Compositions and Methods for Epigenetic Control of B2M Expression

Abstract

Disclosed herein are compositions and methods comprising an epigenetic editor for epigenetic modification of B2M, as well as nucleic acids and vectors encoding the same. Also disclosed are cells modified by epigenetic modification with the epigenetic editor.

Description

Technical Field

[0001] Cross - Reference to Related Applications This application claims the benefit of U.S. Provisional Application No. 63 / 355,061, filed Jun. 23, 2023, entitled "COMPOSITIONS AND METHODS FOR EPIGENETIC REGULATION OF B2M EXPRESSION", under 35 U.S.C. § 119(e). The entire disclosure of each of the above - mentioned applications is hereby incorporated by reference in its entirety into this specification.

[0002] Reference to Sequence Listing Submitted via Electronic Medium The content of the sequence listing submitted via electronic medium (C169870008WO00 - SEQ - AXW.xml; size: 1,879,683 bytes; and creation date: Jun. 23, 2023) is hereby incorporated by reference in its entirety into this specification.

Background Art

[0003] Background Adoptive cell therapy using genetically modified immune cells has emerged as a promising approach for treating cancer, infectious diseases, autoimmune diseases, and other diseases. However, conventional gene manipulation methods are usually based on permanent manipulation at the genomic level of cells, which involves certain risks such as chromosomal translocations, unwanted nucleotide insertions and deletions at the target site, and off - target mutations. There is still a need for an efficient and safe method for genetically engineering immune cells.

Summary of the Invention

[0004] Summary The present disclosure provides systems and compositions for epigenetic modification (referred to herein as "epigenetic editors" or "epigenetic editing systems"), and methods of using said systems and compositions to effect epigenetic modification in B2M, such as host cells and organisms.

[0005] In some aspects, the present disclosure provides a system for suppressing the transcription of the human B2M gene in human cells, optionally human T lymphocytes or human NK cells, wherein this system a) one or more fusion proteins comprising together a DNA methyltransferase (DNMT) domain and / or a domain that recruits DNMT, and a transcriptional repressor domain, wherein the DNMT domain and / or the recruitment domain may comprise a DNMT3A domain and / or a DNMT3L domain, and the recruited DNMT may be DNMT3A, each domain being linked to a DNA binding domain that binds to a target region within the human B2M gene, the target region comprising one or more sequences selected from SEQ ID NOs: 700 - 740, 744, 747 - 749, 752, 753, 757, 758, 760 - 806, 812 - 822, 825, 827, 830, 833, 834, 839 - 841, 843 - 845, 849, 851 - 853, 855, 864, 866 - 877, 879 - 883, 891 - 896, 898 - 900, 903 - 914, 922, 923, 925 - 927, 934, 936, 943 - 947, 949, 951 - 962, 975 - 981, 983, 985, 987 - 989, 995, 997 - 999, 1003 - 1005, and 1007 - 1011 said fusion protein; or b) one or more nucleic acid molecules encoding said one or more fusion proteins and the system may not cause DNA cleavage in the B2M gene. In some embodiments, the DNA binding domain comprises a dead CRISPR Cas (dCas) domain, a ZFP domain, or a TALE domain. For example, the DNA binding domain may comprise a dCas9 domain, and the system may further comprise (i) one or more guide RNAs (e.g., any one of SEQ ID NOs: 1015, 1018-1020, 1023, 1024, 1028, 1029, 1031-1077, 1083-1093, 1096, 1098, 1101, 1104, 1105, 1110-1112, 1114-1116, 1120, 1122-1124, 1126, 1135, 1137-1148, 1150-1154, 1162-1167, 1169-1171, 1174-1185, 1193, 1194, 1196-1198, 1205, 1207, 1214-1218, 1220, 1222-1233, 1246-1252, 1254, 1256, 1258-1260, 1266, 1268-1270, 1274-1276, and 1278-1282), or (ii) a nucleic acid molecule encoding the one or more guide RNAs.

[0006] In some embodiments, the DNA binding domain comprises a dCas9 domain, and the system further comprises (i) two guide RNAs comprising any two of SEQ ID NOs: 1015, 1018-1020, 1023, 1024, 1028, 1029, 1031-1077, 1083-1093, 1096, 1098, 1101, 1104, 1105, 1110-1112, 1114-1116, 1120, 1122-1124, 1126, 1135, 1137-1148, 1150-1154, 1162-1167, 1169-1171, 1174-1185, 1193, 1194, 1196-1198, 1205, 1207, 1214-1218, 1220, 1222-1233, 1246-1252, 1254, 1256, 1258-1260, 1266, 1268-1270, 1274-1276, and 1278-1282, or (ii) a nucleic acid molecule encoding the two guide RNAs.

[0007] In some embodiments, the DNA binding domain comprises a dCas9 domain, and the system further comprises (i) three guide RNAs, any three of SEQ ID NOs: 1015, 1018-1020, 1023, 1024, 1028, 1029, 1031-1077, 1083-1093, 1096, 1098, 1101, 1104, 1105, 1110-1112, 1114-1116, 1120, 1122-1124, 1126, 1135, 1137-1148, 1150-1154, 1162-1167, 1169-1171, 1174-1185, 1193, 1194, 1196-1198, 1205, 1207, 1214-1218, 1220, 1222-1233, 1246-1252, 1254, 1256, 1258-1260, 1266, 1268-1270, 1274-1276, and 1278-1282, or (ii) a nucleic acid molecule encoding the three guide RNAs.

[0008] In some aspects, the present disclosure provides a system for suppressing the transcription of the human B2M gene in human cells, optionally human T lymphocytes or human NK cells, wherein the system a) comprises a DNMT3A domain, a DNMT3L domain, a DNA binding domain, and a transcriptional repressor domain in a fusion protein, or b) a nucleic acid molecule encoding the fusion protein and the system may not cause DNA cleavage in the B2M gene. In some embodiments, the DNA binding domain comprises a dead CRISPR Cas (dCas) domain, a ZFP domain, or a TALE domain. For example, the DNA binding domain may comprise a dCas9 domain, and the system may further comprise (i) one or more guide RNAs (e.g., any one of SEQ ID NOs: 1012-1282), or (ii) a nucleic acid molecule encoding the one or more guide RNAs. ​

[0009] In certain embodiments, the dCas domain comprises a dCas9 sequence, such as a sequence having at least 90% identity to SEQ ID NO: 12 or 13.

[0010] In some embodiments, the DNA binding domain binds to the target sequence of SEQ ID NO: 1283 or 1284.

[0011] In some embodiments, the DNA binding domain comprises a ZFP domain that targets a nucleotide sequence selected from SEQ ID NOs: 700-~740

[0012] In some embodiments, the DNMT3A domain comprises a sequence having at least 90% identity to SEQ ID NO: 574 or 575.

[0013] The DNMT3L domain can comprise, for example, a sequence having at least 90% identity to a sequence selected from SEQ ID NOs: 578-581. In some embodiments, the DNMT3L domain comprises a sequence having at least 90% identity to a sequence selected from SEQ ID NOs: 582-603. In some embodiments, the DNMT3L domain comprises a sequence having at least 90% identity to a sequence selected from SEQ ID NOs: 601-603.

[0014] In some embodiments, the transcriptional repressor domain comprises a sequence having at least 90% identity to a sequence selected from SEQ ID NOs: 33 to 570. In certain embodiments, the transcriptional repressor domain is a KRAB domain derived from KOX1, ZIM3, ZFP28, or ZN627. The KRAB domain can comprise, for example, a sequence having at least 90% identity to a sequence selected from SEQ ID NOs: 89, 116, 245, and 255. In some embodiments, the transcriptional repressor domain comprises a fusion of the N-terminal and C-terminal regions of ZIM3 and KOX1 KRAB, optionally comprising the amino acid sequence of SEQ ID NO: 571 or 572. In certain embodiments, the transcriptional repressor domain is derived from KAP1, MECP2, HP1a / CBX5, HP1b, CBX8, CDYL2, TOX, TOX3, TOX4, EED, EZH2, RBBP4, RCOR1, or SCML2.

[0015] In some embodiments, the system a) comprises a DNMT3A domain, a DNMT3L domain, a transcriptional repressor domain, and a DNA binding domain, wherein optionally one or both of the DNMT3A domain and the DNMT3L domain are human, wherein optionally the DNA binding domain comprises a dead CRISPR Cas domain or a ZFP domain, a fusion protein, or b) a nucleic acid molecule encoding the fusion protein and comprises.

[0016] In certain embodiments, the fusion protein, from N-terminus to C-terminus, comprises a DNMT3A domain, a first peptide linker, a DNMT3L domain, a second peptide linker, a DNA binding domain, a third peptide linker, and a transcriptional repressor domain. For example, the fusion protein, from N-terminus to C-terminus, may comprise a DNMT3A domain, a first peptide linker, a DNMT3L domain, a second peptide linker, a first nuclear localization signal (NLS), a DNA binding domain, a second NLS, a third peptide linker, and a transcriptional repressor domain. The fusion protein, from N-terminus to C-terminus, may comprise a first NLS, a DNMT3A domain, a first peptide linker, a DNMT3L domain, a second peptide linker, a DNA binding domain, a third peptide linker, a transcriptional repressor domain, and a second NLS. The fusion protein, from N-terminus to C-terminus, may comprise first and second NLSs, a DNMT3A domain, a first peptide linker, a DNMT3L domain, a second peptide linker, a DNA binding domain, a third peptide linker, a transcriptional repressor domain, and third and fourth NLSs. In certain embodiments, the transcriptional repressor domain is a KRAB domain, such as the human KOX1, ZFP28, ZN627, or ZIM3 KRAB domain. In certain embodiments, one or both of the second and third peptide linkers are XTEN linkers, which may be selected from XTEN80 (e.g., SEQ ID NO: 643) and XTEN16 (e.g., SEQ ID NO: 638), for example, the second peptide linker is XTEN80 and the third peptide linker is XTEN16.

[0017] In some embodiments, the fusion protein, from N-terminus to C-terminus, may comprise a human DNMT3A domain, a first peptide linker, a human DNMT3L domain, an XTEN80 peptide linker, a first NLS, a dSpCas9 domain, a second NLS, an XTEN16 peptide linker, and a human KOX1 KRAB domain. In certain embodiments, the fusion protein comprises a sequence that is at least 90% identical to SEQ ID NO: 658.

[0018] In some embodiments, the fusion protein comprises, from the N-terminus to the C-terminus, a human DNMT3A domain, a first peptide linker, a human DNMT3L domain, an XTEN80 peptide linker, a first NLS, a ZFP domain, a second NLS, an XTEN16 linker, and a human KOX1 KRAB domain. In certain embodiments, the fusion protein comprises a sequence that is at least 90% identical to SEQ ID NO: 659.

[0019] In some embodiments, the fusion protein comprises, from the N-terminus to the C-terminus, a first and a second NLS, a human DNMT3A domain, a first peptide linker, a human DNMT3L domain, an XTEN80 peptide linker, a dSpCas9 domain, an XTEN16 peptide linker, a human KOX1 KRAB domain, and a third and a fourth NLS. In certain embodiments, the fusion protein may comprise the amino acid sequence of SEQ ID NO: 660 or a sequence that is at least 90% identical thereto.

[0020] In some embodiments, the fusion protein comprises, from the N-terminus to the C-terminus, a first and a second NLS, a human DNMT3A domain, a first peptide linker, a human DNMT3L domain, an XTEN80 peptide linker, a ZFP domain, an XTEN16 peptide linker, a human KOX1 KRAB domain, and a third and a fourth NLS.

[0021] In some embodiments, the fusion protein comprises, from the N-terminus to the C-terminus, a first and a second NLS, a human DNMT3A domain, a first peptide linker, a human DNMT3L domain, an XTEN80 peptide linker, a dSpCas9 domain, an XTEN16 peptide linker, a human ZFP28 KRAB domain, and a third and a fourth NLS. In certain embodiments, the fusion protein may comprise the amino acid sequence of SEQ ID NO: 661 or a sequence that is at least 90% identical thereto.

[0022] In some embodiments, the fusion protein, from the N-terminus to the C-terminus, comprises a first and a second NLS, a human DNMT3A domain, a first peptide linker, a human DNMT3L domain, an XTEN80 peptide linker, a ZFP domain, an XTEN16 peptide linker, a human ZFP28 KRAB domain, and a third and a fourth NLS.

[0023] In some embodiments, the fusion protein, from the N-terminus to the C-terminus, comprises a first and a second NLS, a human DNMT3A domain, a first peptide linker, a human DNMT3L domain, an XTEN80 peptide linker, a dSpCas9 domain, an XTEN16 peptide linker, a human ZN627 KRAB domain, and a third and a fourth NLS. In certain embodiments, the fusion protein may comprise the amino acid sequence of SEQ ID NO: 662 or a sequence that is at least 90% identical thereto.

[0024] In some embodiments, the fusion protein, from the N-terminus to the C-terminus, comprises a first and a second NLS, a human DNMT3A domain, a first peptide linker, a human DNMT3L domain, an XTEN80 peptide linker, a ZFP domain, an XTEN16 peptide linker, a human ZN627 KRAB domain, and a third and a fourth NLS.

[0025] In some embodiments, the fusion protein, from the N-terminus to the C-terminus, comprises a first and a second NLS, a human DNMT3A domain, a first peptide linker, a human DNMT3L domain, an XTEN80 peptide linker, a dSpCas9 domain, an XTEN16 peptide linker, a human ZIM3 KRAB domain, and a third and a fourth NLS. In certain embodiments, the fusion protein may comprise the amino acid sequence of SEQ ID NO: 663 or a sequence that is at least 90% identical thereto, or the amino acid sequence of SEQ ID NO: 667 or a sequence that is at least 90% identical thereto.

[0026] In some embodiments, the fusion protein, from the N-terminus to the C-terminus, comprises a first and a second NLS, a human DNMT3A domain, a first peptide linker, a human DNMT3L domain, an XTEN80 peptide linker, a ZFP domain, an XTEN16 peptide linker, a human ZIM3 KRAB domain, and third and fourth NLSs.

[0027] In some embodiments, at least one of the NLSs in the fusion proteins described herein is an SV40 NLS (e.g., SEQ ID NO: 644).

[0028] In some embodiments, the system a) a first fusion protein comprising a first DNA binding domain and comprising or recruiting a DNMT3A domain, a second fusion protein comprising a second DNA binding domain and comprising or recruiting a DNMT3L domain, a third fusion protein comprising a third DNA binding domain and comprising or recruiting a transcriptional repressor domain, or b) one or more nucleic acid molecules encoding the fusion protein and comprises.

[0029] The present disclosure also provides human cells comprising the systems described herein, and progeny of such cells. In some embodiments, the cells are T lymphocytes or NK cells.

[0030] The present disclosure also provides human cells (which may be ex vivo) modified by the systems described herein, or progeny of such cells. In some embodiments, the cells are T lymphocytes or NK cells.

[0031] The present disclosure also provides a pharmaceutical composition comprising the system described herein and a pharmaceutically acceptable excipient. In some embodiments, the composition comprises lipid nanoparticles (LNPs) comprising the system and / or the DNA binding domain is a dCas domain, and the LNPs further comprise one or more gRNAs.

[0032] The present disclosure also provides a pharmaceutical composition comprising the human cells described herein and a pharmaceutically acceptable excipient.

