and host were plated together. The volume in all wells was filled to 200 μl with TCGM without cytokines.
On day 5 after co-cultivation, half of the medium (about 100 μl) from each well was replaced with fresh medium (TCGM without cytokines).
On day 8 after co-cultivation, the assay plates were stained and analyzed by flow cytometry. For staining purposes, the plates were spun at 600×g for 3 minutes, tapped to remove media, and 100 μl of a 1:100v/v solution of Fc blocker (bioleged, cat. 422302) in FACS buffer was added to each well. Cells were resuspended in Fc blocker and plates incubated for 5 min at room temperature. The antibody cocktail was prepared such that each antibody was present at a 1:100v/v dilution, and 100 μl of this antibody mixture was added to each sample well. The plates were protected from light by covering with aluminum foil and incubated at 2 to 8 ℃ for 20 to 30 minutes. After staining, the plates were spun at 600×g for 3 minutes, tapped to remove the medium and washed with 200 μl FACS buffer. The plates were washed again and the cell pellet resuspended in 70. Mu.L of a 1:200v/v solution of the viability dye 7-AAD (BD Pharmingen, catalog number 51-68981E). The unstained wells were resuspended in 70 μl FACS buffer. The plates were run in rapid mode (60 seconds/well) on a Cytoflex flow cytometer. The results shown in tables 37A and 37B and fig. 13A and 13B (which illustrate data subsets of wild-type, B2M KO, and HLA-A+ciita DKO) confirm that HLA-A+ciita DKO cells elicit minimal CD4 and CD8 responses in allogeneic hosts (HLa-B and C matches), which are similar to those elicited by b2m+ciita DKO cells. For the corresponding hosts, the results for each group have been normalized to the results of proliferation for the 4-fold host group.
TABLE 36 genotypes of T cell donors and PBMC hosts
TABLE 37 proliferation of host CD4+ T cells
TABLE 37 proliferation of host CD8+ T cells
Example 17: sequential delivery of multiple LNP compositions for multiple gene disruption and insertion
T cells are engineered by a series of gene disruptions and insertions. Healthy donor cells were sequentially treated with four LNP compositions, each of which was co-formulated with mRNA encoding Cas9 (SEQ ID NO: 802) and sgRNA targeting TRAC (G013006) (SEQ ID NO: 203), TRBC (G016239) (SEQ ID NO: 211), CIITA (G013675) (SEQ ID NO: 27) or HLA-A (G018995) (comprising the sgRNA of Table ID NO:214, as shown in Table 4). LNP compositions were formulated with lipid a, cholesterol, DSPC and PEG2k-DMG (groups 1 and 2) at the indicated dosages of 35:47.5:15:2.5 molar ratios, respectively, or lipid a, cholesterol, DSPC and PEG2k-DMG (group 3) at the 50:35.5:10:1.5 molar ratios, respectively, according to the groups indicated in table 38. Group 1 and group 2 differ in LNP concentration. The lipid nucleic acid assemblies were formulated at a lipid amine to RNA phosphate (N: P) molar ratio of about 6 and a gRNA to mRNA ratio of 1:2 by weight. TCR-targeting transgenic WT1 sites were specifically integrated into the TRAC cleavage site by using AAV delivery homology directed repair templates. LNP compositions were prepared daily and delivered to T cells as described in table 38.
T cell preparation
T cells from three HLA-A 02:01+ serotypes were isolated from leukopenia products (STEMCELL Technologies) of two healthy donors. T cells were isolated using EasySep human T cell isolation kit (STEMCELL Technologies, catalog No. 17951) following the manufacturer's protocol and cryopreserved using a Cryostor CS10 (STEMCELL Technologies, catalog No. 07930). One day before T cell editing was started, cells were thawed and left overnight in T cell activation medium (TCAM: CTS Optmizer, # A3705001) supplemented with 2.5% human AB serum (Gemini, # 100-512), 1 XGlutaMAX (Thermofilter, # 35050061), 10mM HEPES (Thermofilter, # 15630080), 200U/mL IL-2 (Peprotech, # 200-02), IL-7 (Peprotech, # 200-07) and IL-15 (Peprotech, # 200-15).
LNP treatment and expansion of T cells
LNP compositions were thawed and diluted daily in ApoE-containing medium and delivered to T cells as follows.
TABLE 38 edit sequence for T cell engineering
On day 1, LNP compositions as indicated in Table 38 were incubated in TCAM containing 5 μg/ml rhApoE3 (Peprotection 350-02).At the same time, T cells were harvested, washed, and at 2X 10 6 The individual cells/ml density was resuspended in TCAM with a 1:50 dilution of T cell TransAct human reagent (Miltenyi, 130-111-160). T cells and LNP-ApoE medium were mixed at a 1:1 ratio and plated in flasks overnight.
On day 2, LNP compositions as indicated in Table 38 were incubated at a concentration of 25. Mu.g/mL in TCAM containing 20. Mu.g/mL rhApoE3 (Peprotection, 350-02). The LNP-ApoE solution was then added to the appropriate culture at a ratio of 10:1.
On day 3, TRAC-LNP compositions as indicated in Table 38 were incubated in TCAM containing 5. Mu.g/ml of rhApoE3 (Peprotech 350-02). At the same time, T cells were harvested, washed, and at 1X 10 6 The individual cells/ml density was resuspended in TCAM. T cells and LNP-ApoE medium were mixed at a 1:1 ratio and plated in flasks. WT1 AAV was then run at 3×10 5 The MOI of each GC/cell was added to each group. The DNA-PK inhibitor "Compound 1" was added to each group at a concentration of 0.25. Mu.M.
On day 4, LNP compositions as indicated in Table 38 were incubated in TCAM containing 5 μg/ml rhApoE3 (Peprotection 350-02). At the same time, T cells were harvested, washed, and at 1X 10 6 The individual cells/ml density was resuspended in TCAM. T cells and LNP-ApoE medium were mixed at a 1:1 ratio and plated in flasks.
On days 5 to 13, T cells were transferred to and expanded in T cell expansion medium (TCEM: CTS Optmizer (thermo cleaner # A3705001) supplemented with 5% human AB serum (Gemini # 100-512), 1 XGluthaMAX (thermo cleaner # 35050061), 10mM HEPES (thermo cleaner # 15630080), 200U/mL IL-2 (Peprotech # 200-02), IL-7 (Peprotech # 200-07), IL-15 (Peprotech # 200-15) according to the manufacturer's protocol briefly, and the T cells were expanded for 8 days with medium changes every 2 to 3 days.
After expansion, T cells were edited by flow cytometry analysis to determine HLA-A 02:01 knockdown, HLa-DR-DP-DQ knockdown via CIITA knockdown, WT1-TCR insertion (CD 3 + Vb8 + ) Andexpression of residual endogenous (CD 3) + Vb8 - ) Is a percentage of cells of the cell line. Incubating T cells with a mixture of antibodies targeting: vb8 (bioleged, catalog No. 348104), HLA-A2 (bioleged, catalog No. 343320), HLa-DRDPDQ (bioleged, catalog No. 361712), CD4 (bioleged, catalog No. 300538), CD8 (bioleged, catalog No. 301046), CD3 (bioleged, catalog No. 317336), CCR7 (Biolegend, catalog No. 353214), CD62L (bioleged, catalog No. 304820), CD45RA (Biolege nd, catalog No. 304134), CD45RO (bioleged, catalog No. 304230), CD56 (bioleged, catalog No. 318328), viakrome (Beckman Coulter, catalog No. C36628). Cells were then washed, processed on a Cytoflex LX instrument (Beckman Coulter) and analyzed using FlowJo suite software. T cells were gated according to size and CD4/CD8 status, followed by determination of editing and insertion rate. For cd8+ T cells, the percentages of cells expressing the relevant cell surface proteins after sequential T cell engineering are shown in table 39 and figure 17A, respectively. The percentage of T cells with all expected edits (WT 1-TCR insertion, knockout binding to HLA-A and CIITA) was gated as CD3 + Vb8 + HLA-A - HLA-DRDPDQ - % and shown in fig. 17B. High levels of HLA-A and CIITA knockouts, as well as WT1-TCR insertion, were observed in all groups of edited samples, yielding>75% of fully edited cd8+ T cells. The lower dose (0.65 μg/mL) used with the lipid A35:15:47.5:2.5 composition showed similar efficacy in editing T cells across all targets as the higher dose (2.5 μg/mL) lipid A50:10:35.5:1.5 formulation.
TABLE 39 edit Rate in CD8+T cells
Example 18: CIITA guide RNA screening of BC22n in T cells
Different sgrnas were screened for efficacy of knockout of CIITA gene in human T cells using base editing with C to T. The percentage of T cells negative for MHC class II and/or CD74 protein expression was determined after CIITA editing following electroporation with mRNA and different sgrnas.
18.1T cell preparation
Healthy human donor blood cell line (Hemacare) was obtained commercially, and cells were washed and resuspended inIn PBS/EDTA buffer (Miltenyi Biotec catalog No. 130-070-525) and in MultiMACS TM Treatment was performed in a Cell 24Separator Plus apparatus (Miltenyi Biotec). Use of Stright from +.>CD4/CD8 MicroBead kit human (Miltenyi Biotec catalog No. 130-122-352) isolated T cells via positive selection. T-cells were aliquoted and frozen for storage +. >CS10 (StemCell Technologies catalog number 07930) for future use.
After thawing, T cells were grown at 1.0X10 6 Individual cells/mL were plated in T Cell Growth Medium (TCGM) consisting of CTS OpTmizer T cell expansion SFM and T cell expansion supplement (thermo fisher catalog No. a 1048501), 5% human AB serum (Gemini, catalog No. 100-512), 1 x penicillin-streptomycin, 1 x Glutamax, 10mM HEPES, 200U/mL recombinant human interleukin-2 (Peprotech, catalog No. 200-02), 5ng/mL recombinant human interleukin 7 (Peprotech, catalog No. 200-07) and 5ng/mL recombinant human interleukin 15 (Peprotech, catalog No. 200-15). T cells were allowed to stand in this medium for 24 hours, at which time they were added to T Cell TransAct at a 1:100 volume ratio TM Human agent (Miltenyi, catalog number 130-111-160) was activated. T cells were activated for 48 hours prior to electroporation.
18.2T cell editing with RNA electroporation
A solution containing mRNA encoding BC22n (SEQ ID NO: 972) and UGI (SEQ ID NO: 815) was prepared in P3 buffer. 100. Mu.M CIITA-targeted sgRNA was removed from its storage plate and denatured at 95℃for 2 min and incubated at room temperature for 5 min. T cells were harvested 48 hours after activation, centrifuged, and at 12.5×10 6 The concentration of individual T cells per milliliter was resuspended in P3 electroporation buffer (Lonza). For electroporation, 1X 10 will be 5 The individual T cells were mixed with 20 ng/. Mu.L BC22n mRNA, 20 ng/. Mu.L UGI mRNA and 20pmol sgRNA in a final volume of 20. Mu.L P3 electroporation buffer. This mixture was transferred in duplicate to a 96 well nucleoactor TM The plates were electroporated with the manufacturer's pulse code. The electroporated T cells were then allowed to stand in cytokine-free 80 μ L CTS Optimizer T cell growth medium for 15 minutes, and then transferred to a new flat bottom 96 well plate containing an additional 80 μl of CTS Optimizer T cell growth medium supplemented with 2-fold cytokines. The resulting plates were incubated at 37℃for 10 days. On day 4 after electroporation, cells were split at 1:2 in 2U-shaped plates. One plate was collected for NGS sequencing, while the other plate was supplemented with CTS Optimizer fresh medium containing 1-fold cytokines. This plate was used for flow cytometry on day 7.
18.3 flow cytometry and NGS sequencing
On day 7 post-editing, T cells were analyzed by flow cytometry to determine surface expression of CD74 and HLA-DR, DP, DQ. The results are shown in Table 40. Briefly, T cells were incubated with a mixture of antibodies diluted in cell staining buffer (BioLegend, cat. 420201) for 30 min at 4 ℃. Antibodies against CD3 (BioLegend, catalog No. 317336), CD4 (BioLegend, catalog No. 317434), CD8 (BioLegend, catalog No. 301046) and Viakrome (Beckman Coulter, catalog No. C36628) were diluted 1:100, and antibodies against HLA II-DR (BioLegend, catalog No. 327018), HLA II-DP (BD Biosciences, catalog No. 750872), HLA II-DQ (BioLegend, catalog No. 561504) and CD74 (BioLegend, catalog No. 326808) were diluted 1:50. The cells were then washed, resuspended in 100. Mu.L of cell staining buffer and treated on a Cytoflex flow cytometer (Beckman Coulter). Flow cytometry data was analyzed using FlowJo suite software. T cells are gated based on size, shape, viability, CD8, HLA II-DP, HLA II-DQ, HLA II-DR, and CD74 expression.
Table 40. Percentage of cells negative for surface proteins after genome editing of CIITA with BC22 n. (n=2)
/>
/>
On day 4 post-editing, PCR and subsequent NGS analysis were performed on DNA samples as described in example 1. Table 41 shows CIITA editing results in T cells edited with BC22 n.
Table 41 the average percentages compiled with BC22n at CIITA locus. (n=2)
/>
/>
Example 19: screening CIITA sgRNA dose-responsive to BC22n in T cells
The efficacy of the high efficiency CIITA sgrnas identified in example 18 in base editing in T cells at various primer concentrations was further determined. The efficacy of each was determined for genome editing efficacy by NGS or by flow cytometry destroying surface protein expression of HLA-DR, DP, DQ.
19.1T cell preparation
Healthy human donor blood cell line samples are commercially available (Hemacare) and the cells are washed and resuspendedFloat onIn PBS/EDTA buffer (Miltenyi Biotec catalog No. 130-070-525) and in MultiMACS TM Treatment was performed in a Cell 24Separator Plus apparatus (Miltenyi Biotec). Use of Stright from +.>CD4/CD8 MicroBead kit human (Miltenyi Biotec catalog No. 130-122-352) isolated T cells via positive selection. T-cells were aliquoted and frozen for storage +. >CS10 (StemCell Technologies catalog number 07930) for future use.
After thawing, T cells were grown at 1.0X10 6 Individual cells/mL were plated in T Cell Growth Medium (TCGM) consisting of CTS OpTmizer T cell expansion SFM and T cell expansion supplement (thermo fisher catalog No. a 1048501), 5% human AB serum (Gemini, catalog No. 100-512), 1 x penicillin-streptomycin, 1 x Glutamax, 10mM HEPES, 200U/mL recombinant human interleukin-2 (Peprotech, catalog No. 200-02), 5ng/mL recombinant human interleukin 7 (Peprotech, catalog No. 200-07) and 5ng/mL recombinant human interleukin 15 (Peprotech, catalog No. 200-15). T cells were allowed to stand in this medium for 24 hours, at which time they were added to T Cell TransAct at a 1:100 volume ratio TM Human agent (Miltenyi, catalog number 130-111-160) was activated. T cells were activated for 48 hours prior to electroporation.
19.2T cell editing with RNA electroporation
A solution containing mRNA encoding BC22n (SEQ ID NO: 972) and UGI (SEQ ID NO: 815) was prepared in P3 buffer. 100. Mu.M CIITA-targeted sgRNA was removed from its storage plate and denatured at 95℃for 2 min and incubated at room temperature for 5 min. T cells were harvested 48 hours after activation, centrifuged, and at 12.5×10 6 The concentration of individual T cells per milliliter was resuspended in P3 electroporation buffer (Lonza). Starting from 60pmol in 96-well PCR plates, duplicateEach sgRNA was serially diluted in P3 electroporation buffer at a 1:2 ratio. After dilution, 1X 10 5 Individual T cells, 20 ng/. Mu.l BC22nm mRNA and 20 ng/. Mu.l UGI mRNA were mixed with the sgRNA plates to prepare a final volume of 20. Mu.l P3 electroporation buffer. Transfer the mixture to 4 blocks corresponding to 96 well Nucleofector TM The plates were electroporated with the manufacturer's pulse code. The electroporated T cells were then allowed to stand in cytokine-free 80 μ L CTS Optimizer T cell growth medium for 15 minutes, and then transferred to a new flat bottom 96 well plate containing an additional 80 μl of CTS OpTmizer T cell growth medium supplemented with 2-fold cytokines. The resulting plates were incubated at 37℃for 7 days. On day 4 after electroporation, cells were split 1:2 in two U-shaped bottom plates and one plate was collected for NGS sequencing while the other plate was supplemented with 1-fold cytokine in CTS Optimizer fresh medium. This plate was used for flow cytometry on day 7.