[0033] The present disclosure also provides a method of treating a patient in need thereof, comprising administering to the patient (e.g., intravenously) the system, human cells, or pharmaceutical composition described herein. In some embodiments, the patient has cancer or an autoimmune disease.

[0034] The present disclosure also provides the system, human cells, or pharmaceutical composition described herein for use in treating a patient in need thereof, e.g., in the methods described herein.

[0035] The present disclosure also provides the use of the system or human cells described herein in the manufacture of a therapeutic for a patient in need thereof, e.g., in the methods described herein.

[0036] The present disclosure also provides articles and kits comprising the system or human cells described herein.

[0037] Other features, objects, and advantages of the present invention will be apparent from the following detailed description. However, it should be understood that the detailed description is provided for purposes of illustration only and is not intended to be limiting. Various changes and modifications within the scope of the present invention will be apparent to those skilled in the art from the detailed description.

Brief Description of the Drawings

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DETAILED DESCRIPTION OF THE INVENTION

[0039] Detailed Description The present disclosure provides an epigenetic editor for suppressing the expression of the human B2M gene. By altering the expression of B2M, the editors herein can be used to generate allogeneic cells with reduced alloreactivity (e.g., T cells, NK cells, etc.). Unless otherwise specified, "B2M" (italicized) herein refers to the human B2M gene. The sequence of the human B2M gene can be obtained at Ensembl Accession No. ENSG00000166710. The epigenetic editors of the present invention have several advantages compared to other genome engineering methods, including reversibility, reduced risk of chromosomal translocation, and long-lasting genetically inheritable silencing.

[0040] In some embodiments, the region of the human B2M gene that is the target of epigenetic control is approximately 2 kb in length and is approximately ±1 kb from the B2M TSS. In certain embodiments, the region has the nucleotide sequence of SEQ ID NO: 1284 (below). In some embodiments, the targeted B2M region is approximately 1 kb in length and is approximately ±500 bp from the B2M TSS. In certain embodiments, the region to be targeted has the nucleotide sequence of SEQ ID NO: 1283 (below). The B2M TSS is at #chr15:55039548 of genome GRCh38.

[0041] TIFF2025524456000002.tif85160

[0042] TIFF2025524456000003.tif22160TIFF2025524456000004.tif146163

[0043] In some embodiments, the target site can be 10 - 50 bp in length (e.g., 10 - 40, 10 - 30, 10 - 20, 15 - 30, 15 - 25, or 15 - 20 bp). In some embodiments, the target strand of the target region is the sense strand of the gene. In other embodiments, the target strand of the target region is the antisense strand of the gene.

[0044] In some embodiments, the epigenetic editors described herein include one or more fusion proteins, each of which includes a DNA binding domain linked to one or more effector domains for epigenetic modification. In certain embodiments, when the DNA binding domain is a DNA binding domain guided to a polynucleotide, the epigenetic editor may further include one or more guide polynucleotides. The DNA binding domains, effector domains, and guide polynucleotides of the epigenetic editors described herein may be selected in any functional combination from, for example, those described below.

[0045] The epigenetic editors described herein can be transiently expressed in a host cell or can be integrated into the genome of the host cell, and such cells and their progeny are also contemplated by the present disclosure. Either a transiently expressed epigenetic editor or an integrated epigenetic editor or its components can effect stable epigenetic modification. For example, after introducing an epigenetic editor described herein into a host cell, a target gene in the host cell can be stably or permanently repressed or silenced. In some embodiments, the expression of the target gene is reduced or silenced as compared to the expression level in the absence of the epigenetic editor for at least 1 week, at least 2 weeks, at least 3 weeks, at least 4 weeks, at least 5 weeks, at least 6 weeks, at least 7 weeks, at least 2 months, at least 3 months, at least 4 months, at least 5 months, at least 6 months, at least 1 year, at least 2 years, or for the entire lifespan of the cell or the subject harboring the cell. The epigenetic modification can be inherited by the progeny of the host cell into which the epigenetic editor was introduced.

[0046] I. DNA Binding Domain The epigenetic editors described herein may include one or more DNA binding domains that direct the effector domain of the epigenetic editor to a target sequence within or near the B2M locus. The DNA binding domains described herein can be, for example, DNA binding domains guided by a polynucleotide, zinc finger protein (ZFP) domains, transcription activator-like effector (TALE) domains, meganuclease DNA binding domains, and the like. Examples of DNA binding domains can be found in U.S. Patent No. 11,162,114, which is hereby incorporated by reference in its entirety.

[0047] In some embodiments, the DNA binding domains described herein are encoded by their native coding sequences. In other embodiments, the DNA binding domains are encoded by nucleotide sequences that are codon-optimized for optimal expression in human cells.

[0048] A. DNA Binding Domain Guided by a Polynucleotide In some embodiments, the DNA binding domain herein can be a protein domain that is directed to a target site at the B2M locus by a guide nucleic acid sequence (e.g., a guide RNA sequence). In certain embodiments, the protein domain can be derived from a CRISPR-associated nuclease, e.g., a class I or II CRISPR-associated nuclease. In some embodiments, the protein domain can be derived from a Cas nuclease, e.g., a type II, IIA, IIB, IIC, V, or VI Cas nuclease. In certain embodiments, the protein domain can be derived from a class II Cas nuclease selected from Cas1, Cas1B, Cas2, Cas3, Cas4, Cas5, Cas6, Cas7, Cas8, Cas9, Cas10, Cas14a, Cas14b, Cas14c, CasX, CasY, CasPhi, C2c4, C2c8, C2c9, C2c10, Csy1, Csy2, Csy3, Cse1, Cse2, Csc1, Csc2, Csa5, Csn2, Csm2, Csm3, Csm4, Csm5, Csm6, Cmr1, Cmr3, Cmr4, Cmr5, Cmr6, Csb1, Csb2, Csb3, Csx17, Csx14, Csx10, Csx16, CsaX, Csx3, Csx1, Csx1S, Csf1, Csf2, CsO, Csf4, and homologs and modified versions thereof. "Derived from" is used to mean that the protein domain contains the full-length polypeptide sequence of the parent protein or a variant thereof (e.g., having deletions, insertions, and / or substitutions of amino acid residues). The variant retains the desired function of the parent protein (e.g., the ability to form a complex with the guide nucleic acid sequence and the target DNA).

[0049] In some embodiments, the CRISPR-associated protein domain can be the Cas9 domain described herein. Cas9 can refer to, for example, a polypeptide having at least about 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity and / or sequence similarity to the wild-type Cas9 polypeptide described herein. In some embodiments, the wild-type polypeptide is Cas9 derived from Streptococcus pyogenes (NCBI reference number NC_002737.2 (SEQ ID NO: 1)) and / or UniProt reference number Q99ZW2 (SEQ ID NO: 2). In some embodiments, the wild-type polypeptide is Cas9 derived from Staphylococcus aureus (SEQ ID NO: 3). In some embodiments, the CRISPR-associated protein domain is a Cpf1 domain or protein, or a polypeptide having at least about 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity and / or sequence similarity to the wild-type Cpf1 polypeptide described herein (e.g., Cpf1 derived from Franscisella novicida (UniProt reference number U2UMQ6 or SEQ ID NO: 4)). In certain embodiments, the CRISPR-associated protein domain can be a modified form, fusion, or chimera of the wild-type protein, including changes in one or more amino acid residues, such as deletions, insertions, or substitutions, or any combination thereof.

[0050] The structures of Cas9 sequences and variant Cas9 orthologs have been described for various organisms. Exemplary organisms from which the Cas9 domains in this specification may be derived include Streptococcus pyogenes, Streptococcus thermophilus, Streptococcus sp., Staphylococcus aureus, Listeria innocua, Lactobacillus gasseri, Francisella novicida, Wolinella succinogenes, Sutterella wadsworthensis, Gamma proteobacterium, Neisseria meningitidis, Campylobacter jejuni, Pasteurella multocida, Fibrobacter succinogene, Rhodospirillum rubrum, Nocardiopsis dassonvillei, Streptomyces pristinaespiralis, Streptomyces viridochromogenes, Streptosporangium roseum, Alicyclobacillus acidocaldarius, Bacillus pseudomycoides, Bacillus selenitireducens, Exiguobacterium sibiricum, Lactobacillus delbrueckiidelbrueckii), Lactobacillus salivarius, Lactobacillus buchneri, Treponema denticola, Microscilla marina, Burkholderiales bacterium, Polaromonas naphthalenivorans, Polaromonas sp., Crocosphaera watsonii, Cyanothece sp., Microcystis aeruginosa, Synechococcus sp., Acetohalobium arabaticum, Ammonifex degensii, Caldicelulosiruptor becscii, Candidatus Desulforudis, Clostridium botulinum, Clostridium difficile, Finegoldia magna, Natranaerobius thermophilus, Pelotomaculum thermopropionium, Acidithiobacillus caldus, Acidithiobacillus ferrooxidans, Allochromatium vinosum, Marinobacter sp., Nitrosococcus halophilus, Nitrosococcuswatsoni), Pseudoalteromonas haloplanktis, Ktedonobacter racemifer, Methanohalobium evestigatum, Anabaena variabilis, Nodularia spumigena, Nostoc sp., Arthrospira maxima, Arthrospira platensis, Arthrospira sp., Lyngbya sp., Microcoleus chthonoplastes, Oscillatoria sp., Petrotoga mobilis, Thermosipho africanus, Streptococcus pasteurianus, Neisseria cinerea, Campylobacter lari, Parvibaculum lavamentivorans, Corynebacterium diphtheria, and Acaryochloris marina, but are not limited thereto. The Cas9 sequences also include those derived from the organisms and loci disclosed by Chylinski et al., RNA Biol. (2013) 10(5):726-37.

[0051] In some embodiments, the Cas9 domain is derived from Streptococcus pyogenes (SpCas9). In some embodiments, the Cas9 domain is derived from Staphylococcus aureus (SaCas9).

[0052] Other Cas domains are also contemplated for use in the epigenetic editors herein. These include, for example, those derived from CasX (Cas12E) (e.g., SEQ ID NO: 5), CasY (Cas12d) (e.g., SEQ ID NO: 6), Casφ (CasPhi) (e.g., SEQ ID NO: 7), Cas12f1 (Cas14a) (e.g., SEQ ID NO: 8), Cas12f2 (Cas14b) (e.g., SEQ ID NO: 9), Cas12f3 (Cas14c) (e.g., SEQ ID NO: 10), and C2c8 (e.g., SEQ ID NO: 11).

[0053] Regarding epigenetic editing, a protein domain derived from a nuclease (e.g., Cas9 or Cpf1 domain) may have reduced or no DNA cleavage activity while retaining the ability to form a complex with a guide nucleic acid sequence (e.g., guide RNA) and target DNA, and its nuclease activity may be reduced through mutation or may not have it. For example, the nuclease activity may be reduced by at least 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 99% compared to the wild-type domain. In some embodiments, the CRISPR-related protein domains described herein are catalytically inactive ("dead"). Examples of such domains include, for example, dCas9 ("dead" Cas9), dCpf1, ddCpf1, dCasPhi, ddCas12a, dLbCpf1, and dFnCpf1. The dCas9 protein domain may contain one, two, or more mutations that suppress its nuclease activity compared to, for example, wild-type Cas9. The DNA cleavage domain of Cas9 is known to contain two subdomains, the HNH nuclease subdomain and the RuvC1 subdomain. The HNH subdomain cleaves the strand complementary to the gRNA, while the RuvC1 subdomain cleaves the non-complementary strand. Mutations within these subdomains can silence the nuclease activity of Cas9. For example, the mutations D10A (in the case of RuvC1) and H840A (in the case of HNH) completely inactivate the nuclease activity of SpCas9. Similarly, SaCas9 can be inactivated by the mutations D10A and N580A. In some embodiments, dCas9 contains at least one mutation that reduces or suppresses nuclease activity in the HNH subdomain and / or the RuvC1 subdomain. In some embodiments, dCas9 contains only the RuvC1 subdomain or only the HNH subdomain.It should be understood that any mutation that inactivates the RuvC1 and / or HNH domain, such as insertions, deletions, or single or multiple amino acid substitutions in the RuvC1 domain and / or HNH domain, may be included in the dCas9 herein.

[0054] In some embodiments, the dCas9 protein herein is numbered in the sequence provided by UniProt accession number Q99ZW2 (SEQ ID NO: 2) and contains a mutation at the position corresponding to position D10 (e.g., D10A), position H840 (e.g., H840A), or both in the wild-type SpCas9 sequence. In certain embodiments, dCas9 comprises the amino acid sequence of dSpCas9 (D10A and H840A) (SEQ ID NO: 12).

[0055] In some embodiments, the dCas9 protein described herein contains a mutation at the position corresponding to position D10 (e.g., D10A), position N580 (e.g., N580A), or both in the wild-type SaCas9 sequence (e.g., SEQ ID NO: 3). In certain embodiments, dCas9 comprises the amino acid sequence of dSaCas9 (D10A and N580A) (SEQ ID NO: 13).

[0056] Additional suitable mutations that inactivate Cas9 will be apparent to those skilled in the art based on the present disclosure and knowledge in the art and are within the scope of the present disclosure. Such mutations can include, but are not limited to, D839A, N863A, and / or K603R in SpCas9. The present disclosure contemplates any mutation that reduces or suppresses the nuclease activity of any Cas9 described herein (e.g., a mutation corresponding to any of the Cas9 mutations described herein).

[0057] The dCpf1 protein domain can contain one, two, or more mutations that reduce or inhibit its nuclease activity compared to wild-type Cpf1. The Cpf1 protein has a RuvC-like endonuclease domain similar to the RuvC domain of Cas9, but does not have an HNH endonuclease domain, and the N-terminus of Cpf1 does not have the alpha-helical recognition lobe of Cas9. In some embodiments, dCpf1 contains one or more mutations corresponding to D917A, E1006A, or D1255A numbered in the sequence of the Francisella novicida Cpf1 protein (FnCpf1, SEQ ID NO: 4). In certain embodiments, the dCpf1 protein contains a mutation corresponding to the D917A position, the E1006A position, the D1255A position, the D917A / E1006A position, the D917A / D1255A position, the E1006A / D1255A position, or the D917A / E1006A / D1255A position, or a corresponding mutation in any of the Cpf1 amino acid sequences described herein. In some embodiments, dCpf1 contains the D917A mutation. In a particular embodiment, dCpf1 contains the amino acid sequence of dFnCpf1 (SEQ ID NO: 14).

[0058] Additional nuclease-inactive CRISPR-associated protein domains contemplated herein include, for example, those derived from dNmeCas9 (e.g., SEQ ID NO: 15), dCjCas9 (e.g., SEQ ID NO: 16), dSt1Cas9 (e.g., SEQ ID NO: 17), dSt3Cas9 (e.g., SEQ ID NO: 18), dLbCpf1 (e.g., SEQ ID NO: 19), dAsCpf1 (e.g., SEQ ID NO: 20), denAsCpf1 (e.g., SEQ ID NO: 21), dHFAsCpf1 (e.g., SEQ ID NO: 22), dRVRAsCpf1 (e.g., SEQ ID NO: 23), dRRAsCpf1 (e.g., SEQ ID NO: 24), dCasX (e.g., SEQ ID NO: 25), and dCasPhi (e.g., SEQ ID NO: 26).

[0059] In some embodiments, the Cas9 domains described herein can be high-fidelity Cas9 domains that include, for example, one or more mutations that reduce the electrostatic interaction between the Cas9 domain and the DNA sugar-phosphate backbone to confer increased target binding specificity. In certain embodiments, the high-fidelity Cas9 domain can be nuclease-inactive as described herein.

[0060] The CRISPR-related protein domains described herein can recognize protospacer adjacent motif (PAM) sequences in target genes. The "PAM" sequence is typically a 2-6 bp DNA sequence immediately following the sequence targeted by the CRISPR-related protein domain. The PAM sequence is required for CRISPR protein binding and cleavage, but is not part of the target sequence. The CRISPR-related protein domain can either recognize a naturally occurring or canonical PAM sequence or can have altered PAM specificity. CRISPR-related protein domains that bind to non-canonical PAM sequences are described in the art. For example, Cas9 domains that bind to non-canonical PAM sequences are described in Kleinstiver et al., Nature (2015) 523(7561):481-5 and Kleinstiver et al., Nat Biotechnol. (2015) 33:1293-8. Such Cas9 domains include, for example, those derived from "VRER" SpCas9, "EQR" SpCas9, "VQR" SpCas9, "SpG Cas9", "SpRYCas9", and "KKH" SaCas9. Nuclease-inactive versions of these Cas9 domains, for example, nuclease-inactive VRER SpCas9 (e.g., SEQ ID NO: 27), nuclease-inactive EQR SpCas9 (e.g., SEQ ID NO: 28), nuclease-inactive VQR SpCas9 (e.g., SEQ ID NO: 29), nuclease-inactive SpG Cas9 (e.g., SEQ ID NO: 30), nuclease-inactive SpRY Cas9 (e.g., SEQ ID NO: 31), and nuclease-inactive KKH SaCas9 (e.g., SEQ ID NO: 32) are also contemplated. Another example is Cas9 from Francisella novicida engineered to recognize 5'-YG-3' (where "Y" is a pyrimidine).

[0061] Additional suitable CRISPR-related proteins, orthologs, and variants including nuclease-inactive variants and sequences will be apparent to those skilled in the art based on the present disclosure.

[0062] The guide RNAs that can be used with the CRISPR-related protein domains herein are further described in Section II below.

[0063] B. Zinc Finger Protein Domains In some embodiments, the DNA binding domain of the epigenetic editors described herein comprises a zinc finger protein (ZFP) domain (or “ZF domain” as used herein). A ZFP is a protein that has at least one zinc finger and binds to DNA in a sequence-specific manner. A “zinc finger” (ZF) or “zinc finger motif” (ZF motif) refers to a polypeptide domain that contains a beta-beta-alpha (ββα) type protein fold stabilized by zinc ions. A ZF binds to 2 to 4 nucleotide base pairs, typically 3 or 4 base pairs (consecutive or non-consecutive). Each ZF typically contains approximately 30 amino acids. The ZFP domain may include multiple ZFs that make tandem contacts with their target nucleic acid sequences. The tandem array of ZFs may be engineered to generate an artificial ZFP that binds to a desired nucleic acid target. ZFPs can be rationally designed by using a database that includes triplet (or quadruplet) nucleotide sequences and individual ZF amino acid sequences, where each triplet or quadruplet nucleotide sequence is associated with the amino acid sequence of one or more ZFs that bind to a specific triplet or quadruplet sequence. See, for example, U.S. Pat. Nos. 6,453,242, 6,534,261, and 8,772,453.

[0064] ZFPs are widely present in eukaryotic cells and can belong to, for example, the C2H2 class, CCHC class, PHD class, or RING class. An exemplary motif that characterizes one class of these proteins (the C2H2 class) is -Cys-(X) 2-4 -Cys-(X) 12 -His-(X) 3-5-His-(SEQ ID NO: 657), where X is any independently selected amino acid. In some embodiments, the ZFP domains herein may include a ZF array comprising contiguous C2H2-ZFs each contacting three or more contiguous nucleotides.

[0065] The ZFP domains of the epigenetic editors described herein may include 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, or more ZFs. The ZFP domain may include an array of 2-finger or 3-finger units, e.g., 3, 4, 5, 6, 7, 8, 9, or 10, or more units, where each unit binds to a sub-site in the target sequence. In some embodiments, a ZFP domain comprising at least 3 ZFs recognizes a target DNA sequence of 9 or 10 nucleotides. In some embodiments, a ZFP domain comprising at least 4 ZFs recognizes a target DNA sequence of 12 - 14 nucleotides. In some embodiments, a ZFP domain comprising at least 6 ZFs recognizes a target DNA sequence of 18 - 21 nucleotides.

[0066] In some embodiments, the ZFs in the ZFP domains described herein are connected via a peptide linker. The peptide linker can be of a length of 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or more amino acids. In some embodiments, the linker comprises 5 or more amino acids. In some embodiments, the linker comprises 7 - 17 amino acids. The linker can be flexible or rigid.

[0067] In some embodiments, the zinc finger array has the sequence: TIFF2025524456000005.tif24163 or may have a sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical thereto, where "XXXXXXX" represents the amino acids of the ZF recognition helix that confers DNA binding specificity to the zinc finger, and each X can be independently selected. In the above sequence, "XX" shown in italics can be TR, LR, or LK, and "[linker]" represents a linker sequence. In some embodiments, the linker sequence is TGSQKP (SEQ ID NO: 651), and this linker can be used when the sub-site targeted by the ZF is adjacent. In some embodiments, the linker sequence is TGGGGSQKP (SEQ ID NO: 652), and this linker can be used when there are bases between the sub-sites targeted by the zinc finger. The two indicated linkers may be the same or different. In some embodiments, the length of the linker sequence is at least 5 amino acids at the shortest. In some embodiments, the length of the linker sequence is at most 250 amino acids at the longest.

[0068] The ZFP domains herein can include an array of two or more adjacent ZFs that are directly adjacent to each other (e.g., separated by a short (canonical) linker sequence) or separated by a long flexible or structured polypeptide sequence. In some embodiments, directly adjacent fingers bind to contiguous nucleic acid sequences, i.e., adjacent trinucleotides / triplets. In some embodiments, adjacent fingers cross-link between their respective target triplets, which can help strengthen or enhance the recognition of the target sequence and result in the binding of overlapping sequences. In some embodiments, distal ZFs within the ZFP domain can recognize (or bind to) non-contiguous nucleotide sequences.

[0069] Exemplary B2M target sequences are shown in Table 1 below.

[0070]

Table 1

[0071] In some embodiments, the ZFP domain of the present epigenetic editor binds to a target sequence selected from any one of SEQ ID NOs: 700 to 740. The ZF may include the ZF framework sequence of SEQ ID NO: 650, or any other ZF framework known in the art.

[0072] C. TALE In some embodiments, the DNA binding domain of the epigenetic editor described herein includes a transcription activator-like effector (TALE) domain. The DNA binding domain of TALE includes a highly conserved sequence of about 33-34 amino acids and has repeat variable di-residues (RVDs) that are the centers for the recognition of specific nucleotides at positions 12 and 13. TALE can be engineered to bind to virtually any desired DNA sequence. Methods for programming TALE are known in the art. For example, such methods are described in Carroll et al., Genet Soc Amer. (2011) 188(4):773-82, Miller et al., Nat Biotechnol. (2007) 25(7):778-85, Christian et al., Genetics (2008) 186(2):757-61, Li et al., Nucl Acids Res. (2010) 39(1):359-72, and Moscou et al., Science (2009) 326(5959):1501.

[0073] D. Other DNA Binding Domains Other DNA binding domains are contemplated for the epigenetic editors described herein. In some embodiments, the DNA binding domain includes an Argonaute protein domain, e.g., from Natronobacterium gregoryi (NgAgo). NgAgo is an ssDNA-guided endonuclease that is guided to its target site by 5'-phosphorylated ssDNA (gDNA), where it introduces a double-strand break. In contrast to Cas9, the NgAgo-gDNA system does not require a protospacer adjacent motif (PAM). Thus, the use of nuclease-inactive NgAgo (dNgAgo) can greatly expand the bases that can be targeted. The characterization and use of NgAgo are described, for example, in Gao et al., Nat Biotechnol. (2016) 34(7):768-73, Swarts et al., Nature (2014) 507(7491):258-61, and Swarts et al., Nucl Acids Res. (2015) 43(10):5120-9.

[0074] In some embodiments, the DNA binding domain includes an inactivated nuclease, e.g., an inactivated meganuclease. Additional non-limiting examples of DNA binding domains include the tetracycline-controlled repressor (tetR) DNA binding domain, leucine zipper, helix-loop-helix (HLH) domain, helix-turn-helix domain, β-sheet motif, steroid receptor motif, bZIP domain, homeodomain, and AT hook.

[0075] II. Guide Polynucleotide The epigenetic editors described herein that include a DNA-binding domain guided by a polynucleotide can also include a guide polynucleotide that can form a complex with the DNA-binding domain. The guide polynucleotide can include RNA, DNA, or a mixture of both. For example, when the DNA-binding domain guided by a polynucleotide is a CRISPR-associated protein domain, the guide polynucleotide can be a guide RNA (gRNA). "Guide RNA" or "gRNA" refers to a nucleic acid that can hybridize to a target sequence and direct the binding of a CRISPR-Cas complex to the target sequence. Methods of using guide polynucleotide sequences with programmable DNA-binding proteins (e.g., CRISPR-associated protein domains) for site-specific DNA targeting (e.g., for modifying the genome) are known in the art.

[0076] A guide polynucleotide sequence (e.g., a gRNA sequence) can include two parts: 1) a nucleotide sequence that includes a “targeting sequence” that is complementary to a target nucleic acid sequence (the “target sequence”), e.g., a nucleic acid sequence contained in a genomic target site, and 2) a nucleotide sequence that binds to a DNA binding domain (e.g., a CRISPR-Cas protein domain) that is guided by the polynucleotide. The nucleotide sequence of 1) can include a targeting sequence that is 100% complementary to a genomic nucleic acid sequence, e.g., a nucleic acid sequence contained in a genomic target site, and thus can hybridize to the target nucleic acid sequence. The nucleotide sequence of 1) can be referred to as, for example, a crispr RNA or crRNA. The nucleotide sequence of 2) can be referred to as a scaffold sequence of the guide nucleic acid, e.g., a tracrRNA, or an activation region of the guide nucleic acid, and can include a stem-loop structure. The above-described parts 1) and 2) can fuse to form one single guide (e.g., a single guide RNA or sgRNA), or can be on two separate nucleic acid molecules. In some embodiments, the guide polynucleotide includes parts 1) and 2) connected by a linker. In some embodiments, the guide polynucleotide includes parts 1) and 2) connected by a non-nucleic acid linker, e.g., a peptide linker or a chemical linker.

[0077] Part 2 (scaffold sequence) of the guide polynucleotide described herein can be, for example, that described in Jinek et al., Science (2012) 337:816-21, U.S. Patent Application Publication No. 2016 / 0208288, or U.S. Patent Application Publication No. 2016 / 0200779. Variants of part 2) are also contemplated by the present disclosure. For example, the tetraloop and stemloop of the gRNA scaffold (tracrRNA) sequence can be modified to include an RNA aptamer that can be bound by a specific protein domain. In some embodiments, such modified gRNAs can be used to facilitate the recruitment of a repression or activation domain fused to an RNA aptamer that interacts with a protein.

[0078] The gRNAs provided herein typically include a targeting domain and a binding domain. The targeting domain (also referred to as the “targeting sequence”) can include a nucleic acid sequence that binds to a target site, e.g., a genomic nucleic acid molecule within a cell. The target site can be a double-stranded DNA sequence that includes a PAM sequence and a target sequence that is located directly adjacent to and on the same strand as the PAM sequence. The targeting domain of the gRNA can include an RNA sequence that corresponds to the target sequence, i.e., it is similar to the sequence of the targeting domain and may have one or more mismatches, but typically includes an RNA sequence instead of a DNA sequence. The targeting domain of the gRNA can thus base pair (with complete or partial complementarity) with the sequence of the double-stranded target site that is complementary to the target sequence and thus with the strand that is complementary to the strand containing the PAM sequence. It will be understood that the targeting domain of the gRNA typically does not include a sequence similar to the PAM sequence. It will further be understood that the position of the PAM can be 5' or 3' of the target sequence, depending on the nuclease being utilized. For example, the PAM is typically 3' of the target sequence for Cas9 nuclease and 5' of the target sequence for Cas12a nuclease. For an illustration of the position of the PAM and the mechanism by which the gRNA binds to the target site, see, e.g., FIG. 1 of Vanegas et al., Fungal Biol Biotechnol. (2019) 6:6, which is incorporated herein by reference. For additional illustration and explanation of the mechanism of gRNA targeting to the target site of RNA-guided nucleases, see, e.g., Fu et al., Nat Biotechnol (2014) 32(3):279-84 and Sternberg et al., Nature (2014) 507(7490):62-7, which are each incorporated herein by reference.

[0079] In some embodiments, the targeting domain sequence comprises 17 to 30 nucleotides and is fully complementary to the target sequence (i.e., has no mismatched nucleotides). In some embodiments, however, the targeting domain sequence can include one or more, typically four or fewer, mismatches, e.g., one, two, three, or four mismatches. The targeting domain is part of the gRNA, which being an RNA molecule will typically contain ribonucleotides, while a DNA targeting domain will contain deoxyribonucleotides.

[0080] An exemplary illustration of a Cas9 target site comprising a 22-nucleotide target domain and an NGG PAM sequence, and a gRNA comprising a targeting domain that is fully complementary to the target sequence (and thus base pairs with perfect complementarity to the DNA strand complementary to the strand containing the target sequence and PAM) is provided below. TIFF2025524456000007.tif45160

[0081] An exemplary illustration of a Casl2a target site comprising a 22-nucleotide target domain and a TTN PAM sequence, and a gRNA comprising a targeting domain that is fully complementary to the target sequence (and thus base pairs with perfect complementarity to the DNA strand complementary to the strand containing the target sequence and PAM) is provided below. TIFF2025524456000008.tif44159

[0082] While not wishing to be bound by theory, in at least some embodiments, the length of the targeting domain and its complementarity to the target sequence are thought to contribute to the specificity of the interaction between the gRNA / Cas9 molecular complex and the target nucleic acid. In some embodiments, the targeting domain of the gRNA provided herein is 5 to 50 nucleotides in length. In some embodiments, the targeting domain is 15 to 25 nucleotides in length. In some embodiments, the targeting domain is 18 to 22 nucleotides in length. In some embodiments, the targeting domain is 19 to 21 nucleotides in length. In some embodiments, the targeting domain is 15 nucleotides in length. In some embodiments, the targeting domain is 16 nucleotides in length. In some embodiments, the targeting domain is 17 nucleotides in length. In some embodiments, the targeting domain is 18 nucleotides in length. In some embodiments, the targeting domain is 19 nucleotides in length. In some embodiments, the targeting domain is 20 nucleotides in length. In some embodiments, the targeting domain is 21 nucleotides in length. In some embodiments, the targeting domain is 22 nucleotides in length. In some embodiments, the targeting domain is 23 nucleotides in length. In some embodiments, the targeting domain is 24 nucleotides in length. In some embodiments, the targeting domain is 25 nucleotides in length. In certain embodiments, the targeting domain corresponds exactly, without mismatches, to the target sequence provided herein or a portion thereof. In some embodiments, the targeting domain of the gRNA provided herein includes one mismatch to the target sequence provided herein. In some embodiments, the targeting domain includes two mismatches to the target sequence. In some embodiments, the targeting domain includes three mismatches to the target sequence.