19.3 flow cytometry and NGS sequencing
On day 7 after editing, T cells were analyzed by flow cytometry to determine surface expression of HLA-DR, DP, DQ. Briefly, T cells were incubated with a mixture of antibodies diluted in cell staining buffer (BioLegend, cat. 420201) for 30 min at 4 ℃. Antibodies against CD3 (BioLegend, catalog number 317336), CD4 (BioLegend, catalog number 317434), CD8 (BioLegend, catalog number 301046) and Viakrome (Beckman Coulter, catalog number C36628) were diluted 1:100, and antibodies against HLA II-DR, DP, DQ (BioLegend, catalog number 361714) were diluted 1:50. The cells were then washed, resuspended in 100. Mu.L of cell staining buffer and treated on a Cytoflex flow cytometer (Beckman Coulter). Flow cytometry data was analyzed using FlowJo suite software. T cells are gated on size, shape, viability, CD8 and HLA-DR, DP, DQ.
Table 42 shows CIITA editing results and percentages of T cells negative for HLA-DR, DP, DQ among T cells after base editing with BC22 n.
/>
/>
/>
Example 20: editing human T cells with BC22n, UGI and 91 mer sgrnas
The base editing efficacy of 91-mer sgrnas assessed by NGS and receptor knockouts was compared to that of 100-mer sgrnas formats with the same guide sequence.
The 91 mer sgrnas tested contained a 20 nucleotide guide sequence (as represented by N) and the following guide scaffold: mN nnnnnnnnnnnnnnnnuuagam gmcmum mamma mgm ca aaaauaaggcuaguguguuaucagaaagggcaccgagucggmmum gmu (SEQ ID NO: 1006), wherein A, C, G, U and N are adenine, cytosine, guanine, uracil and any ribonucleotide, respectively, unless otherwise indicated. m indicates 2' O-methyl modification, and * indicating phosphorothioate linkages between nucleotides. Unmodified and modified versions of the primers are provided in table 4 (sequence listing).
Example 20.1.T cell preparation
Healthy human donor blood cell line was obtained commercially (Hemacare) and the cells were washed and resuspended inIn PBS/EDTA buffer (Miltenyi Biotec catalog No. 130-070-525) and in MultiMACS TM Treatment was performed in a Cell 24Separator Plus apparatus (Miltenyi Biotec). Use of Stright from +.>CD4/CD8 MicroBead kit human (Miltenyi Biotec catalog No. 130-122-352) isolated T cells via positive selection. T-cells were aliquoted and frozen for storage +.>CS10 (StemCell Technologies catalog number 07930) for future use.
After thawing, T cells were grown at 1.0X10 6 Individual cells/mL were plated in T Cell Growth Medium (TCGM) consisting of CTS OpTmizer T cell expansion SFM and T cell expansion supplement (thermo fisher catalog No. a 1048501), 5% human AB serum (Gemini, catalog No. 100-512), 1 x penicillin-streptomycin, 1 x Glutamax, 10mM HEPES, 200U/mL recombinant human interleukin-2 (Peprotech, catalog No. 200-02), 5ng/mL recombinant human interleukin 7 (Peprotech, catalog No. 200-07) and 5ng/mL recombinant human interleukin 15 (Peprotech, catalog No. 200-15). T cells were allowed to stand in this medium for 24 hours, at which time they were added to T Cell TransAct at a 1:100 volume ratio TM Human agent (Miltenyi, catalog number 130-111-160) was activated. T cells were activated for 48 hours prior to LNP treatment.
EXAMPLE 20.2T cell LNP treatment and expansion
48 hours after activation, T cells were harvested, centrifuged at 500g for 5 minutes, and at 1X 10 6 The individual T cells/mL were resuspended in T Cell Plating Medium (TCPM), a serum-free version of TCGM, containing 400U/mL recombinant human interleukin-2 (Peprotech, catalog number 200-02), 10ng/mL recombinant human interleukin 7 (Peprotech, catalog number 200-07) and 10ng/mL recombinant human interleukin 15 (Peprotech, catalog number 200-15). mu.L of T cells in TCPM (5X 10) was added per well 4 T cells), in flat bottom 96-well plates.
LNP was prepared as described in example 1 at a ratio of 35:47.5:15:2.5 (lipid A/cholesterol/DSPC/PEG 2 k-DMG). LNP was formulated with a lipid amine to RNA phosphate (N: P) molar ratio of about 6. LNP encapsulates a single RNA species, either sgRNA, BC22n mRNA (SEQ ID NO: 972) or UGI mRNA (SEQ ID No. 815) as described in Table 43.
Tables 43-100 mer and 91 mer sgrnas.
LNP encapsulating sgRNA was diluted to 6.64 μg/mL in T Cell Treatment Medium (TCTM), a TCGM version containing 20ug/mL apoe3 in the absence of interleukins 2, 5 or 7, prior to T cell treatment. These LNPs were incubated at 37℃for 15 min and serially diluted 1:4 with TCTM, which resulted in an 8-point dilution series ranging from 6.64. Mu.g/mL to zero. Similarly, single cargo LNP with BC22n mRNA (SEQ ID NO: 972) or UGI mRNA (SEQ ID NO: 815) was diluted to 3.32 and 1.67 μg/mL, respectively, in TCTM, incubated at 37℃for 15 minutes, and mixed 1:1 by volume with the serially diluted sgRNA LNP in the previous step. Finally, 50 μl of the resulting mixture was added to T cells in a 96-well plate at a 1:1 volume ratio. T cells were incubated at 37 ℃ for 24 hours, at which time they were harvested, centrifuged at 500g for 5 minutes, resuspended in 200 μl TCGM, and returned to the incubator.
Example 20.3 evaluation of edit results by Next Generation Sequencing (NGS)
Four days after LNP treatment, T cells were lysed, PCR amplified for each targeted locus, and subsequent NGS analysis as described in example 1. Table 44 and fig. 18 show the edit level and edit purity of C to T in mass reduced CIITA targeted 100-mer or 91-mer sgRNA treated T cells.
91-mer sgrnas produced higher editing frequencies when delivered at the same concentration when compared to 100-mer versions. No difference in edit purity of C to T was observed between 100 mer and 91 mer sgRNA.
Table 44-average percent editing at CIITA locus in T cells treated with sgrnas of 100 mer (G016086) or 91 mer format (G023521).
Example 20.4 assessment of receptor knockout by flow cytometry
Seven days after LNP treatment, T cells were analyzed by flow cytometry to assess receptor knockdown. T cells were incubated with fixable viability dye (Beckman Coulter, cat. No. C36628) and antibody cocktail targeting HLA-DR, DP, DQ (Biolegend, cat. No. 361714). The cells were then washed and analyzed on a Cytoflex LX instrument (Beckman Coulter) using FlowJo suite software. T cells were gated according to size, viability and CD8 positivity prior to determining any marker expression. The resulting data were plotted on GraphPad Prism v.9.0.2 and analyzed using variable slope (four parameters) nonlinear regression.
As shown in tables 45-46 and FIG. 19, the 91-mer sgRNA tested outperformed the 100-mer version. Targets with lower potency (i.e., higher EC 50) in the 100 mer format (CIITA) appear to benefit most from the use of 91 mer sgrnas.
Table 45-average percentage of CD8+ T cells negative for HLA-DR, DP, DQ surface receptors after treatment with sgRNA targeting CIITA in 100 mer or 91 mer formats, respectively.
TABLE 46 amount of sgRNA (pmol) resulting in 50% loss of receptor expression in CD8+ T cell surfaces (EC 50). The right-most line shows the fold increase in potency achieved by 91-mer sgrnas when compared to 100-mers with the same guide sequence.
Example 21 additional embodiment
The present disclosure further includes the following embodiments.
Embodiment 1 is an engineered cell having reduced or eliminated MHC class II surface expression relative to an unmodified cell comprising a genetic modification in a CIITA gene, wherein the genetic modification comprises at least one nucleotide of an exon within genomic coordinates chr16:10902662-chr16: 10923285.
Embodiment 2 is the engineered cell of embodiment 1, wherein the genetic modification comprises at least 5 consecutive nucleotides within genomic coordinates chr16:10902662-chr16: 10923285.
Embodiment 3 is the engineered cell of any one of the preceding embodiments, wherein the genetic modification comprises at least 6, 7, 8, 9, or 10 consecutive nucleotides within genomic coordinates chr16:10902662-chr16: 10923285.
Embodiment 4 is the engineered cell of any one of the preceding embodiments, wherein the genetic modification comprises substitution of at least one C for T or substitution of at least one a for G within genomic coordinates chr16:10902662-chr16: 10923285.
Embodiment 5 is the engineered cell of any one of the preceding embodiments, wherein the genetic modification comprises at least one nucleotide of an exon within genomic coordinates chr16:10906542-chr16: 10923285.
Embodiment 6 is the engineered cell of any one of the preceding embodiments, wherein the genetic modification comprises at least one nucleotide of an exon within genomic coordinates chr16:10906542-chr16: 10908121.
Embodiment 7 is the engineered cell of any one of the preceding embodiments, wherein the genetic modification comprises at least one nucleotide of an exon within genomic coordinates selected from the group consisting of: chr16:10907539-10907559, chr16:10916426-10916446, chr16:10906907-10906927, chr16:10895702-10895722, chr16:10907757-10907777, chr16:10907623-10907643, chr16:10915626-10915646, chr16:10906756-10906776, chr16:10907476-10907496, chr16:10907385-10907405 and chr16:10923265-10923285.
Embodiment 8 is the engineered cell of any one of the preceding embodiments, wherein the genetic modification comprises at least one nucleotide of an exon within genomic coordinates selected from the group consisting of: chr16:10916432-10916452, chr16:10922444-10922464, chr16:10907924-10907944, chr16:10906985-10907005, chr16:10908073-10908093, chr16:10907433-10907453, chr16:10907979-10907999, chr16:10907139-10907159, chr16:10922435-10922455, chr16:10907384-10907404, chr16:10907434-10907454, chr16:10907119-10907139, chr16:10907539-10907559, chr16:10907810-10907830, chr16:10907315-10907335, chr16:10916426-10916446, chr16:10909138-10909158, chr16:10908101-10908121, chr16:10907790-10907810, chr16:10907787-10907807, and chr16: 10907787-10907807.
Embodiment 9 is the engineered cell of any one of the preceding embodiments, wherein the genetic modification comprises at least one nucleotide of an exon within genomic coordinates selected from the group consisting of: chr16:10906853-10906873, chr16:10922444-10922464, chr16:10907924-10907944, chr16:10907315-10907335, chr16:10916432-10916452, chr16:10907932-10907952, chr16:10915626-10915646, chr16:10907586-10907606, chr16:10916426-10916446, chr16:10907476-10907496, chr16:10907787-10907807, chr16:10907979-10907999, chr16:10906904-10906924 and chr16:10909138-10909158.
Embodiment 10 is the engineered cell of any one of the preceding embodiments, wherein the genetic modification comprises at least one nucleotide of an exon within genomic coordinates selected from the group consisting of: chr16:10895702-10895722, chr16:10916432-10916452, chr16:10907623-10907643, chr16:10907932-10907952, chr16:10906985-10907005, chr16:10915626-10915646, chr16:10907539-10907559, chr16:10916426-10916446, chr16:10907476-10907496, chr16:10907119-10907139, chr16:10907979-10907999 and chr16:10909138-10909158.
Embodiment 11 is the engineered cell of any one of the preceding embodiments, wherein the genetic modification comprises at least one nucleotide of an exon within genomic coordinates selected from the group consisting of: chr16:10906853-10906873, chr16:10906757-10906777, chr16:10895302-10895322, chr16:10907539-10907559, chr16:10907730-10907750, chr16:10895702-10895722.
Embodiment 12 is the engineered cell of any one of the preceding embodiments, wherein the genetic modification comprises at least one nucleotide of an exon within genomic coordinates selected from the group consisting of: chr16:10906853-10906873, chr16:10922444-10922464 and chr16:10916432-10916452.
Embodiment 13 is the engineered cell of any one of the preceding embodiments, wherein the genetic modification comprises at least one nucleotide of an exon within genomic coordinates chr16: 10906853-10906873.
Embodiment 14 is the engineered cell of any one of the preceding embodiments, wherein the genetic modification comprises at least one nucleotide of an exon within genomic coordinates chr16: 10922444-10922464.
Embodiment 15 is the engineered cell of any one of the preceding embodiments, wherein the genetic modification comprises at least one nucleotide of an exon within genomic coordinates chr16:10916432-10916452.
Embodiment 16 is the engineered cell of any one of the preceding embodiments, wherein the genetic modification comprises at least one nucleotide of an exon within genomic coordinates chr16: 10906757-10906777.
Embodiment 17 is the engineered cell of any one of the preceding embodiments, wherein the genetic modification comprises at least one nucleotide of an exon within genomic coordinates chr16: 10895302-10895322.
Embodiment 18 is the engineered cell of any one of the preceding embodiments, wherein the genetic modification comprises at least one nucleotide of an exon within genomic coordinates chr16: 10907539-10907559.
Embodiment 19 is the engineered cell of any one of the preceding embodiments, wherein the genetic modification comprises at least one nucleotide of an exon within genomic coordinates chr16: 10907730-10907750.
Embodiment 20 is the engineered cell of any one of the preceding embodiments, wherein the genetic modification comprises at least one nucleotide of an exon within genomic coordinates chr16: 10895702-10895722.
Embodiment 21 is the engineered cell of any one of the preceding embodiments, wherein the genetic modification comprises at least one nucleotide of an exon within genomic coordinates chr16: 10907932-10907952.
Embodiment 22 is the engineered cell of any one of the preceding embodiments, wherein the genetic modification comprises at least one nucleotide of an exon within genomic coordinates chr16: 10907476-10907496.
Embodiment 23 is the engineered cell of any one of the preceding embodiments, wherein the genetic modification comprises at least one nucleotide of an exon within genomic coordinates chr16: 10909138-10909158.
Embodiment 24 is an engineered cell having reduced or eliminated MHC class II surface expression relative to an unmodified cell comprising a genetic modification in a CIITA gene, wherein the genetic modification comprises an insertion/deletion within genomic coordinates selected from the group consisting of: chr16: chr16: the chr16, chr16, 16, and 16 chr16:10907870-10907890, chr16:10907886-10907906, chr16:10907924-10907944, chr16:10907928-10907948, chr16:10907932-10907952, chr16:10907935-10907955, chr16:10907978-10907998, chr16:10907979-10907999, chr16:10908069-10908089, chr16:10908073-10908093, chr16:10908101-10908121, chr16:10909056-10909076, chr16:10909138-10909158, chr16:10910195-10910215, chr16:10910196-10910216, chr16:10915592-10915612, chr16:10915626-10915646, chr16:10916375-10916395, chr16:10916382-10916402, chr16:10916426-10916446, chr16:10916432-10916452, chr16:10918486-10918506, chr16:10918492-10918512, chr16:10918493-10918513, chr16:10922435-10922455, chr16:10922441-10922461, chr16:10922441-10922461, chr16:10922444-10922464, chr16:10922460-10922480, chr16:10923257-10923277 and chr16:10923265-10923285.