[0083] Methods for designing, selecting, and validating gRNAs are described herein and are known in the art. Software tools can be used to optimize gRNAs corresponding to target DNA sequences, for example, to minimize the overall off-target activity across the genome. For example, a DNA sequence search algorithm can be used to identify target sequences within the crRNA of a gRNA for use with Cas9. Exemplary gRNA design tools include those described in Bae et al., Bioinformatics (2014) 30:1473-5.

[0084] The guide polynucleotides (e.g., gRNAs) described herein can be of various lengths. In some embodiments, the length of the spacer or targeting sequence depends on the CRISPR-associated protein component of the epigenetic editor system being used. For example, Cas proteins from different bacterial species have various optimal targeting sequence lengths. Thus, the spacer sequence can include, for example, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, or more than 50 nucleotides in length. In some embodiments, the spacer includes 10 - 24, 11 - 20, 11 - 16, 18 - 24, 19 - 21, or 20 nucleotides in length. In some embodiments, the guide polynucleotide (e.g., gRNA) is 15 - 100 (e.g., 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50) nucleotides in length and includes a spacer sequence of at least 10 (e.g., 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50) consecutive nucleotides complementary to the target sequence. In some embodiments, the guide polynucleotides described herein can have, for example, 1, 2, 3, 4, 5, 10, 15, 20, 25, 30, 40, 50, or more nucleotides truncated.

[0085] In certain embodiments, the 3' end of the B2M target sequence is directly adjacent to a PAM sequence (e.g., a canonical PAM sequence, e.g., NGG for SpCas9). The degree of complementarity between the targeting sequence (e.g., the spacer sequence of a gRNA) of the guide polynucleotide and the target sequence can be at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%. In certain embodiments, the targeting sequence and the target sequence can be 100% complementary. In other embodiments, the targeting sequence and the target sequence can include, for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 mismatches.

[0086] The guide polynucleotide (e.g., a gRNA) may be modified, for example, by chemical modification and synthetic modification. The modified gRNA can include, for example, one or both of the unlinked phosphate oxygens in the phosphodiester backbone linkage and / or one or more modifications or replacements of the linked phosphate oxygens, modification of the ribose sugar (e.g., the 2'-hydroxyl of the ribose sugar), modification of the phosphate moiety, modification or replacement of a naturally occurring nucleobase, modification or replacement of the ribose-phosphate backbone, modification of the 3' end and / or 5' end of the oligonucleotide, replacement of the terminal phosphate group, or conjugation of a moiety, cap, or linker, or any combination thereof.

[0087] In some embodiments, one or more ribose groups of the gRNA may be modified. Examples of chemical modifications to ribose groups include, but are not limited to, 2'-O-methyl (2'-OMe), 2'-fluoro (2'-F), 2'-deoxy, 2'-O-(2-methoxyethyl) (2'-MOE), 2'-NH2, 2'-O-allyl, 2'-O-ethylamine, 2'-O-cyanoethyl, 2'-O-acetal ester, or bicyclic nucleotides such as locked nucleic acid (LNA), 2'-(5-constrained ethyl (S-cEt)), constrained MOE, or 2'-0,4'-C-aminomethylene bridged nucleic acid (2',4'-BNANC). 2'-O-methyl modification and / or 2'-fluoro modification may increase the binding affinity and / or nuclease stability of the gRNA oligonucleotide.

[0088] In some embodiments, one or more phosphate groups of the gRNA may be chemically modified. Examples of chemical modifications to phosphate groups include, but are not limited to, phosphorothioate (PS), phosphonoacetate (PACE), thiophosphonoacetate (thioPACE), amide, triazole, phosphonate, and phosphotriester modifications. In some embodiments, the guide polynucleotide described herein may include one, two, three, or more PS linkages at or near the 5' end and / or 3' end, and the PS linkages may be continuous or discontinuous.

[0089] In some embodiments, the gRNA herein includes a mixture of ribonucleotides and deoxyribonucleotides and / or one or more PS linkages.

[0090] In some embodiments, one or more nucleobases of the gRNA may be chemically modified. Examples of chemically modified nucleobases include, but are not limited to, 2-thiouridine, 4-thiouridine, N6-methyladenosine, pseudouridine, 2,6-diaminopurine, inosine, thymidine, 5-methylcytosine, 5-substituted pyrimidines, isoguanine, isocytosine, and nucleobases having halogenated aromatic groups. The chemical modification can be performed on the spacer region, the tracr RNA region, the stem-loop, or any combination thereof.

[0091] Table 2 below lists exemplary gRNA target sequences for epigenetic modification of human B2M, as well as the coordinates of the start and end positions of the targeted sites on human chromosome 15 (SEQ indicates the sequence number). This table also shows the distance from the start coordinate of the B2M gene to the TSS coordinate. Exemplary target sequences of the gRNA are listed in Table 3.

[0092] [Table 2] TIFF2025524456000010.tif253160TIFF2025524456000011.tif254159TIFF2025524456000012.tif254159TIFF2025524456000013.tif254159TIFF2025524456000014.tif205159

[0093] [Table 3] TIFF2025524456000016.tif252160TIFF2025524456000017.tif252160TIFF2025524456000018.tif252160TIFF2025524456000019.tif250160TIFF2025524456000020.tif251160TIFF2025524456000021.tif170160

[0094] In some embodiments, the target region of the guide RNA targeting B2M comprises one or more sequences selected from SEQ ID NOs: 700-740, 744, 747-749, 752, 753, 757, 758, 760-806, 812-822, 825, 827, 830, 833, 834, 839-841, 843-845, 849, 851-853, 855, 864, 866-877, 879-883, 891-896, 898-900, 903-914, 922, 923, 925-927, 934, 936, 943-947, 949, 951-962, 975-981, 983, 985, 987-989, 995, 997-999, 1003-1005, and 1007-1011. In some embodiments, the guide RNA targeting B2M comprises any one of SEQ ID NOs: 1015, 1018-1020, 1023, 1024, 1028, 1029, 1031-1077, 1083-1093, 1096, 1098, 1101, 1104, 1105, 1110-1112, 1114-1116, 1120, 1122-1124, 1126, 1135, 1137-1148, 1150-1154, 1162-1167, 1169-1171, 1174-1185, 1193, 1194, 1196-1198, 1205, 1207, 1214-1218, 1220, 1222-1233, 1246-1252, 1254, 1256, 1258-1260, 1266, 1268-1270, 1274-1276, and 1278-1282.

[0095] Any tracr sequence known in the art is contemplated for the gRNA described herein. In some embodiments, the gRNA described herein has a tracr sequence shown in Table 4 below, or a tracr sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to the tracr sequence shown below (SEQ indicates SEQ ID NO).

[0096]

Table 4

[0097] In some embodiments, the gRNA herein is provided directly to the cell (e.g., through an RNP complex together with a CRISPR-related protein domain). In some embodiments, the gRNA is provided to the cell through an expression vector (e.g., a plasmid vector or a viral vector) introduced into the cell, and the cell then expresses the gRNA from the expression vector. Methods for introducing the gRNA and the expression vector into the cell are well known in the art.

[0098] III. Effector Domain The epigenetic editors described herein include one or more effector protein domains (also, as used herein, "epigenetic effector domain" or "effector domain") that effect epigenetic modification of a target gene. An epigenetic editor having one or more effector domains can modulate the expression of a target gene without altering the nucleic acid sequence of the target gene. In some embodiments, the effector domains described herein can provide for repression or silencing of the expression of a target gene, such as B2M, for example, by suppressing transcription or by modifying or remodeling chromatin. Such effector domains are also referred to herein as "repression domains", "repressor domains", or "epigenetic repressor domains". Non-limiting examples of chemical modifications that can be mediated by an effector domain include methylation, demethylation, acetylation, deacetylation, phosphorylation, SUMOylation, and / or ubiquitination of DNA or histone residues.

[0099] In some embodiments, the effector domain of the epigenetic editors described herein can effect histone tail modification, for example, by adding or removing an active mark on the histone tail.

[0100] In some embodiments, the effector domain of the epigenetic editor described herein may include or recruit a transcription-related protein, such as a transcription repressor. The transcription-related protein may be endogenous or exogenous.

[0101] In some embodiments, the effector domain of the epigenetic editor described herein may include a protein that directly or indirectly blocks the access of a transcription factor to a target gene having a target sequence.

[0102] The effector domain can be a full-length protein or a fragment thereof that retains the epigenetic effector function ("functional domain"). The functional domain that can modulate (e.g., suppress) gene expression can also be derived from a larger protein. For example, a functional domain that can reduce target gene expression can be identified based on the sequence of a repressor protein. The amino acid sequence of a protein that modulates gene expression can be obtained from available genome browsers, such as the UCSD Genome Browser or the Ensembl Genome Browser. Protein annotation databases, such as UniProt or Pfam, can be used to identify functional domains within a full-length protein sequence. As a starting point, the largest sequence encompassing all regions identified by different databases can be tested for its modulation activity of gene expression. Then, various truncated forms can be tested to identify the minimal functional unit.

[0103] Variants of the effector domains described herein are also contemplated by the present disclosure. Variants refer to polypeptides having, for example, at least about 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity and / or sequence similarity to the wild-type effector domains described herein. In certain embodiments, the variant retains at least about 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% of the epigenetic effector function of the wild-type effector domain.

[0104] In some embodiments, the effector domains described herein can include fusions of two or more effector domains (e.g., KOX1 KRAB and ZIM3). The effector domains can include, for example, fusions of 2, 3, 4, 5, 6, 7, 8, 9, or 10 effector domains, such as effector domains described herein. In certain embodiments, the effector domain includes a truncated form of one effector domain and a fusion with a second effector domain. In certain embodiments, the effector domain includes a fusion of truncated forms of two effector domains (e.g., a fusion of the N-terminal and C-terminal portions of two effector domains).

[0105] In some embodiments, the epigenetic editors described herein can include one effector domain, two effector domains, three effector domains, four effector domains, five effector domains, six effector domains, seven effector domains, eight effector domains, nine effector domains, ten effector domains, or more. In certain embodiments, the epigenetic editor includes one or more fusion proteins (e.g., one, two, or three fusion proteins) having one or more effector domains (e.g., one, two, or three effector domains), each linked to a DNA binding domain. In some embodiments, the effector domain can induce a combination of epigenetic modifications, such as transcriptional repression and DNA methylation, DNA methylation and histone deacetylation, DNA methylation and histone demethylation, DNA methylation and histone methylation, DNA methylation and histone phosphorylation, DNA methylation and histone ubiquitination, DNA methylation and histone SUMOylation.

[0106] In certain embodiments, the effector domains described herein (e.g., DNMT3A and / or DNMT3L) are encoded by a nucleotide sequence found in the native genome (e.g., human or mouse) for that effector domain. In other embodiments, the effector domains described herein are encoded by a nucleotide sequence that is codon-optimized for optimal expression in human cells.

[0107] The effector domains described herein can include, for example, transcriptional repressors, DNA methyltransferases, and / or histone modifiers, as further detailed below.

[0108] A. Transcriptional Repressors In some embodiments, the epigenetic effector domain described herein mediates the suppression of the expression (e.g., transcription) of a target gene. The effector domain can include, for example, a Kruppel-associated box (KRAB) repressor domain, a repressor element silencing transcription factor (REST) repressor domain, a KRAB-associated protein 1 (KAP1) domain, a MAD domain, a FKHR (forkhead gene in rhabdomyosarcoma) repressor domain, an EGR-1 (early growth response gene product-1) repressor domain, an ets2 repressor factor repressor domain (ERD), a MAD smSIN3 interaction domain (SID), the WRPW motif of a hairy-related basic helix-loop-helix (bHLH) repressor protein, an HP1 alpha chromo shadow repressor domain, an HP1 beta repressor domain, or any combination thereof. The effector domain can recruit, for example, one or more protein domains that suppress the expression of the target gene through a scaffold protein. In some embodiments, the effector domain can recruit or interact with a scaffold protein domain that recruits a PRMT protein, an HDAC protein, a SETDB1 protein, or a NuRD protein domain.

[0109] In some embodiments, the effector domain includes a functional domain derived from a zinc finger repressor protein, such as a KRAB domain. The KRAB domain is found in approximately 400 human ZFP-based transcription factors. Descriptions of the KRAB domain can be found, for example, in Ecco et al., Development (2017) 144(15):2719-29 and Lambert et al., Cell (2018) 172:650-65.

[0110] In certain embodiments, the effector domain comprises a repressor domain (e.g., KRAB) derived from KOX1 / ZNF10, KOX8 / ZNF708, ZNF43, ZNF184, ZNF91, HPF4, HTF10, or HTF34. In some embodiments, the effector domain comprises a repressor domain (e.g., KRAB) derived from ZIM3, ZNF436, ZNF257, ZNF675, ZNF490, ZNF320, ZNF331, ZNF816, ZNF680, ZNF41, ZNF189, ZNF528, ZNF543, ZNF554, ZNF140, ZNF610, ZNF264, ZNF350, ZNF8, ZNF582, ZNF30, ZNF324, ZNF98, ZNF669, ZNF677, ZNF596, ZNF214, ZNF37, ZNF34, ZNF250, ZNF547, ZNF273, ZNF354, ZFP82, ZNF224, ZNF33, ZNF45, ZNF175, ZNF595, ZNF184, ZNF419, ZFP28-1, ZFP28-2, ZNF18, ZNF213, ZNF394, ZFP1, ZFP14, ZNF416, ZNF557, ZNF566, ZNF729, ZIM2, ZNF254, ZNF764, ZNF785, or any combination thereof. For example, the repressor domain can be a KRAB domain derived from KOX1, ZIM3, ZFP28, or ZN627. In certain embodiments, the repressor domain is the ZIM3 KRAB domain. In further embodiments, the effector domain is derived from a human protein, such as human ZIM3, human KOX1, human ZFP28, or human ZN627.

[0111] Exemplary effector domain sequences, or protein sequences containing them, that can reduce or silence target gene expression are provided in Table 5 below (SEQ indicates sequence number). Further examples of repressors and transcriptional repressor domains can be found, for example, in PCT Patent Application Publication No. 2021 / 226077 and Tycko et al., Cell (2020) 183(7):2020-35, each of which is hereby incorporated by reference in its entirety.

[0112]

Table 5

[0113] Any one functional analog of the proteins listed above, i.e., having the same or substantially the same biological function (e.g., 70% or more, 80% or more, 90% or more, 95% or more, or 98% or more of the transcription factor function of the protein) is encompassed by the present disclosure. For example, the functional analog can be an isoform or variant of the proteins listed above, including, for example, a portion of the above proteins with or without additional amino acid residues and / or including mutations to the above proteins. In some embodiments, the functional analog has at least 75, 80, 85, 90, 95, 98, or 99% sequence identity to one of the sequences listed in Table 5. Homologs, orthologs, and mutants of the proteins listed above are also contemplated.

[0114] In certain embodiments, the epigenetic editors described herein include a KRAB domain derived from KOX1, ZIM3, ZFP28, or ZN627, and / or an effector domain derived from KAP1, MECP2, HP1a, HP1b, CBX8, CDYL2, TOX, TOX3, TOX4, EED, EZH2, RBBP4, RCOR1, or SCML2, and optionally, the parent protein is a human protein. In certain embodiments, the epigenetic editors described herein include a domain derived from KOX1, ZIM3, ZFP28, and / or ZN627, and optionally, the parent protein is a human protein. In certain embodiments, the epigenetic editor can include a KRAB domain derived from KOX1 (ZNF10), such as human KOX1. In certain embodiments, the epigenetic editor can include a KRAB domain derived from ZIM3 (ZNF657 or ZNF264), such as human ZIM3. In certain embodiments, the epigenetic editor can include a KRAB domain derived from ZFP28, such as human ZFP28. In certain embodiments, the epigenetic editor can include a KRAB domain derived from ZN627, such as human ZN627. In certain embodiments, the epigenetic editors described herein can include CDYL2, such as human CDYL2 and / or a TOX domain (e.g., a human TOX domain), in combination with a KOX1 KRAB domain (e.g., a human KOX1 KRAB domain).

[0115] In certain embodiments, the epigenetic effector described herein includes a repressor domain (SEQ ID NO: 89) derived from KOX1 / ZNF10. For example, the repressor domain can include the sequence of SEQ ID NO: 89, or a sequence that is at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 89.

[0116] In certain embodiments, the epigenetic effector described herein includes a repressor domain derived from KOX1 / ZNF10, as shown in Table 6 below.

[0117] [Table 6]

[0118] In certain embodiments, the repressor domain can include the amino acid sequence of SEQ ID NO: 565, or a sequence that is at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 565.

[0119] In certain embodiments, the repressor domain can include the amino acid sequence of SEQ ID NO: 566, or a sequence that is at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 566.

[0120] In certain embodiments, the repressor domain can include the amino acid sequence of SEQ ID NO: 567, or a sequence that is at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 567.

[0121] In certain embodiments, the repressor domain can include the amino acid sequence of SEQ ID NO: 568, or a sequence that is at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 568.

[0122] In certain embodiments, the repressor domain can include the amino acid sequence of SEQ ID NO: 569, or a sequence that is at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 569.

[0123] In certain embodiments, the repressor domain can include the amino acid sequence of SEQ ID NO: 570, or a sequence that is at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 570.

[0124] In certain embodiments, the repressor domain can include the amino acid sequence of SEQ ID NO: 571, or a sequence that is at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 571.

[0125] In certain embodiments, the repressor domain can include the amino acid sequence of SEQ ID NO: 572, or a sequence that is at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 572.

[0126] B. DNA methyltransferase In some embodiments, the effector domain of the epigenetic editor described herein alters target gene expression through DNA modification, such as methylation. Highly methylated regions of DNA tend to have lower transcriptional activity than less methylated regions. DNA methylation occurs primarily at CpG sites (truncated form of "C-phosphate-G-" or "cytosine-phosphate-guanine" sites). A number of mammalian genes have a promoter region near or including a CpG island (a nucleic acid region having a high frequency of CpG dinucleotides).

[0127] The effector domains described herein can be, for example, a DNA methyltransferase (DNMT) or its catalytic domain, or can be capable of recruiting a DNA methyltransferase. DNMTs include enzymes that catalyze the transfer of a methyl group to a DNA nucleotide, such as canonical cytosine-5 DNMTs that catalyze the addition of a methyl group to genomic DNA (e.g., DNMT1, DNMT3A, DNMT3B, and DNMT3C). The term also includes non-canonical family members that do not themselves catalyze methylation but recruit (including activating) catalytically active DNMTs, and a non-limiting example of such a DNMT is DNMT3L. See, for example, Lyko, Nat Review (2018) 19:81-92. Unless otherwise indicated, the DNMT domain can refer to a polypeptide domain derived from a catalytically active DNMT (e.g., DNMT1, DNMT3A, and DNMT3B) or a catalytically inactive DNMT (e.g., DNMT3L). DNMTs can suppress the expression of target genes through the recruitment of inhibitory regulatory proteins. In some embodiments, methylation is in a CG (or CpG) dinucleotide sequence. In some embodiments, methylation is in a CHG or CHH sequence, where H is any one of A, T, or C.

[0128] In some embodiments, the DNMTs described herein can be animal DNMTs (e.g., mammalian DNMTs), plant DNMTs, fungal DNMTs, or bacterial DNMTs. Bacterial DNMTs can be obtained from bacterial species (e.g., cocci, bacilli, spiral bacteria, or intracellular gram-positive or gram-negative bacteria). In certain embodiments, the bacterial species is Mycoplasmatales bacterium, Mycoplasma marinum, or Spiroplasma chinense. In certain embodiments, the bacterial species is not M. penetrans, S. monbiae, H. parainfluenzae, A. luteus, H. aegyptius, H. haemolyticus, Moraxella, E. coli, T. aquaticus, C. crescentus, or C. difficile. In certain embodiments, the epigenetic editors described herein include a DNMT domain comprising SEQ ID NO: 601, or a sequence that is at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 601. In certain embodiments, the epigenetic editors described herein include a DNMT domain comprising SEQ ID NO: 602, or a sequence that is at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 602. In certain embodiments, the epigenetic editors described herein include a DNMT domain comprising SEQ ID NO: 603, or a sequence that is at least 75%, 80%, 85%, 90%, 9l%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 603.

[0129] In certain embodiments, the DNMT in the epigenetic editors described herein can include, for example, DNMT1, DNMT3A, DNMT3B, and / or DNMT3C. In some embodiments, the DNMT is a mammalian (e.g., human or mouse) DNMT. In certain embodiments, the DNMT is DNMT3A (e.g., human DNMT3A). In certain embodiments, the epigenetic editor described herein includes a DNMT3A domain comprising the sequence of SEQ ID NO: 574, or a sequence that is at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 574. In certain embodiments, the epigenetic editor described herein includes a DNMT3A domain comprising the sequence of SEQ ID NO: 575, or a sequence that is at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 575. In some embodiments, the DNMT3A domain can have mutations at, for example, position H739 (e.g., H739A or H739E), position R771 (e.g., R771L), and / or position R836 (e.g., R836A or R836Q), or any combination thereof (numbering follows SEQ ID NO: 574).

[0130] In some embodiments, the effector domain described herein can be a DNMT-like domain. As used herein, a "DNMT-like domain" is a regulator of DNMT that can activate or recruit other DNMT domains but does not itself have methylation activity. In some embodiments, the DNMT-like domain is a mammalian (e.g., human or mouse) DNMT-like domain. In certain embodiments, the DNMT-like domain is DNMT3L, which can be, for example, human DNMT3L or mouse DNMT3L. In certain embodiments, the epigenetic editor described herein comprises a DNMT3L domain comprising the sequence of SEQ ID NO: 578, or a sequence that is at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 578. In certain embodiments, the epigenetic editor herein comprises a DNMT3L domain comprising the sequence of SEQ ID NO: 579, or a sequence that is at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 579. In certain embodiments, the epigenetic editor described herein comprises a DNMT3L domain comprising the sequence of SEQ ID NO: 580, or a sequence that is at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 580. In certain embodiments, the epigenetic editor described herein comprises a DNMT3L domain comprising the sequence of SEQ ID NO: 581, or a sequence that is at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 581. In some embodiments, the DNMT3L domain can have mutations corresponding to those at position D226 (e.g., D226V), position Q268 (e.g., Q268K), or both (numbering follows SEQ ID NO: 578).

[0131] In certain embodiments, the epigenetic editors herein can include both DNMT and DNMT-like effector domains. For example, an epigenetic editor can include the DNMT3A-3L domains, and DNMT3A and DNMT3L may be covalently linked. In other embodiments, the epigenetic editors described herein can include an effector domain that includes only the DNMT3A domain (e.g., human DNMT3A) or only a DNMT-like domain (e.g., DNMT3L, which can be human or mouse DNMT3L).

[0132] Table 7 below provides exemplary DNMTs that can be a part of the epigenetic effectors described herein or from which the effector domains of the epigenetic editors described herein can be derived.

[0133] [Table 7] TIFF2025524456000033.tif180168

[0134] Any one of the functional analogs of the proteins listed above, i.e., those having the same or substantially the same biological function (e.g., 70% or more, 80% or more, 90% or more, 95% or more, or 98% or more of the DNA methylation function or mobilization function of the protein) is encompassed by the present disclosure. For example, the functional analog can be an isoform or variant of the protein listed above, including, for example, a portion of the above protein with or without additional amino acid residues and / or including a mutation to the above protein. In some embodiments, the functional analog has at least 75%, 80%, 85%, 90%, 95%, 98%, or 99% sequence identity to one of the sequences listed in Table 7. In some embodiments, the effector domain herein includes only the functional domain of the protein listed above (or a functional analog thereof), such as a catalytic domain or a mobilization domain. In some embodiments, the effector domain herein includes one or more epigenetic effector domains selected from Table 7, or a functional homolog, ortholog, or variant thereof.

[0135] As used herein, a DNMT domain (e.g., a DNMT3A domain or a DNMT3L domain) refers to a protein domain that is identical to a parent protein (e.g., human or mouse DNMT3A or DNMT3L) or a functional analog thereof (e.g., a functional fragment of the parent protein, such as having a catalytic fragment or a mobilization fragment and / or having a mutation that improves the activity of the DNMT protein).

[0136] The epigenetic editor in this specification can bring about methylation in the target gene or chromosome, for example, in 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500, 600, 700, 800, 900, or 1000 or more CpG dinucleotide sequences. The CpG dinucleotide sequence may be located within or near the target gene in a CpG island, or may be located in a region that is not a CpG island. A CpG island generally refers to a nucleic acid sequence or chromosomal region containing a high frequency of CpG dinucleotides. For example, a CpG island may contain at least 50% GC content. A CpG island may have a high observed to predicted CpG ratio, for example, at least 60% observed to predicted CpG ratio. As used herein, the observed to predicted CpG ratio is determined by the number of CpGs × (length of the sequence) / (number of Cs × number of Gs). In some embodiments, the CpG island has an observed to predicted CpG ratio of at least 60%, 70%, 80%, 90%, or more. A CpG island may be, for example, a sequence or region of at least 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, or 800 nucleotides. In some embodiments, only 1, or less than 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 30, 40, or 50 CpG dinucleotides are methylated by the epigenetic editor.

[0137] In some embodiments, the epigenetic editors herein effect methylation in hypomethylated nucleic acid sequences, i.e., sequences in which the methyl group on 5-methylcytosine nucleotides (e.g., at CpGs) may be absent as compared to a standard control. Hypomethylation can occur, for example, in aged cells or cancer (e.g., early stages of neoplasia) as compared to young cells or non-cancerous cells, respectively.

[0138] In some embodiments, the epigenetic editors described herein induce methylation in hypermethylated nucleic acid sequences.

[0139] In some embodiments, methylation can be introduced by an epigenetic editor at sites other than CpG dinucleotides. For example, a target gene sequence can be methylated at the C nucleotide of a CpA, CpT, or CpC sequence. In some embodiments, the epigenetic editor comprises a DNMT3A domain and effects methylation in CpG, CpA, CpT, CpC sequences, or any combination thereof. In some embodiments, the epigenetic editor comprises a DNMT3A domain lacking a regulatory subdomain and maintaining only the catalytic domain. In some embodiments, an epigenetic editor comprising a DNMT3A catalytic domain effects methylation exclusively in CpG sequences. In some embodiments, an epigenetic editor comprising a DNMT3A domain comprising a mutation, e.g., an R836A or R836Q mutation (numbering according to SEQ ID NO: 574), has higher methylation activity in CpA, CpC, and / or CpT sequences as compared to an epigenetic editor comprising a wild-type DNMT3A domain.

[0140] C. Histone modifiers In some embodiments, the effector domain of the epigenetic editor herein mediates histone modifications. Histone modifications play structural and biochemical roles in gene transcription, for example, by forming or disrupting nucleosome structures that bind to histones and prevent gene transcription. Examples of histone modifications include, for example, acetylation, deacetylation, methylation, phosphorylation, ubiquitination, SUMOylation, etc. at their N-terminus (the "histone tail"). These modifications maintain or specifically convert chromatin structure, thereby controlling responses that occur on chromosomal DNA, such as gene expression, DNA replication, DNA repair, etc. Post-translational modifications of histones are epigenetic regulatory mechanisms and are considered essential for gene regulation in eukaryotic cells. Recent studies have shown that chromatin remodeling factors that promote access of transcription factors to DNA by modifying nucleosome structures, such as SWI / SNF, RSC, NURF, NRD, etc., histone acetyltransferases (HATs) that regulate the acetylation state of histones, and histone deacetylases (HDACs) act as important regulatory factors.

[0141] In particular, the unstructured N-terminus of histones can be modified by acetylation, deacetylation, methylation, ubiquitination, phosphorylation, SUMOylation, ribosylation, citrullination, O-GlcN acylation, or crotonylation, or any combination thereof. For example, histone acetyltransferases (HATs) utilize acetyl-CoA as a cofactor and catalyze the transfer of an acetyl group to the epsilon amino group of the lysine side chain. This neutralizes the positive charge of the lysine, weakens the interaction between the histone and DNA, thereby opening the chromosome for transcription factor binding and initiating transcription. Acetylation of lysines K14 and K9 of histone H3 by histone acetyltransferase enzymes can be associated with transcriptional competence in humans. Lysine acetylation can create binding sites for chromatin-modifying enzymes that directly or indirectly regulate transcriptional activation. On the other hand, methylation of lysine 9 of histone H3 can be associated with heterochromatin, or transcriptionally silent chromatin.

[0142] In certain embodiments, the effector domain of the epigenetic editors described herein includes a histone methyltransferase domain. The effector domain can include, for example, a DOT1L domain, a SET domain, an SUV39H1 domain, a G9a / EHMT2 protein domain, an EZH1 domain, an EZH2 domain, a SETDB1 domain, or any combination thereof. In certain embodiments, the effector domain includes the histone-lysine-N-methyltransferase SETDB1 domain.

[0143] In some embodiments, the effector domain includes a histone deacetylase protein domain. In certain embodiments, the effector domain includes an HDAC family protein domain, such as an HDAC1, HDAC3, HDAC5, HDAC7, or HDAC9 protein domain. In certain embodiments, the effector domain includes the nucleosome remodeling and deacetylase complex (NURD), which removes acetyl groups from histones.

[0144] D. Other effector domains In some embodiments, the effector domain comprises a tripartite motif-containing protein (TRIM28, TIF1-beta, or KAP1). In certain embodiments, the effector domain comprises one or more KAP1 proteins. The KAP1 protein in the epigenetic editor herein forms a complex with one or more other effector domains of the epigenetic editor or one or more proteins involved in the modulation of gene expression in the cellular environment. For example, KAP1 can be recruited by the KRAB domain of a transcriptional repressor. The KAP1 protein domain can interact with or recruit one or more protein complexes that reduce or silence gene expression. In some embodiments, KAP1 interacts with or recruits histone deacetylase proteins, histone-lysine methyltransferase proteins, chromatin remodeling proteins, and / or heterochromatin proteins. For example, the KAP1 protein domain can interact with or recruit heterochromatin protein 1 (HP1) proteins, SETDB1 proteins, HDAC proteins, and / or NuRD protein complex components. In some embodiments, the KAP1 protein domain interacts with or recruits the ZFP90 protein (e.g., isoform 2 of ZFP90) and / or the FOXP3 protein. An exemplary KAP1 amino acid sequence is shown in SEQ ID NO: 629.