Embodiment 25 is the engineered cell of embodiment 24, wherein the genetic modification comprises at least one nucleotide of an exon within genomic coordinates selected from the group consisting of: chr16:10916432-10916452 chr16:10922444-10922464, chr16:10907924-10907944, chr16:10906985-10907005, chr16:10908073-10908093, chr16:10907433-10907453, chr16:10907979-10907999, chr16:10907139-10907159, chr16:10922435-10922455, chr16:10907384-10907404, chr16:10907434-10907454, chr16:10907119-10907139, chr16:10907539-10907559, chr16:10907810-10907830, chr37, chr16:10907810-10907830, and chr16: 10907810-10907830.
Embodiment 26 is the engineered cell of embodiment 24 or 25, wherein the genetic modification comprises at least one nucleotide of an exon within genomic coordinates selected from the group consisting of: chr16:10907539-10907559, chr16:10916426-10916446, chr16:10906907-10906927, chr16:10895702-10895722, chr16:10907757-10907777, chr16:10907623-10907643, chr16:10915626-10915646, chr16:10906756-10906776, chr16:10907476-10907496, chr16:10907385-10907405 and chr16:10923265-10923285.
Embodiment 27 is the engineered cell of embodiment 24 or 25, wherein the genetic modification comprises at least one nucleotide of an exon within genomic coordinates selected from the group consisting of: chr16:10906853-10906873, chr16:10922444-10922464, chr16:10907924-10907944, chr16:10907315-10907335, chr16:10916432-10916452, chr16:10907932-10907952, chr16:10915626-10915646, chr16:10907586-10907606, chr16:10916426-10916446, chr16:10907476-10907496, chr16:10907787-10907807, chr16:10907979-10907999, chr16:10906904-10906924 and chr16:10909138-10909158.
Embodiment 28 is the engineered cell of embodiment 24 or 25, wherein the genetic modification comprises at least one nucleotide of an exon within genomic coordinates selected from the group consisting of: chr16:10895702-10895722, chr16:10916432-10916452, chr16:10907623-10907643, chr16:10907932-10907952, chr16:10906985-10907005, chr16:10915626-10915646, chr16:10907539-10907559, chr16:10916426-10916446, chr16:10907476-10907496, chr16:10907119-10907139, chr16:10907979-10907999 and chr16:10909138-10909158.
Embodiment 29 is the engineered cell of embodiment 24 or 25, wherein the genetic modification comprises at least one nucleotide of an exon within genomic coordinates selected from the group consisting of: chr16:10906853-10906873, chr16:10906757-10906777, chr16:10895302-10895322, chr16:10907539-10907559, chr16:10907730-10907750, chr16:10895702-10895722.
Embodiment 30 is the engineered cell of embodiment 24 or 25, wherein the genetic modification comprises at least one nucleotide of an exon within genomic coordinates selected from the group consisting of: chr16:10906853-10906873, chr16:10922444-10922464 and chr16:10916432-10916452.
Embodiment 31 is the engineered cell of any one of embodiments 24 to 30, wherein the genetic modification comprises at least 5 contiguous nucleotides within genomic coordinates.
Embodiment 32 is the engineered cell of any one of embodiments 24 to 31, wherein the genetic modification comprises at least 6, 7, 8, 9, or 10 contiguous nucleotides within genomic coordinates.
Embodiment 33 is the engineered cell of any one of embodiments 24 to 32, wherein the genetic modification comprises substitution of at least one C for T or substitution of at least one a for G within genomic coordinates.
Embodiment 34 is the engineered cell of any one of the preceding embodiments, wherein the mhc class II expression is reduced or eliminated by a gene editing system that binds to a CIITA genomic target sequence comprising at least 5 consecutive nucleotides within genomic coordinates selected from the group consisting of: chr16: chr16: the chr16, chr16, 16, and 16 chr16:10907820-10907840, chr16:10907870-10907890, chr16:10907886-10907906, chr16:10907924-10907944, chr16:10907928-10907948, chr16:10907932-10907952, chr16:10907935-10907955, chr16:10907978-10907998, chr16:10907979-10907999, chr16:10908069-10908089, chr16:10908073-10908093, chr16:10908101-10908121, chr16:10909056-10909076, chr16:10909138-10909158, chr16:10910195-10910215, chr16:10910196-10910216, chr16:10915592-10915612, chr16:10915626-10915646, chr16:10916375-10916395, chr16:10916382-10916402, chr16:10916426-10916446, chr16:10916432-10916452, chr16:10918486-10918506, chr16:10918492-10918512, chr16:10918493-10918513, chr16:10922435-10922455, chr16:10922441-10922461, chr16:10922441-10922461, chr16:10922444-10922464, chr16:10922460-10922480, chr16:10923257-10923277 and chr16:10923265-10923285.
Embodiment 35 is the engineered cell of any one of the preceding embodiments, wherein the mhc class II expression is reduced or eliminated by a gene editing system that binds to a CIITA genomic target sequence comprising at least 5 consecutive nucleotides within genomic coordinates selected from the group consisting of: chr16: chr16: the chr16, chr16, 16, and 16 chr16:10908101-10908121, chr16:10909056-10909076, chr16:10909138-10909158, chr16:10910195-10910215, chr16:10910196-10910216, chr16:10915592-10915612, chr16:10915626-10915646, chr16:10916375-10916395, chr16:10916382-10916402, chr16:10916426-10916446, chr16:10916432-10916452, chr16:10918486-10918506, chr16:10918492-10918512, chr16:10918493-10918513, chr16:10922435-10922455, chr16:10922441-10922461, chr16:10922441-10922461, chr16:10922444-10922464, chr16:10922460-10922480, chr16:10923257-10923277 and chr16:10923265-10923285.
Embodiment 36 is the engineered cell of any one of the preceding embodiments, wherein the mhc class II expression is reduced or eliminated by a gene editing system that binds to a CIITA genomic target sequence comprising at least 5 consecutive nucleotides within genomic coordinates selected from the group consisting of: chr16: chr16: the method comprises the steps of (1), ch16, chr16, chr, chr16, chr, 16 chr16:10908073-10908093 and chr16:10908101-10908121.
Embodiment 37 is the engineered cell of any one of the preceding embodiments, wherein the mhc class II expression is reduced or eliminated by a gene editing system that binds to a CIITA genomic target sequence comprising at least 5 consecutive nucleotides within genomic coordinates selected from the group consisting of: chr16:10916432-10916452, chr16:10922444-10922464, chr16:10907924-10907944, chr16:10906985-10907005, chr16:10908073-10908093, chr16:10907433-10907453, chr16:10907979-10907999, chr16:10907139-10907159, chr16:10922435-10922455, chr16:10907384-10907404, chr16:10907434-10907454, chr16:10907119-10907139, chr16:10907539-10907559, chr16:10907810-10907830, chr16:10907315-10907335, chr16:10916426-10916446, chr16:10909138-10909158, chr16:10908101-10908121, chr16:10907790-10907810, chr16:10907787-10907807, and chr16: 10907787-10907807.
Embodiment 38 is the engineered cell of any one of the preceding embodiments, wherein the mhc class II expression is reduced or eliminated by a gene editing system that binds to a CIITA genomic target sequence comprising at least 5 consecutive nucleotides within genomic coordinates selected from the group consisting of: chr16:10907539-10907559, chr16:10916426-10916446, chr16:10906907-10906927, chr16:10895702-10895722, chr16:10907757-10907777, chr16:10907623-10907643, chr16:10915626-10915646, chr16:10906756-10906776, chr16:10907476-10907496, chr16:10907385-10907405 and chr16:10923265-10923285.
Embodiment 39 is the engineered cell of any one of the preceding embodiments, wherein the mhc class II expression is reduced or eliminated by a gene editing system that binds to a CIITA genomic target sequence comprising at least 5 consecutive nucleotides within genomic coordinates selected from the group consisting of: chr16:10906853-10906873, chr16:10922444-10922464, chr16:10907924-10907944, chr16:10907315-10907335, chr16:10916432-10916452, chr16:10907932-10907952, chr16:10915626-10915646, chr16:10907586-10907606, chr16:10916426-10916446, chr16:10907476-10907496, chr16:10907787-10907807, chr16:10907979-10907999, chr16:10906904-10906924 and chr16:10909138-10909158.
Embodiment 40 is the engineered cell of any one of the preceding embodiments, wherein the mhc class II expression is reduced or eliminated by a gene editing system that binds to a CIITA genomic target sequence comprising at least 5 consecutive nucleotides within genomic coordinates selected from the group consisting of: chr16:10895702-10895722, chr16:10916432-10916452, chr16:10907623-10907643, chr16:10907932-10907952, chr16:10906985-10907005, chr16:10915626-10915646, chr16:10907539-10907559, chr16:10916426-10916446, chr16:10907476-10907496, chr16:10907119-10907139, chr16:10907979-10907999 and chr16:10909138-10909158.
Embodiment 41 is the engineered cell of any one of the preceding embodiments, wherein the mhc class II expression is reduced or eliminated by a gene editing system that binds to a CIITA genomic target sequence comprising at least 5 consecutive nucleotides within genomic coordinates selected from the group consisting of: chr16:10906853-10906873, chr16:10906757-10906777, chr16:10895302-10895322, chr16:10907539-10907559, chr16:10907730-10907750 and chr16:10895702-10895722.
Embodiment 42 is the engineered cell of any one of the preceding embodiments, wherein the mhc class II expression is reduced or eliminated by a gene editing system that binds to a CIITA genomic target sequence comprising at least 5 consecutive nucleotides within genomic coordinates selected from the group consisting of: chr16:10906853-10906873, chr16:10922444-10922464 and chr16:10916432-10916452.
Embodiment 43 is the engineered cell of any one of the preceding embodiments, wherein the MHC class II expression is reduced or eliminated by a gene editing system that binds to a CIITA genomic target sequence comprising at least 5 contiguous nucleotides within genomic coordinates chr16: 10916426-10916446.
Embodiment 44 is the engineered cell of any one of the preceding embodiments, wherein the MHC class II expression is reduced or eliminated by a gene editing system that binds to a CIITA genomic target sequence comprising at least 5 contiguous nucleotides within genomic coordinates chr16: 10906907-10906927.
Embodiment 45 is the engineered cell of any one of the preceding embodiments, wherein the MHC class II expression is reduced or eliminated by a gene editing system that binds to a CIITA genomic target sequence comprising at least 5 contiguous nucleotides within genomic coordinates chr16: 10907757-10907777.
Embodiment 46 is the engineered cell of any one of the preceding embodiments, wherein the MHC class II expression is reduced or eliminated by a gene editing system that binds to a CIITA genomic target sequence comprising at least 5 contiguous nucleotides within genomic coordinates chr16: 10907623-10907643.
Embodiment 47 is the engineered cell of any one of the preceding embodiments, wherein the MHC class II expression is reduced or eliminated by a gene editing system that binds to a CIITA genomic target sequence comprising at least 5 contiguous nucleotides within genomic coordinates chr16: 10915626-10915646.
Embodiment 48 is the engineered cell of any one of the preceding embodiments, wherein the MHC class II expression is reduced or eliminated by a gene editing system that binds to a CIITA genomic target sequence comprising at least 5 contiguous nucleotides within genomic coordinates chr16: 10906756-10906776.
Embodiment 49 is the engineered cell of any one of the preceding embodiments, wherein the MHC class II expression is reduced or eliminated by a gene editing system that binds to a CIITA genomic target sequence comprising at least 5 contiguous nucleotides within genomic coordinates chr16: 10907385-10907405.
Embodiment 50 is the engineered cell of any one of the preceding embodiments, wherein the MHC class II expression is reduced or eliminated by a gene editing system that binds to a CIITA genomic target sequence comprising at least 5 contiguous nucleotides within genomic coordinates chr16: 10923265-10923285.
Embodiment 51 is the engineered cell of any one of the preceding embodiments, wherein the MHC class II expression is reduced or eliminated by a gene editing system that binds to a CIITA genomic target sequence comprising at least 5 contiguous nucleotides within genomic coordinates chr16: 10906853-10906873.
Embodiment 52 is the engineered cell of any one of the preceding embodiments, wherein the MHC class II expression is reduced or eliminated by a gene editing system that binds to a CIITA genomic target sequence comprising at least 5 contiguous nucleotides within genomic coordinates chr16: 10922444-10922464.
Embodiment 53 is the engineered cell of any one of the preceding embodiments, wherein the MHC class II expression is reduced or eliminated by a gene editing system that binds to a CIITA genomic target sequence comprising at least 5 contiguous nucleotides within genomic coordinates chr16: 10916432-10916452.
Embodiment 54 is the engineered cell of any one of the preceding embodiments, wherein the MHC class II expression is reduced or eliminated by a gene editing system that binds to a CIITA genomic target sequence comprising at least 5 contiguous nucleotides within genomic coordinates chr16: 10906757-10906777.
Embodiment 55 is the engineered cell of any one of the preceding embodiments, wherein the MHC class II expression is reduced or eliminated by a gene editing system that binds to a CIITA genomic target sequence comprising at least 5 contiguous nucleotides within genomic coordinates chr16: 10895302-10895322.
Embodiment 56 is the engineered cell of any one of the preceding embodiments, wherein the MHC class II expression is reduced or eliminated by a gene editing system that binds to a CIITA genomic target sequence comprising at least 5 contiguous nucleotides within genomic coordinates chr16: 10907539-10907559.
Embodiment 57 is the engineered cell of any one of the preceding embodiments, wherein the MHC class II expression is reduced or eliminated by a gene editing system that binds to a CIITA genomic target sequence comprising at least 5 contiguous nucleotides within genomic coordinates chr16: 10907730-10907750.
Embodiment 58 is the engineered cell of any one of the preceding embodiments, wherein the MHC class II expression is reduced or eliminated by a gene editing system that binds to a CIITA genomic target sequence comprising at least 5 contiguous nucleotides within genomic coordinates chr16: 10895702-10895722.
Embodiment 59 is the engineered cell of any one of the preceding embodiments, wherein the MHC class II expression is reduced or eliminated by a gene editing system that binds to a CIITA genomic target sequence comprising at least 5 contiguous nucleotides within genomic coordinates chr16: 10907932-10907952.
Embodiment 60 is the engineered cell of any one of the preceding embodiments, wherein the MHC class II expression is reduced or eliminated by a gene editing system that binds to a CIITA genomic target sequence comprising at least 5 contiguous nucleotides within genomic coordinates chr16: 10907476-10907496.
Embodiment 61 is the engineered cell of any one of the preceding embodiments, wherein the MHC class II expression is reduced or eliminated by a gene editing system that binds to a CIITA genomic target sequence comprising at least 5 contiguous nucleotides within genomic coordinates chr16: 10909138-10909158.
Embodiment 62 is the engineered cell of any one of embodiments 34 to 61, wherein the CIITA genomic target sequence comprises at least 10 contiguous nucleotides within genomic coordinates.
Embodiment 63 is the engineered cell of any one of embodiments 34 to 62, wherein the CIITA genomic target sequence comprises at least 15 contiguous nucleotides within genomic coordinates.
Embodiment 64 is the engineered cell of any one of embodiments 34-63, wherein the gene editing system comprises an RNA-guided DNA binding agent.
Embodiment 65 is the engineered cell of embodiment 64, wherein the RNA-guided DNA binding agent comprises a Cas9 protein, such as streptococcus pyogenes Cas9.
Embodiment 66 is the engineered cell of any one of the preceding embodiments, wherein the engineered cell further has reduced or eliminated MHC class I surface expression.
Embodiment 67 is the engineered cell of embodiment 66, wherein the engineered cell comprises a genetic modification in a β -2-microglobulin (B2M) gene.
Embodiment 68 is an engineered cell of embodiment 66, wherein the engineered cell comprises a genetic modification in an HLA-A gene.
Embodiment 69 is the engineered cell of any one of the preceding embodiments, wherein the engineered cell further comprises an exogenous nucleic acid.
Embodiment 70 is the engineered cell of any one of the preceding embodiments, wherein the engineered cell comprises an exogenous nucleic acid encoding a targeting receptor expressed on the surface of the engineered cell.
Embodiment 71 is the engineered cell of embodiment 70, wherein the targeted receptor is a CAR.