[0145] In some embodiments, the effector domain comprises a protein domain that interacts with or is recruited by one or more DNA epigenetic marks. For example, the effector domain can include a methyl-CpG binding protein 2 (MECP2) protein that interacts with methylated DNA nucleotides within a target gene (which may or may not be in the CpG island of the target gene). The MECP2 protein domain in the epigenetic editors described herein can induce a condensed chromatin structure, thereby reducing or silencing the expression of the target gene. In some embodiments, the MECP2 protein domain in the epigenetic editors described herein can interact with a histone deacetylase (e.g., HDAC), thereby suppressing or silencing the expression of the target gene. In some embodiments, the MECP2 protein domain in the epigenetic editors described herein can block access of a transcription factor or transcriptional activator to the target sequence, thereby suppressing or silencing the expression of the target gene. An exemplary MECP2 amino acid sequence is shown in SEQ ID NO: 630.

[0146] As effector domains of the epigenetic editors described in this specification, for example, chromo shadow domain, ubiquitin-2-like Rad60 SUMO-like (Rad60-SLD / SUMO) domain, chromatin organization modifier domain (Chromo) domain, Yaf2 / RYBP C-terminal binding motif domain (YAF2_RYBP), CBX family C-terminal motif domain (CBX7_C), zinc finger C3HC4 type (RING finger) domain (ZF-C3HC4_2), cytochrome b5 domain (Cyt-b5), helix-loop-helix domain (HLH), helix-heparin-helix motif domain (e.g., HHH_3), high mobility group box domain (HMG-box), basic leucine zipper domain (e.g., bZIP_1 or bZIP_2), Myb_DNA binding domain, homeodomain, MYM type zinc finger domain with FCS sequence domain (ZF-FCS), interferon regulatory factor 2-binding protein zinc finger domain (IRF-2BP1_2), SSX repressor domain (SSXRD), B-box type zinc finger domain (ZF-B_box), CXXC zinc finger domain (ZF-CXXC), regulator of chromosome condensation 1 domain (RCC1), SRC homology 3 domain (SH3_9), sterile alpha motif domain (SAM_1), sterile alpha motif domain (SAM_2), sterile alpha motif / pointed domain (SAM_PNT), Vestigial / Tondu family domain (Vg_Tdu), LIM domain, RNA recognition motif domain (RRM_1), paired amphipathic helix domain (PAH), proteasome ATPase OB C-terminal domain (Prot_ATP_ID_OB), nervy homology 2 domain (NHR2), hinge domain of cleavage stimulation factor subunit 2 (CSTF2_hinge), PPAR gamma N-terminal region domain (PPARgamma_N), CDC48 N-terminal domain (CDC48_2), WD40 repeat domain (WD40), Fip1 motif domain (Fip1), PDZ domain (PDZ_6), von Willebrand factor type C domain (VWC), NAB conserved region 1 domain (NCD1), S1RNA binding domain (S1), HNF3 C-terminal domain (HNF_C), Tudor domain (Tudor_2), histone-like transcription factor (CBF / NF-Y), and archaeal histone domain (CBFD_NFYB_HMF), zinc finger protein domain (DUF3669), EGF-like domain (cEGF), GATA zinc finger domain (GATA), TEA / ATTS domain (TEA), phorbol ester / diacylglycerol binding domain (C1-1), polycomb-like MTF2 factor 2 domain (Mtf2_C), transactivation domain of the FOXO protein family (FOXO-TAD), homeobox KN domain (Homeobox_KN), BED zinc finger domain (ZF-BED), zinc finger of C3HC4-type RING domain (ZF-C3HC4_4), RAD51 interaction motif domain (RAD51_interact), p55 binding region of methyl(nethyl)-CpG-binding domain protein MBD (MBDa), notch domain, Raf-like Ras binding domain (RBD), Spin / Ssty family domain (Spin-Ssty), PHD finger domain (PHD_3), low density lipoprotein receptor domain class A (Ldl_recept_a), CS domain, DM DNA binding domain, and QLQ domain are also contemplated.

[0147] In some embodiments, the effector domain is a protein domain that includes the YAF2_RYBP domain, or a homeodomain thereof or any combination thereof. In certain embodiments, the homeodomain of the YAF2_RYBP domain is a PRD domain, an NKL domain, a HOXL domain, or a LIM domain. In specific embodiments, the YAF2_RYBP domain may include the 32 amino acid Yaf2 / RYBP C-terminal binding motif domain (32 aa RYBP).

[0148] In some embodiments, the effector domain comprises a protein domain selected from the group consisting of a SUMO3 domain, a chromodomain derived from mitotic phosphoprotein 8 (MPP8), a chromoshadow domain derived from chromobox 1 (CBX1), and a SAM_1 / SPM domain derived from Scm polycomb group protein homolog 1 (SCMH1).

[0149] In some embodiments, the effector domain comprises an HNF3 C-terminal domain (HNF_C). The HNF_C domain may be derived from FOXA1 or FOXA2. In certain embodiments, the HNF_C domain comprises an EH1 (engrailed homology 1) motif.

[0150] In some embodiments, the effector domain may comprise an interferon regulatory factor 2-binding protein zinc finger domain (IRF-2BP1_2), a Cyt-b5 domain derived from DNA repair factor HERC2 E3 ligase, a variant SH3 domain (SH3_9) derived from Bridging Integrator 1 (BIN1), an HMG box domain derived from transcription factor TOX, or a ZF-C3HC4_2 RING finger domain, a chromodomain-helicase-DNA-binding protein 3 (CHD3) domain, or a ZNF783 domain derived from polycomb component PCGF2.

[0151] IV. Epigenetic Editor For example, provided herein is an epigenetic editor (i.e., an epigenetic editing system) that uses any combination of one or more DNA binding domains and one or more effector domains (e.g., epigenetic repressor domains) described herein to direct epigenetic modifications to a target sequence in a gene of interest. A DNA binding domain (guide polynucleotide, e.g., one that cooperates with those described herein, where the DNA binding domain is a DNA binding domain guided by a polynucleotide) directs an effector domain to epigenetically modify a target sequence, resulting in gene repression or silencing that is persistent and inheritable across cell generations. In some embodiments, the epigenetic editors described herein can reversibly or irreversibly repress or silence genes in a cell.

[0152] In certain embodiments, the epigenetic editors described herein comprise one or more fusion proteins, each of which comprises (1) a DNA binding domain and (2) an effector domain. The effector domain can be on one or more of the fusion proteins comprised by the epigenetic editor. For example, a single fusion protein can comprise all effector domains having a DNA binding domain. Alternatively, the effector domain or a subset thereof can be on separate fusion proteins, each of which has a DNA binding domain (which can be the same or different). The fusion proteins described herein can further comprise one or more linkers (e.g., peptide linkers), detectable tags, nuclear localization signals (NLSs), or any combination thereof. As used herein, "fusion protein" refers to a chimeric protein in which two or more coding sequences (e.g., of a DNA binding domain and / or an effector domain) are directly or indirectly linked, either covalently or non-covalently.

[0153] In some embodiments, the epigenetic editors described herein include two, three, four, five, six, seven, eight, nine, ten, or more effector (e.g., repressor) domains, which may be the same or different. In certain embodiments, two or more of the effector domains function synergistically. Combinations of effector domains can include DNA methylation domains, histone deacetylation domains, histone methylation domains, and / or scaffold domains that recruit any of the above. For example, the epigenetic editors described herein can include one or more transcriptional repressor domains (e.g., KRAB domain, e.g., KOX1, ZIM3, ZFP28, or ZN627 KRAB) in combination with one or more DNA methylation domains (e.g., DNMT domain) and / or recruitment domains (e.g., DNMT3L domain). Such epigenetic editors can include, for example, a KRAB domain, a DNMT3A domain, and a DNMT3L domain. In some embodiments, the epigenetic editor further includes additional effector domains (e.g., KAP1, MECP2, HP1b, CBX8, CDYL2, TOX, TOX3, TOX4, EED, RBBP4, RCOR1, or SCML2 domain). In some embodiments, the additional effector domain is a CDYL2, TOX, TOX3, TOX4, or HP1a domain. For example, the epigenetic editors described herein can include a CDYL2 and / or TOX domain in combination with a KRAB domain (e.g., KOX1 KRAB domain).

[0154] A. Linker The fusion proteins described herein can include one or more linkers that connect the components of the epigenetic editor. The linker can be a peptide linker or a non-peptide linker.

[0155] In some embodiments, one or more linkers utilized in the epigenetic editors provided herein are peptide linkers, i.e., linkers that include a peptide moiety. The peptide linker can be of any length applicable to the epigenetic editor fusion proteins described herein. In some embodiments, the linker can include a peptide of 1 to 200 (e.g., 1 to 80) amino acids. In some embodiments, the linker has a length of 1 to 5, 1 to 10, 1 to 20, 1 to 30, 1 to 40, 1 to 50, 1 to 60, 1 to 80, 1 to 100, 1 to 150, 1 to 200, 5 to 10, 5 to 20, 5 to 30, 5 to 40, 5 to 60, 5 to 80, 5 to 100, 5 to 150, 5 to 200, 10 to 20, 10 to 30, 10 to 40, 10 to 50, 10 to 60, 10 to 80, 10 to 100, 10 to 150, 10 to 200, 20 to 30, 20 to 40, 20 to 50, 20 to 60, 20 to 80, 20 to 100, 20 to 150, 20 to 200, 30 to 40, 30 to 50, 30 to 60, 30 to 80, 30 to 100, 30 to 150, 30 to 200, 40 to 50, 40 to 60, 40 to 80, 40 to 100, 40 to 150, 40 to 200, 50 to 60, 50 to 80, 50 to 100, 50 to 150, 50 to 200, 60 to 80, 60 to 100, 60 to 150, 60 to 200, 80 to 100, 80 to 150, 80 to 200, 100 to 150, 100 to 200, or 150 to 200 amino acids. Longer or shorter linkers are also contemplated. In some embodiments, the peptide linker has a length of 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 25, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or 100 amino acids. For example, the peptide linker can have a length of 4, 5, 16, 20, 24, 27, 32, 40, 64, 92, or 104 amino acids. The peptide linker can be a flexible linker or a rigid linker.In certain embodiments, the peptide linker comprises any one of the amino acid sequences of SEQ ID NOs: 631-637 and 664-666, or a sequence that is at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical thereto.

[0156] In certain embodiments, the peptide linker is an XTEN linker. Such a linker may comprise a portion of the XTEN sequence, which is an unstructured hydrophilic polypeptide consisting of only the residues G, S, P, T, E, and A (Schellenberger et al., Nat Biotechnol (2009) 27(1):1186-90). As used herein, the term "XTEN" refers to a recombinant peptide or polypeptide lacking hydrophobic amino acid residues. XTEN linkers are typically unstructured and contain a limited set of natural amino acids. Fusion of XTEN to a protein alters its hydrodynamic properties and reduces the clearance and rate of degradation of the fusion protein. These XTEN fusion proteins are produced using recombinant techniques without the need for chemical modification and are degraded by natural pathways. XTEN linkers can be, for example, 5, 10, 16, 20, 26, or 80 amino acids in length. In some embodiments, the XTEN linker is 16 amino acids in length. In some embodiments, the XTEN linker is 80 amino acids in length. In certain embodiments, the XTEN linker can be XTEN10, XTEN16, XTEN20, or XTEN80. In certain embodiments, the XTEN linker may comprise any one of the amino acid sequences of SEQ ID NOs: 638-643, or a sequence that is at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical thereto. In certain embodiments, the XTEN linker comprises the amino acid sequence of SEQ ID NO: 638. In certain embodiments, the XTEN linker comprises the amino acid sequence of SEQ ID NO: 643.

[0157] In some embodiments, one or more linkers utilized in the epigenetic editors provided herein are non-peptide linkers. For example, the linker can be a carbon-carbon bond, a disulfide bond, or a carbon-heteroatom bond. In certain embodiments, the linker is the carbon-nitrogen bond of an amide linkage. In certain embodiments, the linker is a cyclic or acyclic, substituted or unsubstituted, or branched or unbranched aliphatic or heteroaliphatic linker.

[0158] In some embodiments, one or more linkers utilized in the epigenetic editors provided herein are polymers (e.g., polyethylene, polyethylene glycol, polyamide, polyester, etc.). The linker can include, for example, monomers, dimers, or polymers of aminoalkanoic acids, aminoalkanoic acids (e.g., glycine, ethanoic acid, alanine, beta-alanine, 3-aminopropanoic acid, 4-aminobutanoic acid, 5-pentanoic acid, etc.), monomers, dimers, or polymers of aminohexanoic acid (Ahx), or polyethylene glycol moieties (PEG), or aryl or heteroaryl moieties. In certain embodiments, the linker can be based on a carbocyclic moiety (e.g., cyclopentane or cyclohexane) or a phenyl ring. The linker can include a functionalized moiety that facilitates the attachment of a nucleophile (e.g., thiol, amino) from a peptide to the linker. Any electrophile can be used as part of the linker. Exemplary electrophiles include, but are not limited to, activated esters, activated amides, alkyl halides, aryl halides, acyl halides, and isothiocyanates.

[0159] The lengths and mobilities of various linkers can be utilized between any two components of an epigenetic editor (e.g., between an effector domain (e.g., a repressor domain) and a DNA-binding domain (e.g., a Cas9 domain), between a first effector domain and a second effector domain, etc.). The linker can range from a very flexible linker, such as a glycine / serine-rich linker, to a more rigid linker in order to achieve an optimal length for effector domain activity with respect to a specific application. In some embodiments, the more flexible linker is a glycine / serine-rich linker (GS-rich linker), with more than 45% (e.g., more than 48%, more than 50%, more than 55%, more than 60%, more than 70%, more than 80%, or more than 90%) of the residues being glycine or serine residues. Non-limiting examples of GS-rich linkers are (GGGGS)n (SEQ ID NO: 1285), (G)n (SEQ ID NO: 1288), and the W linker (SEQ ID NO: 637). In some embodiments, the more rigid linker is of the form (EAAAK)n (SEQ ID NO: 1286), (SGGS)n (SEQ ID NO: 1287), and (XP)n (SEQ ID NO: 1289). In the above formulas for flexible linkers and rigid linkers, n can be any integer from 1 to 30. In some embodiments, n is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15. In some embodiments, the linker contains a (GGS)n motif, where n is 1, 3, or 7 (SEQ ID NO: 1290). In some embodiments, the linker contains a (GGGGS)n motif, where n is 4 (SEQ ID NO: 636).

[0160] In some embodiments, the linker in the epigenetic editor described herein includes a nuclear localization signal having an amino acid sequence of any one of SEQ ID NOs: 644 to 649, for example. In some embodiments, the linker in the epigenetic editor described herein includes an expression tag, such as a detectable tag, such as green fluorescent protein.

[0161] B. Nuclear localization signal The fusion proteins described herein may contain one or more nuclear localization signals and, in certain embodiments, may contain two or more nuclear localization signals. For example, the fusion protein may contain one, two, three, four, five, six, seven, eight, nine, ten, or more nuclear localization signals. As used herein, a "nuclear localization signal" (NLS) is an amino acid sequence that directs a protein to the nucleus. In certain embodiments, the NLS may be the SV40 NLS (e.g., having the amino acid sequence of SEQ ID NO: 644). The fusion protein may contain the NLS at its N-terminus, C-terminus, or both, and / or the NLS may be embedded in the middle of the fusion protein (e.g., at the N-terminus or C-terminus of the DNA binding domain or effector domain).

[0162] In some embodiments, the fusion protein may contain two NLSs. The fusion protein may contain two NLSs at its N-terminus or C-terminus. The fusion protein may contain one NLS located at its N-terminus and one NLS embedded in the middle of the fusion protein, or one NLS located at its C-terminus and one NLS embedded in the middle of the fusion protein. The fusion protein may contain two NLSs embedded in the middle of the fusion protein.

[0163] In some embodiments, the fusion protein may contain four NLSs. The fusion protein may contain at least two (e.g., two, three, or four) NLSs at its N-terminus or C-terminus. The fusion protein may contain at least one (e.g., one, two, three, or four) NLS embedded in the middle of the fusion protein. In certain embodiments, the fusion protein may contain two NLSs at its N-terminus and two NLSs at its C-terminus.

[0164] The NLS described in this specification can be an endogenous NLS sequence. In certain embodiments, the NLS described in this specification comprises the amino acid sequence of any one of SEQ ID NOs: 644 to 649, or a sequence that is at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to a selected sequence. In a particular embodiment, the NLS comprises the amino acid sequence of SEQ ID NO: 644. Additional NLSs are known in the art.

[0165] In some embodiments, an epigenetic editor comprising a fusion protein having at least one NLS at the N-terminus and at least one NLS at the C-terminus can increase the efficiency of the epigenetic editor by at least 5%, at least 10%, at least 15%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 100%, at least 200%, at least 300%, at least 400%, at least 500%, at least 600%, at least 700%, at least 800%, at least 900%, at least 1,000%, at least 5,000%, at least 10,000%, at least 50,000%, at least 100,000%, or more, compared to an epigenetic editor having a corresponding fusion protein that does not have at least one NLS at the N-terminus and at least one NLS at the C-terminus.

[0166] In some embodiments, an epigenetic editor comprising a fusion protein having two NLSs at the N-terminus and two NLSs at the C-terminus can increase the efficiency of the epigenetic editor by at least 5%, at least 10%, at least 15%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 100%, at least 200%, at least 300%, at least 400%, at least 500%, at least 600%, at least 700%, at least 800%, at least 900%, at least 1,000%, at least 5,000%, at least 10,000%, at least 50,000%, at least 100,000%, or more compared to an epigenetic editor having a corresponding fusion protein that does not have two NLSs at the N-terminus and two NLSs at the C-terminus.

[0167] C. Tag The epigenetic editors provided herein can include one or more additional sequences (tags) for tracking, detecting, and localizing the editors. In some embodiments, the epigenetic editor includes one, two, three, four, five, six, seven, eight, nine, ten, or more detectable tags. Each of the detectable tags may be the same or different.

[0168] For example, an epigenetic editor fusion protein may include a cytoplasmic localization sequence, an external transport sequence, such as a nuclear export sequence, or other localization sequences, as well as a sequence tag useful for solubilization, purification, or detection of the fusion protein. Suitable protein tags provided herein include, but are not limited to, biotin carboxylase carrier protein (BCCP) tag, myc tag, calmodulin tag, FLAG tag, hemagglutinin (HA) tag, polyhistidine tag (also referred to as histidine tag or His tag), maltose binding protein (MBP) tag, nus tag, glutathione-S-transferase (GST) tag, green fluorescent protein (GFP) tag, thioredoxin tag, S tag, Softag (e.g., Softag 1 or Softag 3), strep tag, biotin ligase tag, FlAsH tag, V5 tag, and SBP tag. Additional suitable sequences will be apparent to those skilled in the art.

[0169] D. Composition of the Fusion Protein The fusion proteins of the epigenetic editors described herein may be structured with their components in different configurations. For example, the DNA binding domain may be at the C-terminus, at the N-terminus, or between two or more epigenetic effector domains or additional domains. In some embodiments, the DNA binding domain is at the C-terminus of the epigenetic editor. In some embodiments, the DNA binding domain is at the N-terminus of the epigenetic editor. In some embodiments, the DNA binding domain is linked to one or more nuclear localization signals. In some embodiments, the DNA binding domain is flanked by epigenetic effector domains or additional domains. In some embodiments, when "DBD" represents the DNA binding domain and "ED" represents the effector domain, the epigenetic editor is -N']-[ED1]-[DBD]-[ED2]-[C' -N']-[ED1]-[DBD]-[ED2]-[ED3]-[C' -N']-[ED1]-[ED2]-[DBD]-[ED3]-[C' Or -N']-[ED1]-[ED2]-DBD]-[ED3]-[ED4]-[C' Includes the configuration of.

[0170] In some embodiments, the epigenetic editor includes a DNA binding domain (DBD), a DNA methyltransferase (DNMT) domain, and a transcriptional repressor (the "repressor") domain that suppresses or silences the expression of the target gene. The DBD, DNMT, and transcriptional repressor domains can be any of those described herein in any combination. The DBD, DNMT domain, and repressor domain can be in any configuration having any of said domains at the N-terminus, C-terminus, or central part of the fusion protein. In some embodiments, the epigenetic editor is N']-[DNMT domain]-[DBD]-[repressor domain]-[C' N']-[repressor domain]-[DBD]-[DNMT domain]-[C' N']-[DNMT domain]-[repressor domain]-[DBD]-[C' Or N']-[repressor domain]-[DNMT domain]-[DBD]-[C' Includes a fusion protein having the configuration of.

[0171] In some embodiments, the connecting structure “]-[” in any one of the epigenetic editor structures is a linker, such as a peptide linker, a detectable tag, a peptide bond, a nuclear localization signal, and / or a promoter or regulatory sequence. In the epigenetic editor structure, the plurality of connecting structures “]-[” may be the same, or each may be a different linker, tag, NLS, or peptide bond. In some embodiments, the DNMT domain may include any one of the domains in Table 7, or any combination or homolog thereof. In certain embodiments, the DNMT domain includes DNMT3A or a truncated version thereof, DNMT3L or a truncated version thereof, or both. In certain embodiments, the DBD is a DNA binding domain (e.g., dCas9) or a ZFP domain guided by a catalytically inactive polynucleotide. In certain embodiments, the repressor domain includes any one of the domains shown in Table 5 or 6, or any combination or homolog thereof. For example, the repressor domain can be a KRAB domain. In certain embodiments, the repressor domain is the ZFP28, ZN627, KAP1, MeCP2, HP1b, CBX8, CDYL2, TOX, Tox3, Tox4, EED, RBBP4, RCOR1, or SCML2 domain, or a fusion of two of the above domains (e.g., a fusion of the N-terminal and C-terminal regions of ZIM3 and KOX1 KRAB). In certain embodiments, the repressor domain is a KRAB domain derived from ZFP28, ZN627, ZIM3, or KOX1.

[0172] In some embodiments, the epigenetic editor is N']-[DNMT3A-DNMT3L]-[DBD]-[repressor]-[C' N']-[repressor]-[DBD]-[DNMT3A-DNMT3L]-[C' N']-[repressor]-[DBD]-[DNMT3A]-[C' N']-[DNMT3A]-[DBD]-[repressor]-[C' N']-[repressor]-[DBD]-[DNMT3A]-[DNMT3L]-[C' N']-[DNMT3A]-[DNMT3L]-[DBD]-[repressor]-[C' N']-[DNMT3A]-[DBD]-[C' N']-[DBD]-[DNMT3A]-[C' N']-[DNMT3L]-[DBD]-[C' N']-[DBD]-[DNMT3L]-[C' comprises a configuration selected from, wherein [DNMT3A-DNMT3L] indicates that the DNMT3A and DNMT3L domains are directly fused via a peptide bond, and the connecting structure]-[is any one of the linkers described herein, a detectable tag, an affinity domain, a peptide bond, a nuclear localization signal, a promoter, and / or a regulatory sequence. The DBD, repressor, DNMT3A, and DNMT3L domains can be any of those described herein in any combination. For example, the DNMT3A and DNMT3L domains can be selected from those in Table 7. In certain embodiments, the DBD is a CRISPR-related protein domain (e.g., dCas9) or a ZFP domain, the repressor domain is a KRAB domain derived from KOX1, ZIM3, ZFP28, or ZN627, the DNMT3A domain is a human DNMT3A domain, the DNMT3L domain is a human or mouse DNMT3L domain, and any combination of these components is also contemplated by the present disclosure.

[0173] In some embodiments, the epigenetic editor is N']-[DNMT-3A]-[DBD]-[SETDB1]-[C' N']-[DNMT3A]-[DNMT3L]-[DBD]-[SETDB1]-[C' N']-[DNMT3A-DNMT3L]-[DBD]-[SETDB1]-[C' N']-[SETDB1]-[DBD]-[DNMT3A]-[DNMT3L]-[C' N']-[SETDB1]-[DBD]-[DNMT3A]-[C' comprises a configuration selected from wherein [DNMT3A-DNMT3L] indicates that the DNMT3A and DNMT3L domains are directly fused via a peptide bond, and the connecting structure]-[is any one of the linkers described herein, a detectable tag, an affinity domain, a peptide bond, a nuclear localization signal, a promoter, and / or a regulatory sequence. The DBD, SETDB1, DNMT3A, and DNMT3L domains can be any of those described herein in any combination. In certain embodiments, the DBD is a CRISPR-associated protein domain (e.g., dCas9) or a ZFP domain, the SETDB1 domain is derived from human SETDB1, ZIM3, ZFP28, or ZN627, the DNMT3A domain is a human DNMT3A domain, the DNMT3L domain is a human or mouse DNMT3L domain, and any combination of these components is also contemplated by the present disclosure.

[0174] Specific constructs contemplated herein include DNMT3A-DNMT3L-XTEN80-NLS-dCas9-NLS-XTEN16-KOX1 KRAB (Configuration 1), DNMT3A-DNMT3L-XTEN80-NLS-ZFP domain-NLS-XTEN16-KOX1 KRAB (Configuration 2), NLS-DNMT3A-DNMT3L-XTEN80-dCas9-XTEN16-KOX1 KRAB-NLS (Configuration 3), NLS-DNMT3A-DNMT3L-XTEN80-ZFP domain-XTEN16-KOX1 KRAB-NLS (Configuration 4), NLS-NLS-DNMT3A-DNMT3L-XTEN80-dCas9-XTEN16-KOX1 KRAB-NLS-NLS (Configuration 5), and NLS-NLS-DNMT3A-DNMT3L-XTEN80-ZFP domain-XTEN16-KOX1 KRAB-NLS-NLS (Configuration 6) include. DNMT3L and DNMT3A can be derived from human parent proteins, mouse parent proteins, or any combination thereof. In certain embodiments, DNMT3L and DNMT3A are derived from mouse and human parent proteins (mDNMT3L and hDNMT3A), respectively. In certain embodiments, DNMT3L and DNMT3A are both derived from human parent proteins (hDNMT3L and hDNMT3A). In some embodiments, dCas9 is dSpCas9. In some embodiments, KOX1 is human KOX1. Also contemplated are any of Configurations 1-6 in which the KOX1 KRAB domain is replaced by the ZFP28, ZN627, or ZIM3 KRAB domain. In some embodiments, ZFP28, ZN627, and ZIM3 are human ZFP28, ZN627, and ZIM3, respectively. In certain embodiments, the fusion construct has the configuration: NLS-NLS-hDNMT3A-hDNMT3L-XTEN80-dCas9-XTEN16-KOX1 KRAB-NLS-NLS (Configuration 7), NLS-NLS-DNMT3A-DNMT3L-XTEN80-ZFP domain-XTEN16-KOX1 KRAB-NLS-NLS (Configuration 8), NLS-NLS-hDNMT3A-hDNMT3L-XTEN80-dCas9-XTEN16-ZFP28 KRAB-NLS-NLS (Configuration 9), NLS-NLS-DNMT3A-DNMT3L-XTEN80-ZFP domain-XTEN16-ZFP28 KRAB-NLS-NLS (Configuration 10), NLS-NLS-hDNMT3A-hDNMT3L-XTEN80-dCas9-XTEN16-ZN627 KRAB-NLS-NLS (Configuration 11), NLS-NLS-DNMT3A-DNMT3L-XTEN80-ZFP domain-XTEN16-ZN627 KRAB-NLS-NLS (Configuration 12), NLS-NLS-hDNMT3A-hDNMT3L-XTEN80-dCas9-XTEN16-ZIM3 KRAB-NLS-NLS (Configuration 13), or NLS-NLS-DNMT3A-DNMT3L-XTEN80-ZFP domain-XTEN16-ZIM3 KRAB-NLS-NLS (Configuration 14) may have.

[0175] In certain embodiments, the fusion constructs described herein may have Configuration 1 and may include the sequence of SEQ ID NO: 658, or a sequence that is at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical thereto. In SEQ ID NO: 658 below, the XTEN linker is underlined, the W linker is bold, underlined, and italicized, the NLS sequence is bold, the DNMT3A sequence is italicized, the DNMT3L sequence is underlined and italicized, the dCas9 domain is bold and italicized, and the KOX1 KRAB domain is underlined and bold.

[0176] TIFF2025524456000034.tif198161

[0177] In certain embodiments, the fusion constructs described herein may have Configuration 2 and may include the sequence of SEQ ID NO: 659, or a sequence that is at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical thereto. In SEQ ID NO: 659 below, the XTEN linker is shown underlined, the W linker is shown in bold, underlined, and italic, the NLS sequence is shown in bold and underlined, the DNMT3A sequence is shown in italic, the DNMT3L sequence is shown underlined and italic, the ZFP domain is shown in bold, and the KOX1 KRAB domain is shown underlined and bold. The variable amino acids represented by X are the amino acids of the DNA recognition helix of the zinc finger, and the italicized XX can be any of TR, LR, or LK.

[0178] TIFF2025524456000035.tif87161

[0179] In certain embodiments, the six "XXXXXXX" regions in SEQ ID NO: 659 include the amino acid sequences that form zinc fingers. In the above sequence, [Linker] represents the linker sequence. In some embodiments, one or both of the linker sequences can be TGSQKP (SEQ ID NO: 651). In some embodiments, one or both of the linker sequences can be TGGGGSQKP (SEQ ID NO: 652). In some embodiments, one linker sequence can have the amino acid sequence of SEQ ID NO: 651, and the other linker sequence can have the amino acid sequence of SEQ ID NO: 652.

[0180] In certain embodiments, the fusion constructs described herein may have Configuration 7 and may include the sequence of SEQ ID NO: 660, or a sequence that is at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical thereto.

[0181] In certain embodiments, the fusion constructs described herein may have Configuration 9 and may include the sequence of SEQ ID NO: 661, or a sequence that is at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical thereto.

[0182] In certain embodiments, the fusion constructs described herein may have Configuration 11 and may include the sequence of SEQ ID NO: 662, or a sequence that is at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical thereto.

[0183] In certain embodiments, the fusion constructs described herein may have Configuration 13 and may include the sequence of SEQ ID NO: 663, or a sequence that is at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical thereto.