Embodiment 72 is the engineered cell of embodiment 70, wherein the targeting receptor is a TCR.
Embodiment 73 is the engineered cell of embodiment 70, wherein the targeting receptor is a WT1TCR.
Embodiment 74 is the engineered cell of any one of the preceding embodiments, wherein the engineered cell further comprises an exogenous nucleic acid encoding a polypeptide secreted by the engineered cell.
Embodiment 75 is the engineered cell of any one of the preceding embodiments, wherein the engineered cell is an immune cell.
Embodiment 76 is the engineered cell of any of the preceding embodiments, wherein the engineered cell is a monocyte, macrophage, mast cell, dendritic cell, or granulocyte.
Embodiment 77 is the engineered cell of any one of the preceding embodiments, wherein the engineered cell is a lymphocyte.
Embodiment 78 is the engineered cell of embodiment 77, wherein the engineered cell is a T cell.
Embodiment 79 is an engineered cell of embodiment 78, wherein the engineered cell further has reduced or eliminated expression of an endogenous T Cell Receptor (TCR) protein relative to an unmodified cell.
Embodiment 80 is an engineered cell of any one of embodiments 78 to 79, wherein the cell has reduced or eliminated expression of a TRAC protein relative to an unmodified cell.
Embodiment 81 is the engineered cell of any one of embodiments 78 to 80, wherein the cell reduces expression of a TRBC protein relative to an unmodified cell.
Embodiment 82 is a pharmaceutical composition comprising the engineered cell of any one of the preceding embodiments.
Embodiment 83 is a cell population comprising the engineered cell of any one of the preceding embodiments.
Embodiment 84 is a pharmaceutical composition comprising a population of cells, wherein the population of cells comprises the engineered cell of any one of the preceding embodiments.
Embodiment 85 is the cell population of embodiment 83 or the pharmaceutical composition of embodiment 84, wherein the cell population is at least 65% MHC class II negative as measured by flow cytometry.
Embodiment 86 is the cell population of embodiment 83 or the pharmaceutical composition of embodiment 84, wherein the cell population is at least 70% MHC class II negative as measured by flow cytometry.
Embodiment 87 is the cell population of embodiment 83 or the pharmaceutical composition of embodiment 84, wherein the cell population is at least 80% MHC class II negative as measured by flow cytometry.
Embodiment 88 is the cell population of embodiment 83 or the pharmaceutical composition of embodiment 84, wherein the cell population is at least 90% MHC class II negative as measured by flow cytometry.
Embodiment 89 is the cell population of embodiment 83 or the pharmaceutical composition of embodiment 84, wherein the cell population is at least 92% MHC class II negative as measured by flow cytometry.
Embodiment 90 is the cell population of embodiment 83 or the pharmaceutical composition of embodiment 84, wherein the cell population is at least 93% MHC class II negative as measured by flow cytometry.
Embodiment 91 is the cell population of embodiment 83 or the pharmaceutical composition of embodiment 84, wherein the cell population is at least 94% MHC class II negative as measured by flow cytometry.
Embodiment 92 is the cell population of embodiment 83 or the pharmaceutical composition of embodiment 84, wherein the cell population is at least 95% MHC class II negative as measured by flow cytometry.
Embodiment 93 is the cell population of embodiment 83 or the pharmaceutical composition of embodiment 84, wherein the cell population is at least 96% MHC class II negative as measured by flow cytometry.
Embodiment 94 is the cell population of embodiment 83 or the pharmaceutical composition of embodiment 84, wherein the cell population is at least 97% MHC class II negative as measured by flow cytometry.
Embodiment 95 is the cell population of embodiment 83 or the pharmaceutical composition of embodiment 84, wherein the cell population is at least 98% MHC class II negative as measured by flow cytometry.
Embodiment 96 is the cell population of embodiment 83 or the pharmaceutical composition of embodiment 84, wherein the cell population is at least 99% MHC class II negative as measured by flow cytometry.
Embodiment 97 is the cell population or pharmaceutical composition of any one of embodiments 83-96, wherein the cell population is at least 95% endogenous TCR protein negative as measured by flow cytometry.
Embodiment 98 is the cell population or pharmaceutical composition of any one of embodiments 83-96, wherein the cell population is at least 97% negative for endogenous TCR protein as measured by flow cytometry.
Embodiment 99 is the cell population or pharmaceutical composition of any one of embodiments 83-96, wherein the cell population is at least 98% negative for endogenous TCR protein as measured by flow cytometry.
Embodiment 100 is a cell population or pharmaceutical composition of any one of embodiments 83-96, wherein the cell population is at least 99% endogenous TCR protein negative as measured by flow cytometry.
Embodiment 101 is a method of administering an engineered cell, population of cells, or pharmaceutical composition of any one of the preceding embodiments to a subject in need thereof.
Embodiment 102 is a method of administering the engineered cell, cell population, or pharmaceutical composition of any of the preceding embodiments as Adoptive Cell Transfer (ACT) therapy to a subject.
Embodiment 103 is a method of making an engineered cell having reduced or eliminated surface expression of an MHC class II protein relative to an unmodified cell, comprising contacting the cell with a composition comprising: (a) CIITA guide RNA comprising (i) a guide sequence selected from the group consisting of SEQ ID NOS: 1-117; (ii) At least 17, 18, 19 or 20 contiguous nucleotides of a sequence selected from the group consisting of SEQ ID NOS: 1-117; (iii) A leader sequence having at least 95%, 90% or 85% identity to a sequence selected from the group consisting of SEQ ID NOS.1-117; (iv) A sequence of 10 contiguous nucleotides ± 10 nucleotides comprising the genomic coordinates listed in table 2; (v) At least 17, 18, 19 or 20 consecutive nucleotides of the sequence from (iv); or (vi) a leader sequence having at least 95%, 90% or 85% identity to a sequence selected from (v); and (b) optionally an RNA-guided DNA binding agent or a nucleic acid encoding an RNA-guided DNA binding agent.
Embodiment 104 is a method of reducing or eliminating surface expression of MHC class II proteins in an engineered cell relative to an unmodified cell comprising contacting the cell with a composition comprising: (a) CIITA guide RNA comprising (i) a guide sequence selected from the group consisting of SEQ ID NOS: 1-117; (ii) At least 17, 18, 19 or 20 contiguous nucleotides of a sequence selected from the group consisting of SEQ ID NOS: 1-117; (iii) A leader sequence having at least 95%, 90% or 85% identity to a sequence selected from the group consisting of SEQ ID NOS.1-117; (iv) A sequence of 10 contiguous nucleotides ± 10 nucleotides comprising the genomic coordinates listed in table 2; (v) At least 17, 18, 19 or 20 consecutive nucleotides of the sequence from (iv); or (vi) a leader sequence having at least 95%, 90% or 85% identity to a sequence selected from (v); and (b) optionally an RNA-guided DNA binding agent or a nucleic acid encoding an RNA-guided DNA binding agent.
Embodiment 105 is the method of embodiment 103 or 104, wherein the CIITA guide RNA comprises (i) a guide sequence selected from the group consisting of SEQ ID NOs 32, 64, 67, 68, 74, 76, 84, 86, 90, 91, and 115; (ii) At least 17, 18, 19 or 20 consecutive nucleotides of a sequence selected from the group consisting of SEQ ID NOs 32, 64, 67, 68, 74, 76, 84, 86, 90, 91 and 115; or (iii) a guide sequence having at least 95%, 90% or 85% identity to a sequence selected from the group consisting of SEQ ID NOs 32, 64, 67, 68, 74, 76, 84, 86, 90, 91 and 115.
Embodiment 106 is the method of any one of embodiments 103-105, further comprising reducing or eliminating surface expression of MHC class I proteins in the cell relative to the unmodified cell.
Embodiment 107 is the method of any one of embodiments 103-106, further comprising reducing or eliminating surface expression of a B2M protein in a cell relative to an unmodified cell.
Embodiment 108 is the method of any one of embodiments 103-107, further comprising reducing or eliminating surface expression of an HLA-A protein in a cell relative to an unmodified cell.
Embodiment 109 is the method of any one of embodiments 103-108, further comprising reducing or eliminating surface expression of a TCR protein in the cell relative to an unmodified cell.
Embodiment 110 is the method of any one of embodiments 103-109, further comprising contacting the cell with an exogenous nucleic acid.
Embodiment 111 is the method of any one of embodiments 103-110, further comprising contacting the cell with a DNA-dependent protein kinase inhibitor (DNAPKi).
Embodiment 112 is the method of embodiment 111, wherein the DNAPKi is compound 1.
Embodiment 113 is the method of embodiment 110, further comprising contacting the cell with an exogenous nucleic acid encoding a targeting receptor.
Embodiment 114 is the method of embodiment 110, further comprising contacting the cell with an exogenous nucleic acid encoding a polypeptide secreted by the cell.
Embodiment 115 is the engineered cell, cell population, pharmaceutical composition, or method of any of the preceding embodiments, wherein the cell is an allogeneic cell.
Embodiment 116 the engineered cell, population of cells, pharmaceutical composition, or method of any one of the preceding embodiments, wherein the cell is a human cell.
Embodiment 117 is the engineered cell, cell population, pharmaceutical composition, or method of any of the preceding embodiments, wherein the cell is a primary cell.
Embodiment 118 is the engineered cell, cell population, pharmaceutical composition, or method of any one of the preceding embodiments, wherein the cell is a cd4+ T cell.
Embodiment 119 is the engineered cell, cell population, pharmaceutical composition, or method of any one of the preceding embodiments, wherein the cell is a cd8+ T cell.
Embodiment 120 is the engineered cell, cell population, pharmaceutical composition, or method of any one of the preceding embodiments, wherein the cell is a memory T cell.
Embodiment 121 is the engineered cell, cell population, pharmaceutical composition, or method of any one of the preceding embodiments, wherein the cell is a B cell.
Embodiment 122 is the engineered cell, cell population, pharmaceutical composition, or method of any of the preceding embodiments, wherein the cell is a plasma B cell.
Embodiment 123 is the engineered cell, cell population, pharmaceutical composition, or method of any of the preceding embodiments, wherein the cell is a memory B cell.
Embodiment 124 is the engineered cell, population of cells, pharmaceutical composition, or method of any of the preceding embodiments, wherein the cell is a Hematopoietic Stem Cell (HSC).
Embodiment 125 is the engineered cell, cell population, pharmaceutical composition, or method of any of the preceding embodiments, wherein the cell is an activated cell.
Embodiment 126 is the engineered cell, cell population, pharmaceutical composition, or method of any of the preceding embodiments, wherein the cell is a non-activated cell.
Embodiment 127 is the engineered cell, population of cells, pharmaceutical composition or method of any one of the preceding embodiments, comprising or contacting a cell with an exogenous nucleic acid, wherein the exogenous nucleic acid encodes an NK cell inhibitor molecule.
Embodiment 128 is the engineered cell, population of cells, pharmaceutical composition, or method of any one of the preceding embodiments, comprising or contacting a cell with an exogenous nucleic acid, wherein the exogenous nucleic acid encodes an NK cell inhibitor molecule, wherein the NK cell inhibitor molecule binds to an inhibitory receptor on an NK cell.
Embodiment 129 is the engineered cell, population of cells, pharmaceutical composition, or method of any one of the preceding embodiments, comprising or contacting a cell with an exogenous nucleic acid, wherein the exogenous nucleic acid encodes an NK cell inhibitor molecule, wherein the NK cell inhibitor molecule binds to NKG2A on an NK cell.
Embodiment 130 is the engineered cell, population of cells, pharmaceutical composition, or method of any one of the preceding embodiments, comprising or contacting a cell with an exogenous nucleic acid, wherein the exogenous nucleic acid encodes an NK cell inhibitor molecule, wherein the NK cell inhibitor molecule is a non-classical MHC class I molecule.
Embodiment 131 is the engineered cell, population of cells, pharmaceutical composition, or method of any one of the preceding embodiments, comprising or contacting a cell with an exogenous nucleic acid, wherein the exogenous nucleic acid encodes an NK cell inhibitor molecule, wherein the NK cell inhibitor molecule is HLA-E.
Embodiment 132 is the engineered cell, cell population, pharmaceutical composition, or method of any one of the preceding embodiments, comprising or contacting a cell with an exogenous nucleic acid, wherein the exogenous nucleic acid encodes an NK cell inhibitor molecule, wherein the NK cell inhibitor molecule is a fusion protein.
Embodiment 133 is the engineered cell, population of cells, pharmaceutical composition, or method of any one of the preceding embodiments, comprising or contacting a cell with an exogenous nucleic acid, wherein the exogenous nucleic acid encodes an NK cell inhibitor molecule, wherein the NK cell inhibitor molecule is a fusion protein comprising HLA-E and B2M.
Embodiment 134 is the engineered cell, cell population, pharmaceutical composition, or method of any of the preceding embodiments, comprising or contacting a cell with an exogenous nucleic acid encoding a polypeptide secreted by the cell, wherein the secreted polypeptide is an antibody or antibody fragment.
Embodiment 135 is an engineered cell, population of cells, pharmaceutical composition, or method of any of the preceding embodiments comprising or contacting a cell with an exogenous nucleic acid encoding a polypeptide secreted by the cell, wherein the secreted polypeptide is a full length IgG antibody.
Embodiment 136 is the engineered cell, cell population, pharmaceutical composition, or method of any of the preceding embodiments, comprising or contacting a cell with an exogenous nucleic acid encoding a polypeptide secreted by the cell, wherein the secreted polypeptide is a single chain antibody.
Embodiment 137 is the engineered cell, cell population, pharmaceutical composition, or method of any of the preceding embodiments, comprising or contacting a cell with an exogenous nucleic acid encoding a polypeptide secreted by the cell, wherein the secreted polypeptide is a neutralizing antibody.
Embodiment 138 is the engineered cell, cell population, pharmaceutical composition, or method of any of the preceding embodiments, comprising or contacting a cell with an exogenous nucleic acid encoding a polypeptide secreted by the cell, wherein the secreted polypeptide is an enzyme.
Embodiment 139 is the engineered cell, cell population, pharmaceutical composition, or method of any of the preceding embodiments, comprising or contacting a cell with an exogenous nucleic acid encoding a polypeptide secreted by the cell, wherein the secreted polypeptide is a cytokine.
Embodiment 140 is the engineered cell, cell population, pharmaceutical composition, or method of any of the preceding embodiments, comprising or contacting a cell with an exogenous nucleic acid encoding a polypeptide secreted by the cell, wherein the secreted polypeptide is a fusion protein.
Embodiment 141 is the engineered cell, cell population, pharmaceutical composition, or method of any of the preceding embodiments, comprising or contacting a cell with an exogenous nucleic acid encoding a polypeptide secreted by the cell, wherein the secreted polypeptide comprises a soluble receptor. The engineered cell, population of cells, pharmaceutical composition, or method of any one of the preceding embodiments, comprising or contacting a cell with an exogenous nucleic acid encoding a targeting receptor, wherein the targeting receptor is a T Cell Receptor (TCR).
Embodiment 142 is the engineered cell, population of cells, pharmaceutical composition, or method of any one of the preceding embodiments, comprising or contacting a cell with an exogenous nucleic acid encoding a targeting receptor, wherein the targeting receptor is a genetically modified TCR.
Embodiment 143 is the engineered cell, population of cells, pharmaceutical composition, or method of any one of the preceding embodiments, comprising or contacting a cell with an exogenous nucleic acid encoding a targeting receptor, wherein the targeting receptor is a WT1 TCR.
Embodiment 144 is the engineered cell, population of cells, pharmaceutical composition, or method of any of the preceding embodiments, comprising or contacting a cell with an exogenous nucleic acid encoding a targeting receptor, wherein the targeting receptor is a CAR.
Embodiment 145 is the engineered cell, population of cells, pharmaceutical composition, or method of any one of the preceding embodiments, wherein CIITA guide RNA is provided to the cell in a vector.
Embodiment 146 is the engineered cell, cell population, pharmaceutical composition, or method of any of the preceding embodiments, wherein the CIITARNA-directed DNA binding agent is provided to the cell in a vector, optionally in the same vector as the CIITARNA-directed RNA.
Embodiment 147 is the engineered cell, cell population, pharmaceutical composition, or method of any of the preceding embodiments, wherein the exogenous nucleic acid is provided to the cell in a vector.
Embodiment 148 is the engineered cell, cell population, pharmaceutical composition, or method of any of the preceding embodiments, wherein the vector is a viral vector.
Embodiment 149 is the engineered cell, cell population, pharmaceutical composition, or method of any one of the preceding embodiments, wherein the vector is a lentiviral vector.
Embodiment 150 is the engineered cell, cell population, pharmaceutical composition, or method of any one of the preceding embodiments, wherein the vector is AAV.
Embodiment 151 is the engineered cell, cell population, pharmaceutical composition, or method of any of the preceding embodiments, wherein the vector is a non-viral vector.
Embodiment 152 is the engineered cell, cell population, pharmaceutical composition, or method of any one of the preceding embodiments, wherein the gene editing system components are provided to the cell in a lipid nucleic acid assembly composition.
Embodiment 153 is the engineered cell, cell population, pharmaceutical composition, or method of any of the preceding embodiments, wherein guide RNA is provided to the cell in a lipid nucleic acid assembly composition, optionally in the same lipid nucleic acid assembly composition as the RNA-guided DNA binding agent.
Embodiment 154 is the engineered cell, cell population, pharmaceutical composition, or method of any of the preceding embodiments, wherein the exogenous nucleic acid is provided to the cell in a lipid nucleic acid assembly composition.
Embodiment 155 is the engineered cell, cell population, pharmaceutical composition, or method of any of the preceding embodiments, wherein the lipid nucleic acid assembly composition is a Lipid Nanoparticle (LNP).
Embodiment 156 is the engineered cell, cell population, pharmaceutical composition, or method of any of the preceding embodiments, wherein the exogenous nucleic acid line is integrated into the genome of the cell.
Embodiment 157 is the engineered cell, cell population, pharmaceutical composition, or method of any of the preceding embodiments, wherein the exogenous nucleic acid is integrated into the genome of the cell by Homologous Recombination (HR).
Embodiment 158 is an engineered cell, cell population, pharmaceutical composition, or method of any of the preceding embodiments, wherein an exogenous nucleic acid is integrated into a safe harbor locus in the cell genome.
Embodiment 159 is the engineered cell, cell population, pharmaceutical composition, or method of any one of the preceding embodiments, wherein the CIITA guide RNA comprises SEQ ID No. 1.
Embodiment 160 is the engineered cell, cell population, pharmaceutical composition, or method of any of the preceding embodiments, wherein the CIITA guide RNA comprises SEQ ID No. 2.
Embodiment 161 is the engineered cell, cell population, pharmaceutical composition, or method of any of the preceding embodiments, wherein the CIITA guide RNA comprises SEQ ID No. 3.
Embodiment 162 is the engineered cell, cell population, pharmaceutical composition, or method of any of the preceding embodiments, wherein the CIITA guide RNA comprises SEQ ID No. 4.
Embodiment 163 is the engineered cell, cell population, pharmaceutical composition, or method of any of the preceding embodiments, wherein the CIITA guide RNA comprises SEQ ID No. 5.
Embodiment 164 is the engineered cell, cell population, pharmaceutical composition, or method of any of the preceding embodiments, wherein the CIITA guide RNA comprises SEQ ID No. 6.
Embodiment 165 is the engineered cell, cell population, pharmaceutical composition, or method of any of the preceding embodiments, wherein the CIITA guide RNA comprises SEQ ID No. 7.
Embodiment 166 is the engineered cell, cell population, pharmaceutical composition, or method of any of the preceding embodiments, wherein the CIITA guide RNA comprises SEQ ID No. 8.
Embodiment 167 is the engineered cell, cell population, pharmaceutical composition, or method of any of the preceding embodiments, wherein the CIITA guide RNA comprises SEQ ID No. 9.
Embodiment 168 is the engineered cell, cell population, pharmaceutical composition, or method of any of the preceding embodiments, wherein the CIITA guide RNA comprises SEQ ID No. 10.
Embodiment 169 is the engineered cell, cell population, pharmaceutical composition or method of any one of the preceding embodiments, wherein the CIITA guide RNA comprises SEQ ID No. 11.
Embodiment 170 is the engineered cell, cell population, pharmaceutical composition, or method of any one of the preceding embodiments, wherein the CIITA guide RNA comprises SEQ ID No. 12.
Embodiment 171 is the engineered cell, cell population, pharmaceutical composition, or method of any one of the preceding embodiments, wherein the CIITA guide RNA comprises SEQ ID No. 13.
Embodiment 172 is the engineered cell, cell population, pharmaceutical composition, or method of any one of the preceding embodiments, wherein the CIITA guide RNA comprises SEQ ID No. 14.
Embodiment 173 is the engineered cell, cell population, pharmaceutical composition, or method of any of the preceding embodiments, wherein the CIITA guide RNA comprises SEQ ID No. 15.
Embodiment 174 is the engineered cell, cell population, pharmaceutical composition, or method of any of the preceding embodiments, wherein the CIITA guide RNA comprises SEQ ID No. 16.
Embodiment 175 is the engineered cell, cell population, pharmaceutical composition, or method of any one of the preceding embodiments, wherein the CIITA guide RNA comprises SEQ ID No. 17.
Embodiment 176 is the engineered cell, cell population, pharmaceutical composition, or method of any one of the preceding embodiments, wherein the CIITA guide RNA comprises SEQ ID No. 18.
Embodiment 177 is the engineered cell, cell population, pharmaceutical composition or method of any of the preceding embodiments, wherein the CIITA guide RNA comprises SEQ ID No. 19.
Embodiment 178 is the engineered cell, cell population, pharmaceutical composition, or method of any one of the preceding embodiments, wherein the CIITA guide RNA comprises SEQ ID No. 20.
Embodiment 179 is the engineered cell, cell population, pharmaceutical composition, or method of any one of the preceding embodiments, wherein the CIITA guide RNA comprises SEQ ID No. 21.
Embodiment 180 is the engineered cell, cell population, pharmaceutical composition, or method of any one of the preceding embodiments, wherein the CIITA guide RNA comprises SEQ ID No. 22.
Embodiment 181 is the engineered cell, cell population, pharmaceutical composition, or method of any of the preceding embodiments, wherein the CIITA guide RNA comprises SEQ ID No. 23.
Embodiment 182 is the engineered cell, cell population, pharmaceutical composition, or method of any of the preceding embodiments, wherein the CIITA guide RNA comprises SEQ ID No. 24.
Embodiment 183 is the engineered cell, cell population, pharmaceutical composition, or method of any of the preceding embodiments, wherein the CIITA guide RNA comprises SEQ ID No. 25.
Embodiment 184 is the engineered cell, cell population, pharmaceutical composition, or method of any of the preceding embodiments, wherein the CIITA guide RNA comprises SEQ ID No. 26.
Embodiment 185 is the engineered cell, cell population, pharmaceutical composition, or method of any one of the preceding embodiments, wherein the CIITA guide RNA comprises SEQ ID No. 27.
Embodiment 186 is the engineered cell, cell population, pharmaceutical composition, or method of any of the preceding embodiments, wherein the CIITA guide RNA comprises SEQ ID No. 28.
Embodiment 187 is the engineered cell, cell population, pharmaceutical composition, or method of any preceding embodiment, wherein the CIITA guide RNA comprises SEQ ID No. 29.
Embodiment 188 is the engineered cell, cell population, pharmaceutical composition or method of any of the preceding embodiments, wherein the CIITA guide RNA comprises SEQ ID No. 30.
Embodiment 189 is an engineered cell, cell population, pharmaceutical composition, or method of any one of the preceding embodiments, wherein the CIITA guide RNA comprises SEQ ID No. 31.
Embodiment 190 is the engineered cell, cell population, pharmaceutical composition, or method of any of the preceding embodiments, wherein the CIITA guide RNA comprises SEQ ID No. 32.
Embodiment 191 is the engineered cell, cell population, pharmaceutical composition, or method of any of the preceding embodiments, wherein the CIITA guide RNA comprises SEQ ID No. 33.
Embodiment 192 is the engineered cell, cell population, pharmaceutical composition, or method of any one of the preceding embodiments, wherein the CIITA guide RNA comprises SEQ ID No. 34.
Embodiment 193 is the engineered cell, cell population, pharmaceutical composition or method of any preceding embodiment, wherein the CIITA guide RNA comprises SEQ ID No. 35.
Embodiment 194 is the engineered cell, cell population, pharmaceutical composition, or method of any of the preceding embodiments, wherein the CIITA guide RNA comprises SEQ ID No. 36.
Embodiment 195 is the engineered cell, cell population, pharmaceutical composition, or method of any of the preceding embodiments, wherein the CIITA guide RNA comprises SEQ ID No. 37.
Embodiment 196 is the engineered cell, cell population, pharmaceutical composition, or method of any one of the preceding embodiments, wherein the CIITA guide RNA comprises SEQ ID No. 38.
Embodiment 197 is the engineered cell, cell population, pharmaceutical composition, or method of any of the preceding embodiments, wherein the CIITA guide RNA comprises SEQ ID No. 39.
Embodiment 198 is the engineered cell, cell population, pharmaceutical composition, or method of any one of the preceding embodiments, wherein the CIITA guide RNA comprises SEQ ID No. 40.
Embodiment 199 is the engineered cell, cell population, pharmaceutical composition, or method of any one of the preceding embodiments, wherein the CIITA guide RNA comprises SEQ ID No. 41.
Embodiment 200 is the engineered cell, cell population, pharmaceutical composition, or method of any one of the preceding embodiments, wherein the CIITA guide RNA comprises SEQ ID No. 42.
Embodiment 201 is the engineered cell, cell population, pharmaceutical composition, or method of any one of the preceding embodiments, wherein the CIITA guide RNA comprises SEQ ID No. 43.
Embodiment 202 is the engineered cell, cell population, pharmaceutical composition, or method of any one of the preceding embodiments, wherein the CIITA guide RNA comprises SEQ ID No. 44.
Embodiment 203 is the engineered cell, cell population, pharmaceutical composition, or method of any of the preceding embodiments, wherein the CIITA guide RNA comprises SEQ ID No. 45.
Embodiment 204 is the engineered cell, cell population, pharmaceutical composition, or method of any of the preceding embodiments, wherein the CIITA guide RNA comprises SEQ ID No. 46.
Embodiment 205 is the engineered cell, cell population, pharmaceutical composition, or method of any one of the preceding embodiments, wherein the CIITA guide RNA comprises SEQ ID No. 47.
Embodiment 206 is the engineered cell, cell population, pharmaceutical composition, or method of any of the preceding embodiments, wherein the CIITA guide RNA comprises SEQ ID No. 48.
Embodiment 207 is an engineered cell, cell population, pharmaceutical composition, or method of any of the preceding embodiments, wherein the CIITA guide RNA comprises SEQ ID No. 49.
Embodiment 208 is the engineered cell, cell population, pharmaceutical composition, or method of any one of the preceding embodiments, wherein the CIITA guide RNA comprises SEQ ID No. 50.
Embodiment 209 is the engineered cell, cell population, pharmaceutical composition, or method of any of the preceding embodiments, wherein the CIITA guide RNA comprises SEQ ID No. 51.
Embodiment 210 is the engineered cell, cell population, pharmaceutical composition, or method of any one of the preceding embodiments, wherein the CIITA guide RNA comprises SEQ ID No. 52.
Embodiment 211 is the engineered cell, cell population, pharmaceutical composition, or method of any one of the preceding embodiments, wherein the CIITA guide RNA comprises SEQ ID No. 53.
Embodiment 212 is the engineered cell, cell population, pharmaceutical composition, or method of any one of the preceding embodiments, wherein the CIITA guide RNA comprises SEQ ID No. 54.
Embodiment 213 is the engineered cell, cell population, pharmaceutical composition or method of any of the preceding embodiments, wherein the CIITA guide RNA comprises SEQ ID No. 55.
Embodiment 214 is the engineered cell, cell population, pharmaceutical composition, or method of any one of the preceding embodiments, wherein the CIITA guide RNA comprises SEQ ID No. 56.
Embodiment 215 is the engineered cell, cell population, pharmaceutical composition or method of any of the preceding embodiments, wherein the CIITA guide RNA comprises SEQ ID NO 57.
Embodiment 216 is the engineered cell, cell population, pharmaceutical composition, or method of any one of the preceding embodiments, wherein the CIITA guide RNA comprises SEQ ID No. 58.
Embodiment 217 is the engineered cell, cell population, pharmaceutical composition, or method of any of the preceding embodiments, wherein the CIITA guide RNA comprises SEQ ID No. 59.
Embodiment 218 is the engineered cell, cell population, pharmaceutical composition, or method of any one of the preceding embodiments, wherein the CIITA guide RNA comprises SEQ ID No. 60.
Embodiment 219 is the engineered cell, cell population, pharmaceutical composition, or method of any one of the preceding embodiments, wherein the CIITA guide RNA comprises SEQ ID No. 61.
Embodiment 220 is the engineered cell, cell population, pharmaceutical composition, or method of any of the preceding embodiments, wherein the CIITA guide RNA comprises SEQ ID No. 62.
Embodiment 221 is the engineered cell, cell population, pharmaceutical composition, or method of any of the preceding embodiments, wherein the CIITA guide RNA comprises SEQ ID No. 63.
Embodiment 222 is the engineered cell, cell population, pharmaceutical composition, or method of any one of the preceding embodiments, wherein the CIITA guide RNA comprises SEQ ID No. 64.
Embodiment 223 is the engineered cell, cell population, pharmaceutical composition, or method of any of the preceding embodiments, wherein the CIITA guide RNA comprises SEQ ID No. 65.
Embodiment 224 is the engineered cell, cell population, pharmaceutical composition, or method of any one of the preceding embodiments, wherein the CIITA guide RNA comprises SEQ ID No. 66.
Embodiment 225 is the engineered cell, cell population, pharmaceutical composition, or method of any one of the preceding embodiments, wherein the CIITA guide RNA comprises SEQ ID No. 67.
Embodiment 226 is the engineered cell, cell population, pharmaceutical composition, or method of any one of the preceding embodiments, wherein the CIITA guide RNA comprises SEQ ID No. 68.
Embodiment 227 is the engineered cell, cell population, pharmaceutical composition, or method of any one of the preceding embodiments, wherein the CIITA guide RNA comprises SEQ ID No. 69.
Embodiment 228 is the engineered cell, cell population, pharmaceutical composition or method of any of the preceding embodiments, wherein the CIITA guide RNA comprises SEQ ID No. 70.
Embodiment 229 is the engineered cell, cell population, pharmaceutical composition or method of any of the preceding embodiments, wherein the CIITA guide RNA comprises SEQ ID No. 71.
Embodiment 230 is the engineered cell, cell population, pharmaceutical composition, or method of any of the preceding embodiments, wherein the CIITA guide RNA comprises SEQ ID No. 72.
Embodiment 231 is an engineered cell, cell population, pharmaceutical composition, or method of any of the preceding embodiments, wherein the CIITA guide RNA comprises SEQ ID No. 73.
Embodiment 232 is the engineered cell, cell population, pharmaceutical composition, or method of any one of the preceding embodiments, wherein the CIITA guide RNA comprises SEQ ID No. 74.
Embodiment 233 is the engineered cell, cell population, pharmaceutical composition, or method of any of the preceding embodiments, wherein the CIITA guide RNA comprises SEQ ID No. 75.
Embodiment 234 is the engineered cell, cell population, pharmaceutical composition, or method of any one of the preceding embodiments, wherein the CIITA guide RNA comprises SEQ ID No. 76.
Embodiment 235 is the engineered cell, cell population, pharmaceutical composition, or method of any of the preceding embodiments, wherein the CIITA guide RNA comprises SEQ ID No. 77.
Embodiment 236 is the engineered cell, cell population, pharmaceutical composition or method of any preceding embodiment, wherein the CIITA guide RNA comprises SEQ ID No. 78.
Embodiment 237 is the engineered cell, cell population, pharmaceutical composition, or method of any one of the preceding embodiments, wherein the CIITA guide RNA comprises SEQ ID No. 79.
Embodiment 238 is the engineered cell, cell population, pharmaceutical composition, or method of any of the preceding embodiments, wherein the CIITA guide RNA comprises SEQ ID No. 80.
Embodiment 239 is the engineered cell, cell population, pharmaceutical composition, or method of any of the preceding embodiments, wherein the CIITA guide RNA comprises SEQ ID No. 81.
Embodiment 240 is the engineered cell, cell population, pharmaceutical composition, or method of any one of the preceding embodiments, wherein the CIITA guide RNA comprises SEQ ID No. 82.
Embodiment 241 is the engineered cell, cell population, pharmaceutical composition, or method of any of the preceding embodiments, wherein the CIITA guide RNA comprises SEQ ID No. 83.
Embodiment 242 is the engineered cell, cell population, pharmaceutical composition, or method of any of the preceding embodiments, wherein the CIITA guide RNA comprises SEQ ID No. 84.
Embodiment 243 is the engineered cell, cell population, pharmaceutical composition, or method of any of the preceding embodiments, wherein the CIITA guide RNA comprises SEQ ID No. 85.
Embodiment 244 is the engineered cell, cell population, pharmaceutical composition, or method of any one of the preceding embodiments, wherein the CIITA guide RNA comprises SEQ ID No. 86.
Embodiment 245 is the engineered cell, cell population, pharmaceutical composition, or method of any of the preceding embodiments, wherein the CIITA guide RNA comprises SEQ ID No. 87.
Embodiment 246 is the engineered cell, cell population, pharmaceutical composition, or method of any of the preceding embodiments, wherein the CIITA guide RNA comprises SEQ ID No. 88.
Embodiment 247 is the engineered cell, cell population, pharmaceutical composition, or method of any one of the preceding embodiments, wherein the CIITA guide RNA comprises SEQ ID No. 89.
Embodiment 248 is the engineered cell, cell population, pharmaceutical composition, or method of any of the preceding embodiments, wherein the CIITA guide RNA comprises SEQ ID No. 90.
Embodiment 249 is the engineered cell, cell population, pharmaceutical composition, or method of any of the preceding embodiments, wherein the CIITA guide RNA comprises SEQ ID No. 91.
Embodiment 250 is the engineered cell, cell population, pharmaceutical composition, or method of any of the preceding embodiments, wherein the CIITA guide RNA comprises SEQ ID No. 92.
Embodiment 251 is an engineered cell, cell population, pharmaceutical composition, or method of any of the preceding embodiments, wherein the CIITA guide RNA comprises SEQ ID No. 93.
Embodiment 252 is the engineered cell, cell population, pharmaceutical composition, or method of any of the preceding embodiments, wherein the CIITA guide RNA comprises SEQ ID No. 94.
Embodiment 253 is the engineered cell, cell population, pharmaceutical composition, or method of any one of the preceding embodiments, wherein the CIITA guide RNA comprises SEQ ID No. 95.
Embodiment 254 is the engineered cell, cell population, pharmaceutical composition, or method of any of the preceding embodiments, wherein the CIITA guide RNA comprises SEQ ID No. 96.
Embodiment 255 is the engineered cell, cell population, pharmaceutical composition, or method of any one of the preceding embodiments, wherein the CIITA guide RNA comprises SEQ ID No. 97.
Embodiment 256 is the engineered cell, cell population, pharmaceutical composition, or method of any one of the preceding embodiments, wherein the CIITA guide RNA comprises SEQ ID No. 98.
Embodiment 257 is an engineered cell, cell population, pharmaceutical composition, or method of any one of the preceding embodiments, wherein the CIITA guide RNA comprises SEQ ID NO 99.
Embodiment 258 is the engineered cell, cell population, pharmaceutical composition, or method of any of the preceding embodiments, wherein the CIITA guide RNA comprises SEQ ID No. 100.
Embodiment 259 is an engineered cell, cell population, pharmaceutical composition or method of any of the preceding embodiments, wherein the CIITA guide RNA comprises SEQ ID No. 101.
Embodiment 260 is the engineered cell, cell population, pharmaceutical composition, or method of any of the preceding embodiments, wherein the CIITA guide RNA comprises SEQ ID No. 102.
Embodiment 261 is an engineered cell, cell population, pharmaceutical composition, or method of any one of the preceding embodiments, wherein the CIITA guide RNA comprises SEQ ID No. 103.
Embodiment 262 is the engineered cell, cell population, pharmaceutical composition, or method of any one of the preceding embodiments, wherein the CIITA guide RNA comprises SEQ ID No. 104.
Embodiment 263 is the engineered cell, cell population, pharmaceutical composition, or method of any of the preceding embodiments, wherein the CIITA guide RNA comprises SEQ ID No. 105.
Embodiment 264 is the engineered cell, cell population, pharmaceutical composition, or method of any of the preceding embodiments, wherein the CIITA guide RNA comprises SEQ ID No. 106.
Embodiment 265 is the engineered cell, cell population, pharmaceutical composition or method of any of the preceding embodiments, wherein the CIITA guide RNA comprises SEQ ID No. 107.
Embodiment 266 is the engineered cell, cell population, pharmaceutical composition, or method of any of the preceding embodiments, wherein the CIITA guide RNA comprises SEQ ID No. 108.
Embodiment 267 is the engineered cell, cell population, pharmaceutical composition, or method of any of the preceding embodiments, wherein the CIITA guide RNA comprises SEQ ID No. 109.
Embodiment 268 is the engineered cell, cell population, pharmaceutical composition, or method of any one of the preceding embodiments, wherein the CIITA guide RNA comprises SEQ ID No. 110.
Embodiment 269 is an engineered cell, cell population, pharmaceutical composition, or method of any of the preceding embodiments, wherein the CIITA guide RNA comprises SEQ ID No. 111.
Embodiment 270 is the engineered cell, cell population, pharmaceutical composition, or method of any of the preceding embodiments, wherein the CIITA guide RNA comprises SEQ ID No. 112.
Embodiment 271 is an engineered cell, cell population, pharmaceutical composition, or method of any of the preceding embodiments, wherein the CIITA guide RNA comprises SEQ ID No. 113.
Embodiment 272 is the engineered cell, cell population, pharmaceutical composition, or method of any of the preceding embodiments, wherein the CIITA guide RNA comprises SEQ ID No. 114.
Embodiment 273 is an engineered cell, cell population, pharmaceutical composition, or method of any of the preceding embodiments, wherein the CIITA guide RNA comprises SEQ ID No. 115.
Embodiment 274 is the engineered cell, cell population, pharmaceutical composition or method of any of the preceding embodiments, wherein the CIITA guide RNA comprises SEQ ID No. 116.
Embodiment 275 is the engineered cell, cell population, pharmaceutical composition, or method of any of the preceding embodiments, wherein the CIITA guide RNA comprises SEQ ID No. 117.
Embodiment 276 is an engineered cell, cell population, pharmaceutical composition, or method of any of the preceding embodiments, wherein the CIITA guide RNA comprises at least one modification.
Embodiment 277 is the engineered cell, cell population, pharmaceutical composition, or method of any of the preceding embodiments, wherein the CIITA guide RNA comprises at least one modification, wherein the at least one modification comprises a 2 '-O-methyl (2' -O-Me) modified nucleotide.
Embodiment 278 is the engineered cell, cell population, pharmaceutical composition, or method of any of the preceding embodiments, wherein the CIITA guide RNA comprises at least one modification comprising Phosphorothioate (PS) linkages between nucleotides.
Embodiment 279 is the engineered cell, cell population, pharmaceutical composition, or method of any one of the preceding embodiments, wherein the CIITA guide RNA comprises at least one modification comprising a 2 '-fluoro (2' -F) modified nucleotide.
Embodiment 280 is the engineered cell, cell population, pharmaceutical composition, or method of any of the preceding embodiments, wherein the CIITA guide RNA comprises at least one modification comprising a modification at one or more of the first five nucleotides at the 5' end of the guide RNA.
Embodiment 281 is the engineered cell, cell population, pharmaceutical composition or method of any of the preceding embodiments, wherein the CIITA guide RNA comprises at least one modification comprising a modification at one or more of the last five nucleotides of the 3' end of the guide RNA.
Embodiment 282 is the engineered cell, cell population, pharmaceutical composition, or method of any of the preceding embodiments, wherein the CIITA guide RNA comprises at least one modification comprising a PS bond between the first four nucleotides of the guide RNA.
Embodiment 283 is the engineered cell, cell population, pharmaceutical composition, or method of any of the preceding embodiments, wherein the CIITA guide RNA comprises at least one modification comprising a PS bond between the last four nucleotides of the guide RNA.
Embodiment 284 is the engineered cell, cell population, pharmaceutical composition, or method of any one of the preceding embodiments, wherein the CIITA guide RNA comprises at least one modification comprising a 2'-O-Me modified nucleotide at the first three nucleotides of the 5' end of the guide RNA.
Embodiment 285 is the engineered cell, cell population, pharmaceutical composition, or method of any of the preceding embodiments, wherein the CIITA guide RNA comprises at least one modification comprising a 2'-O-Me modified nucleotide at the last three nucleotides of the 3' end of the guide RNA.
Embodiment 286 is the engineered cell or cell population of any of the preceding embodiments, comprising a genetic modification comprising an insertion/deletion within a genomic region targeted by CIITA guide RNA.
Embodiment 287 is the engineered cell or cell population of any of the preceding embodiments, comprising a genetic modification comprising a substitution of C for T or a substitution of a for G in a genomic region targeted by CIITA guide RNA.
Embodiment 288 is an engineered cell, cell population, pharmaceutical composition or method of any of the preceding embodiments for expressing a TCR specific for a polypeptide expressed by a cancer cell.
Embodiment 289 is an engineered cell, population of cells, pharmaceutical composition, or method of any one of the preceding embodiments, for administration to a subject as Adoptive Cell Transfer (ACT) therapy.
Embodiment 290 is the engineered cell, cell population, pharmaceutical composition or method of any of the preceding embodiments for use in treating a subject having cancer.
Embodiment 291 is an engineered cell, population of cells, pharmaceutical composition, or method of any of the preceding embodiments for use in treating a subject having an infectious disease.
Embodiment 292 is the engineered cell, cell population, pharmaceutical composition or method of any one of the preceding embodiments for use in treating a subject suffering from an autoimmune disease.
Embodiment 293 is the engineered cell, cell population, pharmaceutical composition, or method of any of the preceding embodiments, wherein the genetic modification comprises an insertion/deletion.
Embodiment 294 is the engineered cell, cell population, pharmaceutical composition, or method of any of the preceding embodiments, wherein the genetic modification comprises substitution of C to T.
Embodiment 295 is the engineered cell, cell population, pharmaceutical composition, or method of any of the preceding embodiments, wherein the genetic modification comprises a substitution of a to G.
Embodiment 296 is the engineered cell, population of cells, pharmaceutical composition, or method of any one of the preceding embodiments, wherein the cell is homozygous for HLA-B and homozygous for HLA-C.
Embodiment 297 is the engineered cell, population of cells, pharmaceutical composition, or method of any one of the preceding embodiments, wherein the cell further comprises a genetic modification in an HLA-A gene, wherein the cell is homozygous for HLa-B and homozygous for HLa-C, and wherein the genetic modification in the HLA-A gene comprises at least one nucleotide within genomic coordinates selected from the group consisting of: (a) chr6: 29942854-chr6: 29942913 and (b) chr6: 29943518-chr6: 29943619.
Embodiment 298 is the engineered cell, population of cells, pharmaceutical composition, or method of any one of the preceding embodiments, wherein the cell further comprises a genetic modification in an HLA-A gene, and wherein the genetic modification in the HLA-A gene comprises at least one nucleotide within genomic coordinates selected from the group consisting of: chr6:29942864 to chr6:29942903.
Embodiment 299 is an engineered cell, population of cells, pharmaceutical composition, or method of any of the preceding embodiments, wherein the cell further comprises a genetic modification in an HLA-A gene, and wherein the genetic modification in the HLA-A gene comprises at least one nucleotide within genomic coordinates selected from the group consisting of: chr6:29943528 to chr6:29943609.
Embodiment 300 is the engineered cell, cell population, pharmaceutical composition, or method of any one of the preceding embodiments, wherein the cell further comprises a genetic modification in an HLA-A gene, and wherein the genetic modification in the HLA-A gene comprises at least one nucleotide within genomic coordinates selected from the group consisting of: chr6:29942864-29942884; chr6:29942868-29942888; chr6:29942876-29942896; chr6:29942877-29942897; chr6:29942883-29942903; chr6:29943126-29943146; chr6:29943528-29943548; chr6:29943529-29943549; chr6:29943530-29943550; chr6:29943537-29943557; chr6:29943549-29943569; chr6:29943589-29943609; and chr6:29944026-29944046.
Embodiment 301 is the engineered cell, cell population, pharmaceutical composition, or method of any of the preceding embodiments, wherein the cell further comprises a genetic modification in an HLA-A gene, and wherein the genetic modification in an HLA-A gene comprises an insertion/deletion within genomic coordinates selected from the group consisting of: chr6:29942864-29942884; chr6:29942868-29942888; chr6:29942876-29942896; chr6:29942877-29942897; chr6:29942883-29942903; chr6:29943126-29943146; chr6:29943528-29943548; chr6:29943529-29943549; chr6:29943530-29943550; chr6:29943537-29943557; chr6:29943549-29943569; chr6:29943589-29943609; and chr6:29944026-29944046.
Embodiment 302 is an engineered cell, population of cells, pharmaceutical composition, or method of any one of the preceding embodiments, wherein HLA-A expression of the cell is reduced or eliminated by a gene editing system that binds to an HLA-A genomic target sequence comprising at least 5 contiguous nucleotides within genomic coordinates selected from the group consisting of: chr6:29942864-29942884; chr6:29942868-29942888; chr6:29942876-29942896; chr6:29942877-29942897; chr6:29942883-29942903; chr6:29943126-29943146; chr6:29943528-29943548; chr6:29943529-29943549; chr6:29943530-29943550; chr6:29943537-29943557; chr6:29943549-29943569; chr6:29943589-29943609; and chr6:29944026-29944046.
Embodiment 303 is a method of making an engineered cell having reduced or eliminated surface expression of MHC class II proteins and HLA-A proteins relative to an unmodified cell, the method comprising: (a) Contacting the cell with a CIITA guide RNA, wherein the guide RNA comprises a guide sequence selected from SEQ ID NOs 1-117; (b) Contacting the cell with HLA-A guide RNA, wherein HLA-A guide RNA comprises a guide sequence selected from any one of SEQ ID NOs 2001-2095; and (c) optionally contacting the cell with an RNA-guided DNA binding agent or a nucleic acid encoding an RNA-guided DNA binding agent; thereby reducing or eliminating the surface expression of MHC class II proteins and HLA-A proteins in the cell relative to the unmodified cell.
Embodiment 304 is the method of embodiment 303, wherein the CIITA guide RNA comprises a sequence selected from SEQ ID NOs 32, 64, 67, 68, 74, 76, 84, 86, 90, 91 and 115.
Embodiment 305 is the method of embodiment 303 or 304, comprising contacting a cell with an RNA-guided DNA binding agent or a nucleic acid encoding an RNA-guided DNA binding agent, optionally wherein the RNA-guided DNA binding agent comprises streptococcus pyogenes Cas9.
Embodiment 306 is the engineered cell, cell population, pharmaceutical composition, or method of any of the preceding embodiments, wherein HLA-B is selected from any of the following HLA-B alleles: HLA-B.times.07:02; HLA-B08:01; HLA-B44:02; HLA-B35:01; HLA-B40:01; HLA-B57:01; HLA-B14:02; HLA-B15:01; HLA-B13:02; HLA-B44:03; HLA-B38:01; HLA-B18:01; HLA-B44:03; HLA-B51:01; HLA-B49:01; HLA-B15:01; HLA-B18:01; HLA-B27:05; HLA-B35:03; HLA-B18:01; HLA-B52:01; HLA-B51:01; HLA-B37:01; HLA-B53:01; HLA-B55:01; HLA-B44:02; HLA-B44:03; HLA-B35:02; HLA-B15:01; and HLA-B40:02.
Embodiment 307 is an engineered cell, cell population, pharmaceutical composition, or method of any one of the preceding embodiments, wherein HLA-C is selected from any one of the following HLA-C alleles: HLA-C.times.07:02; HLA-C.times.07:01; HLA-C05:01; HLA-C.times.04:01HLA-C.times.03:04; HLA-C06:02; HLA-C08:02; HLA-C.times.03:03; HLA-C06:02; HLA-C16:01; HLA-C12:03; HLA-C.times.07:01; HLA-C04:01; HLA-C15:02; HLA-C.times.07:01; HLA-C03:04; HLA-C12:03; HLA-C02:02; HLA-C04:01; HLA-C05:01; HLA-C12:02; HLA-C14:02; HLA-C06:02; HLA-C04:01; HLA-C.times.03:03; HLA-C07:04; HLA-C.times.07:01; HLA-C04:01; HLA-C04:01; and HLA-C02:02.
Embodiment 308 is the engineered cell, cell population, pharmaceutical composition, or method of any one of the preceding embodiments, wherein the HLA-B allele is selected from any one of the following HLA-B alleles: HLA-B.times.07:02; HLA-B08:01; HLA-B44:02; HLA-B35:01; HLA-B40:01; HLA-B57:01; HLA-B14:02; HLA-B15:01; HLA-B13:02; HLA-B44:03; HLA-B38:01; HLA-B18:01; HLA-B44:03; HLA-B51:01; HLA-B49:01; HLA-B15:01; HLA-B18:01; HLA-B27:05; HLA-B35:03; HLA-B18:01; HLA-B52:01; HLA-B51:01; HLA-B37:01; HLA-B53:01; HLA-B55:01; HLA-B44:02; HLA-B44:03; HLA-B35:02; HLA-B15:01; and HLA-B40:02; and the HLA-C allele is selected from any one of the following HLA-C alleles: HLA-C.times.07:02; HLA-C.times.07:01; HLA-C05:01; HLA-C.times.04:01HLA-C.times.03:04; HLA-C06:02; HLA-C08:02; HLA-C.times.03:03; HLA-C06:02; HLA-C16:01; HLA-C12:03; HLA-C.times.07:01; HLA-C04:01; HLA-C15:02; HLA-C.times.07:01; HLA-C03:04; HLA-C12:03; HLA-C02:02; HLA-C04:01; HLA-C05:01; HLA-C12:02; HLA-C14:02; HLA-C06:02; HLA-C04:01; HLA-C.times.03:03; HLA-C07:04; HLA-C.times.07:01; HLA-C04:01; HLA-C04:01; and HLA-C02:02.
Embodiment 309 is an engineered cell, population of cells, pharmaceutical composition, or method of any of the preceding embodiments, wherein HLA-B and HLA-C alleles are selected from any of the following HLA-B and HLA-C alleles: HLA-B.times.07:02 and HLA-C.times.07:02; HLA-B.times.08:01 and HLA-C.times.07:01; HLA-B44:02 and HLA-C05:01; HLA-B35:01 and HLA-C04:01; HLA-B.times.40:01 and HLA-C.times.03:04; HLA-B57:01 and HLA-C06:02; HLA-B14:02 and HLA-C08:02; HLA-B15:01 and HLA-C03:03; HLA-B13:02 and HLA-C06:02; HLA-B44:03 and HLA-C16:01; HLA-B.times.38:01 and HLA-C.times.12:03; HLA-B18:01 and HLA-C07:01; HLA-B44:03 and HLA-C04:01; HLA-B.times.51:01 and HLA-C.times.15:02; HLA-B.times.49:01 and HLA-C.times.07:01; HLA-B15:01 and HLA-C03:04; HLA-B18:01 and HLA-C12:03; HLA-B.times.27:05 and HLA-C.times.02:02; HLA-B35:03 and HLA-C04:01; HLA-B18:01 and HLA-C05:01; HLA-B52:01 and HLA-C12:02; HLA-B.times.51:01 and HLA-C.times.14:02; HLA-B37:01 and HLA-C06:02; HLA-B53:01 and HLA-C04:01; HLA-B55:01 and HLA-C03:03; HLA-B44:02 and HLA-C07:04; HLA-B44:03 and HLA-C07:01; HLA-B35:02 and HLA-C04:01; HLA-B15:01 and HLA-C04:01; and HLA-B.times.40:02 and HLA-C.times.02:02.
Embodiment 310 is the engineered cell, cell population, pharmaceutical composition, or method of any one of the preceding embodiments, wherein HLA-B and HLA-C alleles are HLA-B x 07:02 and HLA-C07: 02.
embodiment 311 is the engineered cell, cell population, pharmaceutical composition, or method of any one of the preceding embodiments, wherein HLA-B and HLA-C alleles are HLA-B x 08:01 and HLA-C07: 01.
embodiment 312 is the engineered cell, cell population, pharmaceutical composition, or method of any one of the preceding embodiments, wherein HLA-B and HLA-C alleles are HLA-B44: 02 and HLA-C05: 01.
embodiment 313 is the engineered cell, cell population, pharmaceutical composition, or method of any one of the preceding embodiments, wherein HLA-B and HLA-C alleles are HLA-B x 35:01 and HLA-C04: 01.
Claims (73)
1. an engineered cell having reduced or eliminated MHC class II surface expression relative to an unmodified cell comprising a genetic modification in a CIITA gene, wherein the genetic modification comprises at least one nucleotide of an exon within genomic coordinates chr16:10902662-chr16: 10923285.
2. The engineered cell of claim 1, wherein the genetic modification comprises at least 5, 6, 7, 8, 9, or 10 consecutive nucleotides within genomic coordinates chr16:10902662-chr16: 10923285.
3. The engineered cell of claim 1 or 2, wherein the genetic modification comprises substitution of at least one C for T or substitution of at least one a for G within genomic coordinates chr16:10902662-chr16: 10923285.
4. The engineered cell of any one of claims 1 to 3, wherein the genetic modification comprises at least one nucleotide of an exon within genomic coordinates chr16:10906542-chr16: 10923285.
5. The engineered cell of any one of claims 1 to 4, wherein the genetic modification comprises at least one nucleotide of an exon within genomic coordinates chr16:10906542-chr16: 10908121.
6. The engineered cell of any one of claims 1 to 5, wherein the genetic modification comprises at least one nucleotide of an exon within genomic coordinates selected from the group consisting of: chr16:10907539-10907559, chr16:10916426-10916446, chr16:10906907-10906927, chr16:10895702-10895722, chr16:10907757-10907777, chr16:10907623-10907643, chr16:10915626-10915646, chr16:10906756-10906776, chr16:10907476-10907496, chr16:10907385-10907405 and chr16:10923265-10923285.
7. The engineered cell of any one of claims 1 to 6, wherein the genetic modification comprises at least one nucleotide of an exon within genomic coordinates selected from the group consisting of: chr16:10916432-10916452, chr16:10922444-10922464, chr16:10907924-10907944, chr16:10906985-10907005, chr16:10908073-10908093, chr16:10907433-10907453, chr16:10907979-10907999, chr16:10907139-10907159, chr16:10922435-10922455, chr16:10907384-10907404, chr16:10907434-10907454, chr16:10907119-10907139, chr16:10907539-10907559, chr16:10907810-10907830, chr16:10907315-10907335, chr16:10916426-10916446, chr16:10909138-10909158, chr16:10908101-10908121, chr16:10907790-10907810, chr16:10907787-10907807, ch16: 10907787-10907807, chr16:10907787-10907807, and chr16: 10907787-10907807.
8. An engineered cell having reduced or eliminated MHC class II surface expression relative to an unmodified cell, comprising a genetic modification in a CIITA gene, wherein the genetic modification comprises an insertion/deletion within genomic coordinates selected from the group consisting of: chr16: chr16: the chr16, chr16, 16, and 16 chr16:10907870-10907890, chr16:10907886-10907906, chr16:10907924-10907944, chr16:10907928-10907948, chr16:10907932-10907952, chr16:10907935-10907955, chr16:10907978-10907998, chr16:10907979-10907999, chr16:10908069-10908089, chr16:10908073-10908093, chr16:10908101-10908121, chr16:10909056-10909076, chr16:10909138-10909158, chr16:10910195-10910215, chr16:10910196-10910216, chr16:10915592-10915612, chr16:10915626-10915646, chr16:10916375-10916395, chr16:10916382-10916402, chr16:10916426-10916446, chr16:10916432-10916452, chr16:10918486-10918506, chr16:10918492-10918512, chr16:10918493-10918513, chr16:10922435-10922455, chr16:10922441-10922461, chr16:10922441-10922461, chr16:10922444-10922464, chr16:10922460-10922480, chr16:10923257-10923277 and chr16:10923265-10923285.
9. The engineered cell of claim 8, wherein the genetic modification comprises at least one nucleotide of an exon within genomic coordinates selected from the group consisting of: chr16:10916432-10916452 chr16:10922444-10922464, chr16:10907924-10907944, chr16:10906985-10907005, chr16:10908073-10908093, chr16:10907433-10907453, chr16:10907979-10907999, chr16:10907139-10907159, chr16:10922435-10922455, chr16:10907384-10907404, chr16:10907434-10907454, chr16:10907119-10907139, chr16:10907539-10907559, chr16:10907810-10907830, chr37, chr16:10907810-10907830, and chr16: 10907810-10907830.
10. The engineered cell of claim 8 or 9, wherein the genetic modification comprises at least one nucleotide of an exon within genomic coordinates selected from the group consisting of: chr16:10907539-10907559, chr16:10916426-10916446, chr16:10906907-10906927, chr16:10895702-10895722, chr16:10907757-10907777, chr16:10907623-10907643, chr16:10915626-10915646, chr16:10906756-10906776, chr16:10907476-10907496, chr16:10907385-10907405 and chr16:10923265-10923285.
11. The engineered cell of any one of claims 8 to 10, wherein the genetic modification comprises at least 5, 6, 7, 8, 9, or 10 consecutive nucleotides within the genomic coordinates.
12. The engineered cell of any one of claims 8 to 11, wherein the genetic modification comprises a substitution of at least one C for T or a substitution of at least one a for G within the genomic coordinates.
13. The engineered cell of any one of claims 1 to 12, wherein the MHC class II expression is reduced or eliminated by a gene editing system that binds to a CIITA genomic target sequence comprising at least 5 consecutive nucleotides within genomic coordinates selected from the group consisting of: chr16: chr16: the chr16, chr16, 16, and 16 chr16:10907820-10907840, chr16:10907870-10907890, chr16:10907886-10907906, chr16:10907924-10907944, chr16:10907928-10907948, chr16:10907932-10907952, chr16:10907935-10907955, chr16:10907978-10907998, chr16:10907979-10907999, chr16:10908069-10908089, chr16:10908073-10908093, chr16:10908101-10908121, chr16:10909056-10909076, chr16:10909138-10909158, chr16:10910195-10910215, chr16:10910196-10910216, chr16:10915592-10915612, chr16:10915626-10915646, chr16:10916375-10916395, chr16:10916382-10916402, chr16:10916426-10916446, chr16:10916432-10916452, chr16:10918486-10918506, chr16:10918492-10918512, chr16:10918493-10918513, chr16:10922435-10922455, chr16:10922441-10922461, chr16:10922441-10922461, chr16:10922444-10922464, chr16:10922460-10922480, chr16:10923257-10923277 and chr16:10923265-10923285.
14. The engineered cell of any one of claims 1 to 13, wherein the MHC class II expression is reduced or eliminated by a gene editing system that binds to a CIITA genomic target sequence comprising at least 5 consecutive nucleotides within genomic coordinates selected from the group consisting of: chr16: chr16: the chr16, chr16, 16, and 16 chr16:10908101-10908121, chr16:10909056-10909076, chr16:10909138-10909158, chr16:10910195-10910215, chr16:10910196-10910216, chr16:10915592-10915612, chr16:10915626-10915646, chr16:10916375-10916395, chr16:10916382-10916402, chr16:10916426-10916446, chr16:10916432-10916452, chr16:10918486-10918506, chr16:10918492-10918512, chr16:10918493-10918513, chr16:10922435-10922455, chr16:10922441-10922461, chr16:10922441-10922461, chr16:10922444-10922464, chr16:10922460-10922480, chr16:10923257-10923277 and chr16:10923265-10923285.
15. The engineered cell of any one of claims 1 to 14, wherein the MHC class II expression is reduced or eliminated by a gene editing system that binds to a CIITA genomic target sequence comprising at least 5 consecutive nucleotides within genomic coordinates selected from the group consisting of: chr16: chr16: the method comprises the steps of (1), ch16, chr16, chr, chr16, chr, 16 chr16:10908073-10908093 and chr16:10908101-10908121.
16. The engineered cell of any one of claims 1 to 15, wherein the MHC class II expression is reduced or eliminated by a gene editing system that binds to a CIITA genomic target sequence comprising at least 5 consecutive nucleotides within genomic coordinates selected from the group consisting of: chr16:10916432-10916452, chr16:10922444-10922464, chr16:10907924-10907944, chr16:10906985-10907005, chr16:10908073-10908093, chr16:10907433-10907453, chr16:10907979-10907999, chr16:10907139-10907159, chr16:10922435-10922455, chr16:10907384-10907404, chr16:10907434-10907454, chr16:10907119-10907139, chr16:10907539-10907559, chr16:10907810-10907830, chr16:10907315-10907335, chr16:10916426-10916446, chr16:10909138-10909158, chr16:10908101-10908121, chr16:10907790-10907810, chr16:10907787-10907807, and chr16: 10907787-10907807.
17. The engineered cell of any one of claims 1 to 16, wherein the MHC class II expression is reduced or eliminated by a gene editing system that binds to a CIITA genomic target sequence comprising at least 5 consecutive nucleotides within genomic coordinates selected from the group consisting of: chr16:10907539-10907559, chr16:10916426-10916446, chr16:10906907-10906927, chr16:10895702-10895722, chr16:10907757-10907777, chr16:10907623-10907643, chr16:10915626-10915646, chr16:10906756-10906776, chr16:10907476-10907496, chr16:10907385-10907405 and chr16:10923265-10923285.
18. The engineered cell of any one of claims 13 to 17, wherein the CIITA genomic target sequence comprises at least 10 or at least 15 contiguous nucleotides within the genomic coordinates.
19. The engineered cell of any one of claims 13-18, wherein the gene editing system comprises an RNA-guided DNA binding agent, optionally wherein the RNA-guided DNA binding agent comprises a Cas9 protein, such as streptococcus pyogenes Cas9.
20. The engineered cell of any one of claims 1 to 19, wherein the engineered cell further has reduced or eliminated MHC class I surface expression.
21. The engineered cell of claim 20, wherein the engineered cell comprises a genetic modification in a beta-2-microglobulin (B2M) gene.
22. The engineered cell of claim 20, wherein the engineered cell comprises a genetic modification in an HLA-A gene.
23. The engineered cell of any one of claims 1 to 22, wherein the engineered cell comprises an exogenous nucleic acid encoding a targeting receptor expressed on the surface of the engineered cell.
24. The engineered cell of claim 23, wherein the targeting receptor is a CAR, T Cell Receptor (TCR), or WT1 TCR.
25. The engineered cell of any one of claims 1 to 24, wherein the engineered cell further comprises an exogenous nucleic acid encoding a polypeptide secreted by the engineered cell.
26. The engineered cell of any one of claims 1 to 25, wherein the engineered cell is a T cell and further has reduced or eliminated expression of an endogenous T Cell Receptor (TCR) protein relative to an unmodified cell.
27. The engineered cell of claim 26, wherein the cell has reduced or eliminated expression of a TRAC protein or a TRBC protein relative to an unmodified cell.
28. A pharmaceutical composition comprising the engineered cell of any one of claims 1 to 27.
29. A population of cells comprising the engineered cell of any one of claims 1 to 27.
30. A pharmaceutical composition comprising a population of cells, wherein the population of cells comprises the engineered cell of any one of claims 1 to 27.
31. The population of cells of claim 29 or the pharmaceutical composition of claim 30, wherein the population of cells is at least 65%, at least 70%, at least 80%, at least 90%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% mhc class II negative as measured by flow cytometry.
32. The cell population or pharmaceutical composition of any one of claims 29 to 31, wherein the cell population is at least 95%, at least 97%, at least 98% or at least 99% endogenous TCR protein negative as measured by flow cytometry.
33. A method of administering the engineered cell, cell population or pharmaceutical composition of any one of claims 1 to 32 to a subject in need thereof.
34. A method of administering the engineered cell, cell population, or pharmaceutical composition of any one of claims 1 to 33 as Adoptive Cell Transfer (ACT) therapy to a subject.
35. A method of making an engineered cell having reduced or eliminated surface expression of an MHC class II protein relative to an unmodified cell, the method comprising contacting the cell with a composition comprising:
CIITA guide RNA comprising
i) A guide sequence selected from SEQ ID NOS.1-117;
ii) at least 17, 18, 19 or 20 consecutive nucleotides of the sequence selected from SEQ ID NO. 1-117;
iii) A leader sequence having at least 95%, 90% or 85% identity to a sequence selected from the group consisting of SEQ ID NOS.1-117;
iv) a sequence of 10 consecutive nucleotides + -10 nucleotides comprising the genomic coordinates listed in table 2;
v) at least 17, 18, 19 or 20 consecutive nucleotides of the sequence from (iv); or (b)
vi) a leader sequence having at least 95%, 90% or 85% identity to a sequence selected from (v); and
b. optionally an RNA-guided DNA binding agent or a nucleic acid encoding an RNA-guided DNA binding agent.
36. A method of reducing or eliminating surface expression of MHC class II proteins in an engineered cell relative to an unmodified cell, comprising contacting the cell with a composition comprising:
CIITA guide RNA comprising
i) A guide sequence selected from SEQ ID NOS.1-117;
ii) at least 17, 18, 19 or 20 consecutive nucleotides of the sequence selected from SEQ ID NO. 1-117;
iii) A leader sequence having at least 95%, 90% or 85% identity to a sequence selected from the group consisting of SEQ ID NOS.1-117;
iv) a sequence of 10 consecutive nucleotides + -10 nucleotides comprising the genomic coordinates listed in table 2;
v) at least 17, 18, 19 or 20 consecutive nucleotides of the sequence from (iv); or (b)
vi) a leader sequence having at least 95%, 90% or 85% identity to a sequence selected from (v); and
b. optionally an RNA-guided DNA binding agent or a nucleic acid encoding an RNA-guided DNA binding agent.
37. The method of claim 35 or 36, wherein the CIITA guide RNA comprises
i) A guide sequence selected from the group consisting of SEQ ID NOs 32, 64, 67, 68, 74, 76, 84, 86, 90, 91 and 115;
ii) at least 17, 18, 19 or 20 consecutive nucleotides of a sequence selected from the group consisting of SEQ ID NOs 32, 64, 67, 68, 74, 76, 84, 86, 90, 91 and 115; or (b)
iii) A guide sequence having at least 95%, 90% or 85% identity to a sequence selected from the group consisting of SEQ ID NOs 32, 64, 67, 68, 74, 76, 84, 86, 90, 91 and 115.
38. The method of any one of claims 35 to 37, further comprising reducing or eliminating surface expression of MHC class I proteins in the cell relative to an unmodified cell.
39. The method of any one of claims 35 to 38, further comprising reducing or eliminating surface expression of B2M protein in the cell relative to an unmodified cell.
40. The method of any one of claims 35 to 39, further comprising reducing or eliminating surface expression of HLA-A protein in the cell relative to an unmodified cell.
41. The method of any one of claims 35 to 40, further comprising reducing or eliminating surface expression of TCR proteins in an unmodified cell relative to the cell.
42. The method of any one of claims 35 to 41, further comprising contacting the cell with an exogenous nucleic acid.
43. The method of any one of claims 35 to 42, further comprising contacting the cell with a DNA-dependent protein kinase inhibitor (DNAPKi).
44. The method of claim 43, wherein the DNAPKi is Compound 1.
45. The method of claim 42, further comprising contacting the cell with an exogenous nucleic acid encoding a target receptor or a polypeptide secreted by the cell.
46. The engineered cell, population of cells, pharmaceutical composition, or method of any one of claims 1 to 45, comprising or contacting the cell with an exogenous nucleic acid, wherein the exogenous nucleic acid encodes an NK cell inhibitor molecule.
47. The engineered cell, population of cells, pharmaceutical composition, or method of any one of claims 1 to 46, comprising or contacting the cell with an exogenous nucleic acid, wherein the exogenous nucleic acid encodes an NK cell inhibitor molecule, wherein the NK cell inhibitor molecule binds to an inhibitory receptor on an NK cell; the NK cell inhibitor molecule binds to NKG2A on NK cells; the NK cell inhibitor molecule is a non-classical MHC class I molecule; the NK cell inhibitor molecule is HLA-E; the NK cell inhibitor molecule is a fusion protein; or the NK cell inhibitor molecule is a fusion protein comprising HLA-E and B2M.
48. The engineered cell, cell population, pharmaceutical composition, or method of any one of claims 1-47, comprising or contacting the cell with an exogenous nucleic acid encoding a polypeptide secreted by the cell, wherein the secreted polypeptide is an antibody or antibody fragment.
49. The engineered cell, cell population, pharmaceutical composition, or method of any one of claims 1 to 48, comprising or contacting the cell with an exogenous nucleic acid encoding a polypeptide secreted by the cell, wherein the secreted polypeptide is a full-length IgG antibody, a single-chain antibody, or a neutralizing antibody.
50. The engineered cell, cell population, pharmaceutical composition, or method of any one of claims 1-49, comprising or contacting the cell with an exogenous nucleic acid encoding a polypeptide secreted by the cell, wherein the secreted polypeptide is an enzyme, cytokine, or fusion protein.
51. The engineered cell, cell population, pharmaceutical composition, or method of any one of claims 1 to 50, comprising or contacting the cell with an exogenous nucleic acid encoding a polypeptide secreted by the cell, wherein the secreted polypeptide comprises a soluble receptor.
52. The engineered cell, population of cells, pharmaceutical composition, or method of any one of claims 1 to 51, comprising or contacting the cell with an exogenous nucleic acid encoding a targeting receptor, wherein the targeting receptor is a T Cell Receptor (TCR), a genetically modified TCR, WT1 TCR, or CAR.
53. The engineered cell, cell population, pharmaceutical composition or method of any one of claims 23 to 52, wherein the CIITA guide RNA, the RNA-guided DNA binding agent and/or the exogenous nucleic acid is provided to the cell in a vector, optionally wherein the CIITA guide RNA and the RNA-guided DNA binding agent are provided in the same vector.
54. The engineered cell, cell population, pharmaceutical composition, or method of any one of claims 1 to 53, wherein the exogenous nucleic acid is provided to the cell in a vector, optionally wherein the vector is a viral vector or a non-viral vector.
55. The engineered cell, cell population, pharmaceutical composition, or method of claim 54, wherein the vector is a lentiviral vector or an AAV.
56. The engineered cell, cell population, pharmaceutical composition, or method of any one of claims 1-55, wherein a gene editing system component is provided to the cell in a lipid nucleic acid assembly composition.
57. The engineered cell, cell population, pharmaceutical composition, or method of any one of claims 1 to 56, wherein the guide RNA or the exogenous nucleic acid is provided to the cell in a lipid nucleic acid assembly composition, optionally in the same lipid nucleic acid assembly composition as an RNA-guided DNA binding agent.
58. The engineered cell, cell population, pharmaceutical composition, or method of claim 56 or 57, wherein the lipid nucleic acid assembly composition is a Lipid Nanoparticle (LNP).
59. The engineered cell, cell population, pharmaceutical composition, or method of any one of claims 35 to 58, wherein
(i) Wherein the CIITA guide RNA is a single guide RNA comprising any of the sequences SEQ ID NOs 335-426 and 1008 or a sequence having at least 99%, 98%, 97%, 96%, 95%, 94%, 93%, 92%, 91% or 90% identity to any of the sequences SEQ ID NOs 335-426 and 1008;
(ii) The CIITA guide RNA comprises any of the sequences SEQ ID NOs 32, 64, 67, 68, 74, 76, 84, 86, 90, 91 and 115;
(iii) Wherein the CIITA guide RNA is a single guide RNA comprising any of the sequences SEQ ID NOs 341, 373, 376, 377, 383, 385, 393, 395, 399, 400 and 424 or a sequence having at least 99%, 98%, 97%, 96%, 95%, 94%, 93%, 92%, 91% or 90% identity to any of the sequences SEQ ID NOs 341, 373, 376, 377, 383, 385, 393, 395, 399, 400 and 424.
60. The engineered cell, cell population, pharmaceutical composition, or method of any one of claims 35 to 59, wherein the CIITA guide RNA comprises at least one modification, wherein the at least one modification comprises (i) a 2' -O-methyl (2 ' -O-Me) modified nucleotide, (ii) a Phosphorothioate (PS) linkage between nucleotides, (iii) a 2' -fluoro (2 ' -F) modified nucleotide, (iv) a modification at one or more of the five foremost nucleotides of the 5' end of the guide RNA, (v) a modification at one or more of the five rearmost nucleotides of the 3' end of the guide RNA, (vi) a PS linkage between the four foremost nucleotides of the guide RNA, (vii) a PS linkage between the four rearmost nucleotides of the guide RNA, (viii) a 2' -O-Me modified nucleotide at the three foremost nucleotides of the 5' end of the guide RNA, (ix) a modification at one or more of the three foremost nucleotides of the 3' end of the guide RNA, (Me) a combination of (ix).
61. The engineered cell or cell population of any one of claims 35-60, comprising a genetic modification comprising an insertion/deletion within a genomic region targeted by the CIITA guide RNA.
62. The engineered cell or cell population of any one of claims 35-61, comprising a genetic modification comprising a substitution of C for T or a substitution of a for G within a genomic region targeted by the CIITA guide RNA.
63. An engineered cell, cell population, pharmaceutical composition or method of any one of claims 1 to 62 for expressing a TCR specific for a polypeptide expressed by a cancer cell.
64. The engineered cell, cell population, pharmaceutical composition, or method of any one of claims 1-63, for administration to a subject as Adoptive Cell Transfer (ACT) therapy.
65. The engineered cell, cell population, pharmaceutical composition or method of any one of claims 1 to 64, for use in treating a subject suffering from cancer, infectious disease or autoimmune disease.
66. The engineered cell, cell population, pharmaceutical composition, or method of any one of claims 1-65, wherein the genetic modification comprises an insertion/deletion.
67. The engineered cell, cell population, pharmaceutical composition, or method of any one of claims 1-66, wherein the genetic modification comprises substitution of C to T.
68. The engineered cell, cell population, pharmaceutical composition, or method of any one of claims 1-67, wherein the genetic modification comprises substitution of a to G.
69. The engineered cell, cell population, pharmaceutical composition, or method of any one of claims 1-68, wherein the cell is homozygous for HLA-B and homozygous for HLA-C.
70. The engineered cell, cell population, pharmaceutical composition, or method of any one of claims 1 to 69, wherein the cell further comprises a genetic modification in an HLA-A gene, wherein the cell is homozygous for HLa-B and homozygous for HLa-C, and wherein the genetic modification in the HLA-A gene comprises at least one nucleotide within genomic coordinates selected from the group consisting of:
chr6:29942854 to chr6:29942913 and
chr6:29943518 to chr6:29943619.
71. The engineered cell, cell population, pharmaceutical composition, or method of any one of claims 1 to 70, wherein the cell further comprises a genetic modification in an HLA-A gene, and wherein the genetic modification in the HLA-A gene comprises at least one nucleotide within genomic coordinates selected from the group consisting of: chr6: 29942864-chr6: 29942903 and chr6: 29943528-chr6: 29943609.
72. A method of making an engineered cell having reduced or eliminated surface expression of MHC class II proteins and HLA-A proteins relative to an unmodified cell, the method comprising:
a. contacting the cell with a CIITA guide RNA, wherein the guide RNA comprises a guide sequence selected from SEQ ID NOs 1-117;
b. contacting the cell with HLA-A guide RNA, wherein the HLA-A guide RNA comprises a guide sequence selected from any one of SEQ ID NOs 2001-2095; and
c. optionally contacting the cell with an RNA-guided DNA binding agent or a nucleic acid encoding an RNA-guided DNA binding agent;
thereby reducing or eliminating the surface expression of MHC class II proteins and HLA-A proteins in the cell relative to an unmodified cell.
73. The method of claim 72, wherein the CIITA guide RNA comprises a sequence selected from the group consisting of SEQ ID NOs 32, 64, 67, 68, 74, 76, 84, 86, 90, 91 and 115.
Applications Claiming Priority (6)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| US63/130,098 | 2020-12-23 | ||
| US63/251,002 | 2021-09-30 | ||
| US63/254,971 | 2021-10-12 | ||
| US202163288502P | 2021-12-10 | 2021-12-10 | |
| US63/288,502 | 2021-12-10 | ||
| PCT/US2021/064933 WO2022140587A1 (en) | 2020-12-23 | 2021-12-22 | Compositions and methods for genetically modifying ciita in a cell |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN116783285A true CN116783285A (en) | 2023-09-19 |
Family
ID=88006719
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202180092210.6A Pending CN116783285A (en) | 2020-12-23 | 2021-12-22 | Compositions and methods for genetically modifying CIITA in cells |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN116783285A (en) |
-
2021
- 2021-12-22 CN CN202180092210.6A patent/CN116783285A/en active Pending
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| JP2022512703A (en) | Compositions and Methods for Immunotherapy | |
| KR20230017783A (en) | In vitro cell delivery methods | |
| US20240016934A1 (en) | Compositions and Methods for Reducing MHC Class II in a Cell | |
| US20240024478A1 (en) | Compositions and Methods for Reducing HLA-A in a Cell | |
| CA3227964A1 (en) | Method for producing genetically modified cells | |
| JP2024506016A (en) | T cell immunoglobulin and mucin domain 3 (TIM3) compositions and methods for immunotherapy | |
| US20240139323A1 (en) | Compositions and Methods for Genetically Modifying CIITA in a Cell | |
| CN116783285A (en) | Compositions and methods for genetically modifying CIITA in cells | |
| TW202409271A (en) | Compositions and methods for reducing mhc class i in a cell | |
| CN116802274A (en) | Compositions and methods for reducing MHC class II in cells | |
| CN116745406A (en) | Compositions and methods for reducing HLA-A in cells | |
| US20230383252A1 (en) | Natural Killer Cell Receptor 2B4 Compositions and Methods for Immunotherapy | |
| WO2023245109A2 (en) | Compositions and methods for genomic editing | |
| TW202413631A (en) | Compositions and methods for genomic editing | |
| WO2023245113A1 (en) | Methods and compositions for genetically modifying a cell | |
| WO2024047561A1 (en) | Biomaterials and processes for immune synapse modulation of hypoimmunogenicity | |
| TW202334396A (en) | Cd38 compositions and methods for immunotherapy | |
| CN117940153A (en) | Programmed cell death protein 1 (PD 1) compositions and methods for cell-based therapies |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| REG | Reference to a national code |
Ref country code: HK Ref legal event code: DE Ref document number: 40094124 Country of ref document: HK |