[0184] In some embodiments, the fusion constructs described herein (e.g., any one of the fusion constructs of Configurations 1-14) are within an expression construct that includes a WPRE sequence, a polyadenylation site, or both. In certain embodiments, the WPRE sequence is in the 3' untranslated region. In certain embodiments, the WPRE sequence is upstream of the poly-adenylation site. In certain embodiments, the expression construct includes a fusion construct (e.g., any one of Configurations 1-14) and a WPRE sequence in the 3' untranslated region upstream of the polyadenylation site.

[0185] Multiple fusion proteins can be used to effect activation or repression of a target gene or multiple target genes. For example, an epigenetic editor fusion protein comprising a DNA binding domain (e.g., a dCas9 domain) and an effector domain may be co-delivered with two or more guide polynucleotides (e.g., gRNAs) each targeting a different target DNA sequence. The two target sites of the DNA binding domain can be the same or in proximity to each other, or can be separated by, for example, about 100 base pairs, about 200 base pairs, about 300 base pairs, about 400 base pairs, about 500 base pairs, about 600 base pairs, or more. Additionally, when targeting double-stranded DNA, e.g., an endogenous locus, the guide polynucleotide may target the same strand or different strands (one or more plus strands and / or one or more minus strands).

[0186] In some embodiments, an epigenetic editor targeting B2M is used in combination with an epigenetic editor targeting TRAC, TRBC, CIITA, PDCD1, TIM-3, TIGIT, LAG3, CTLA4, AAVS1, CCR5, TET2, TGFBR2, A2AR, CISH, PTPN11, PTPN6, PTPA, PTPN2, JUNB, TOX, TOX2, NR4A1, NR4A2, NR4A3, MAP4K1, REL, IRF4, DGKA, PIK3CD, HLA-A, USP16, DCK, FAS, or any combination thereof.

[0187] V. Target Sequences The epigenetic editors herein can be directed to a target sequence in B2M to effect epigenetic modification of the B2M gene.

[0188] As used herein, "target sequence", "target site", or "target region" is a nucleic acid sequence present in a gene of interest. In some instances, the target sequence may be outside of but in the vicinity of the gene of interest, where gene expression is suppressed by methylation of the target sequence or binding of a repressor to the target sequence. In some embodiments, the target sequence can be a hypomethylated or hypermethylated nucleic acid sequence.

[0189] The target sequence can be in any part of the target gene. In some embodiments, the target sequence is part of or in the vicinity of a non-coding sequence of the gene. In some embodiments, the target sequence is part of an exon of the gene. In some embodiments, the target sequence is part of or in the vicinity of a transcriptional regulatory sequence of the gene, such as a promoter or enhancer. In some embodiments, the target sequence is adjacent to, overlapping with, or encompassing a CpG island. In certain embodiments, the target sequence is within about 3000, 2900, 2800, 2700, 2600, 2500, 2400, 2300, 2200, 2100, 2000, 1900, 1800, 1700, 1600, 1500, 1400, 1300, 1200, 1100, 1000, 900, 800, 700, 600, 500, 400, 300, 200, or 100 base pairs (bp) adjacent to the B2M TSS. In certain embodiments, the target sequence is within 500 bp adjacent to the B2M TSS. In certain embodiments, the target sequence is within 1000 bp adjacent to the B2M TSS.

[0190] In some embodiments, the target sequence can hybridize to a guide polynucleotide sequence (e.g., gRNA) that forms a complex with a fusion protein comprising a DNA binding domain (e.g., a CRISPR protein, such as dCas9) and an effector domain guided by the polynucleotide. The guide polynucleotide sequence can be designed to have complementarity to the target sequence or identity to the opposite strand of the target sequence. In some embodiments, the guide polynucleotide comprises a spacer sequence that is about 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the protospacer sequence in the target sequence. In certain embodiments, the guide polynucleotide comprises a spacer sequence that is 100% identical to the protospacer sequence in the target sequence.

[0191] In some embodiments, where the DNA binding domain of the epigenetic editor described herein is a zinc finger array, the target sequence can be recognized by the zinc finger array.

[0192] In some embodiments, where the DNA binding domain of the epigenetic editor described herein is a TALE, the target sequence can be recognized by the TALE.

[0193] The target sequences described herein can be specific to one copy of the target gene or to one allele of the target gene. Thus, epigenetic modification and modulation of its expression can be specific to one copy or one allele of the target gene. For example, an epigenetic editor can suppress the expression of a particular copy (e.g., a copy associated with a disease or condition, or one having a mutation associated with a disease or condition) of the target gene recognized by the DNA binding domain.

[0194] In some embodiments, the target B2M genomic region can be included within the sequence set forth in SEQ ID NO: 1283 or 1284, with or without a terminal A.

[0195] VI. Epigenetic Modification The epigenetic editors described herein can effect sequence-specific epigenetic modifications (e.g., changes in chemical modifications) of a target gene having a target sequence. Such epigenetic modulation can be safer and more readily reversible than, for example, modulation resulting from gene editing using the generation of DNA double-strand breaks. In some embodiments, the epigenetic modulation can reduce or silence the target gene. In some embodiments, the modification is at a specific site of the target sequence. In some embodiments, the modification is at a specific allele of the target gene. Thus, the epigenetic modification can result in modulation (e.g., reduction) of the expression of one copy of the target gene having a specific allele, and not for other copies of the target gene. In some embodiments, the specific allele is associated with a disease, condition, or disorder.

[0196] In some embodiments, the epigenetic modification reduces or halts transcription of the target gene having the target sequence. In some embodiments, the epigenetic modification reduces or halts transcription of a copy of the target gene having a specific allele recognized by the epigenetic editor. In some embodiments, the epigenetic editor reduces the expression level of the protein encoded by the target gene or eliminates its expression. In some embodiments, the epigenetic editor reduces the expression level of the protein encoded by a copy of the target gene having a specific allele recognized by the epigenetic editor or eliminates its expression. The target B2M gene can be epigenetically modified in vitro, ex vivo, or in vivo.

[0197] The effector domains of the epigenetic editors described herein can alter (e.g., install or remove) chemical modifications in the nucleotides of the target gene or in the histones associated with the target gene. The chemical modifications can be altered in a single nucleotide or a single histone, or in 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 1500, 2000, 2500, 3000, or more nucleotides.

[0198] In some embodiments, the effector domain of the epigenetic editor described herein can modify CpG dinucleotides within the target gene. In some embodiments, all CpG dinucleotides within 2000 bp, within 1500 bp, within 1000 bp, within 500 bp, or within 200 bp (e.g., within the modification site described herein) adjacent to the target sequence are modified according to the modification types described herein as compared to the gene in its original state or in an equivalent cell that has not been contacted with the epigenetic editor. In some embodiments, at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, 50, 60, 70, 80, 90, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, or more of the CpG dinucleotides are modified as compared to the gene in its original state or in an equivalent cell that has not been contacted with the epigenetic editor. In some embodiments, at least 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% of the CpG dinucleotides are modified as compared to the gene in its original state or in an equivalent cell that has not been contacted with the epigenetic editor. In some embodiments, a single CpG dinucleotide is modified as compared to the gene in its original state or in an equivalent cell that has not been contacted with the epigenetic editor.

[0199] The effector domain of the epigenetic editor described herein can alter the histone modification state of histones associated or bound to a target gene. For example, the effector domain can install a modification on one or more lysine residues of the histone tail of a histone associated with a target gene. In some embodiments, the effector domain can result in the deacetylation of one or more histone tails of a histone associated with a target gene, thereby reducing or silencing the expression of the target gene. In some embodiments, the histone modification state is a methylation state. For example, the effector domain can result in H3K9, H3K27, or H4K20 methylation (e.g., one or more of H3K9me2, H3K9me3, H3K27me2, H3K27me3, and H4K20me3 methylation) in one or more histone tails associated with a target gene, thereby reducing or silencing the expression of the target gene.

[0200] In some embodiments, all histone tails of histones bound to DNA nucleotides within 2000 bp, within 1500 bp, within 1000 bp, within 500 bp, or within 200 bp adjacent to the target sequence are altered according to the modification types described herein as compared to the chromosome in its original state or the chromosome in equivalent cells that have not been contacted with the epigenetic editor. In some embodiments, at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 105, 110, 115, 120, or more histone tails of the bound histones are altered as compared to the chromosome in its original state or the chromosome in equivalent cells that have not been contacted with the epigenetic editor. In some embodiments, at least 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% of the histone tails of the bound histones are altered as compared to the chromosome in its original state or the chromosome in equivalent cells that have not been contacted with the epigenetic editor. For example, a single histone tail of a bound histone can be altered as compared to the chromosome in its original state or the chromosome in equivalent cells that have not been contacted with the epigenetic editor. As another example, a single bound histone octamer can be altered as compared to the chromosome in its original state or the chromosome in equivalent cells that have not been contacted with the epigenetic editor.

[0201] Chemical modifications placed on the target gene DNA nucleotides or histone residues can be those in the target sequence in the target gene or in its vicinity. In some embodiments, the effector domain of the epigenetic editor described herein changes the chemical modification state of nucleotides or histone tails attached to nucleotides 100 to 200, 200 to 300, 300 to 400, 400 to 55, 500 to 600, 600 to 700, or 700 to 800 nucleotides 5' or 3' to the target sequence in the target gene. In some embodiments, the effector domain changes the chemical modification state of one nucleotide or histone tail attached to a nucleotide within 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 1100, 1200, 1300, 1400, 1500, 1600, 1700, 1800, 1900, or 2000 nucleotides adjacent to the target sequence. As used herein, "adjacent to" refers to the nucleotide positions from the 5' end to the 5' terminus and from the 3' end to the 3' terminus of a particular sequence, e.g., the target sequence.

[0202] In some embodiments, the effector domain mediates or induces a change in the chemical modification of nucleotides distal from the target sequence or histone tails bound to the nucleotides. Such modifications can be initiated in the vicinity of the target sequence and subsequently spread to one or more nucleotides in the target gene distal from the target sequence. For example, the effector domain can initiate a change in the chemical modification state of one or more nucleotides within 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 300, 400, or 500 nucleotides adjacent to the target sequence or one or more histone residues bound to one or more nucleotides, and the change in the chemical modification state can spread from the target sequence in the target gene to at least 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 1100, 1200, 1300, 1400, 1500, 1600, 1700, 1800, 1900, 2000, 2500, 3000, or more nucleotides either upstream or downstream. In certain embodiments, the chemical modification can be initiated in less than 2, 3, 5, 10, 20, 30, 40, 50, or 100 nucleotides in the target gene and can spread to at least 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 2000, or more nucleotides in the target gene. In some embodiments, the chemical modification spreads to the nucleotides throughout the target gene. Additional proteins or transcription factors, such as transcription repressors, methyltransferases, or transcription regulatory scaffold proteins, can be involved in the spread of the chemical modification. Alternatively, the epigenetic editor alone can be involved.

[0203] In some embodiments, the epigenetic editor described herein reduces the expression of a target gene by at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 95%, at least about 99%, or more, as measured by transcription of the target gene in a cell, tissue, or subject, compared to a control cell, control tissue, or control subject (e.g., in the absence of the epigenetic editor). In some embodiments, the epigenetic editor described herein reduces the expression of a copy of a target gene by at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 95%, at least about 99%, or more, as measured by transcription of the copy of the target gene in a cell, tissue, or subject, compared to a control cell, control tissue, or control subject. In certain embodiments, the copy of the target gene has a specific sequence or allele recognized by the epigenetic editor. In specific embodiments, the epigenetically modified copy encodes a functional protein, and thus the epigenetic editors disclosed herein can reduce or halt protein expression and / or function.For example, the epigenetic editors described herein can reduce the expression and / or function of a protein encoded by a target gene in a cell, tissue, or subject by at least one-third, at least one-fourth, at least one-fifth, at least one-sixth, at least one-seventh, at least one-eighth, at least one-ninth, at least one-tenth, at least one-eleventh, at least one-twelfth, at least one-thirteenth, at least one-fourteenth, at least one-fifteenth, at least one-twentieth, at least one-twenty-fifth, at least one-thirtieth, at least one-thirty-fifth, at least one-fortieth, at least one-forty-fifth, at least one-fiftieth, at least one-sixtieth, at least one-seventieth, at least one-eightieth, at least one-ninetieth, or at least one-hundredth, as compared to a control cell, control tissue, or control subject.

[0204] Modulation of target gene expression can be assayed by determining any parameter that is indirectly or directly affected by the expression of the target gene. Such parameters include, for example, changes at the RNA or protein level, changes in protein activity, changes in product level, changes in downstream gene expression, changes in the transcription or activity of a reporter gene, such as luciferase, CAT, beta-galactosidase, or GFP, changes in signal transduction, changes in phosphorylation and dephosphorylation, changes in receptor-ligand interactions, changes in a second messenger, such as cGMP, cAMP, IP3, and Ca2 +Changes in concentration, changes in cell growth, changes in angiogenesis, and / or changes in any functional effect of gene expression can be mentioned. The measurement can be performed in vitro, in vivo, and / or ex vivo, and can be carried out by conventional methods, for example, measurement at the RNA or protein level, measurement of RNA stability, and / or identification of downstream or reporter gene expression. The readout can be, for example, chemiluminescence, fluorescence, colorimetric reaction, antibody binding, inducible marker, ligand binding assay, changes in intracellular second messengers such as cGMP and inositol trisphosphate (IP3), changes in intracellular calcium levels, cytokine release, etc.

[0205] Methods for determining the expression level of a gene, for example, a target of an epigenetic editor, may include, for example, determining the transcript level of the gene by reverse transcription PCR, quantitative RT-PCR, droplet digital PCR (ddPCR), Northern blot, RNA sequencing, DNA sequencing (for example, sequencing of complementary deoxyribonucleic acid (cDNA) obtained from RNA), next-generation (Next-Gen) sequencing, nanopore sequencing, pyrosequencing, or nanostring sequencing. The level of the protein expressed from the gene can be determined by, for example, Western blotting, enzyme-linked immunosorbent assay, mass spectrometry, immunohistochemistry, or flow cytometry analysis. The level of the gene expression product may be normalized to an internal standard, for example, total messenger ribonucleic acid (mRNA), or the expression level of a specific gene, for example, a housekeeping gene.

[0206] In some embodiments, the action of an epigenetic editor in modulating target gene expression can be tested using a reporter system. For example, an epigenetic editor may be designed to target a reporter gene that encodes a reporter protein, such as a fluorescent protein. The expression of the reporter gene in such a model system can be monitored, for example, by flow cytometry, fluorescence-activated cell sorting (FACS), or fluorescence microscopy. In some embodiments, a cell population can be transfected with a vector having the reporter gene. The vector can be constructed such that the reporter gene is expressed when the vector is transfected into the cell. Suitable reporter genes include genes that encode fluorescent proteins, such as green, yellow, cherry, cyan, or orange fluorescent proteins. A cell population having a reporter system can be transfected with DNA, mRNA, or a vector encoding an epigenetic editor that targets the reporter gene.

[0207] VII. Epigenetically Modified Cells In one aspect, the present disclosure provides cells modified using one or more of the epigenetic editors described herein. In some embodiments, one or more nucleic acid molecules encoding the one or more epigenetic editors or one or more of their components are applied to the cells. Any type of cell can be modified as described herein. The cells can be modified in vitro, in vivo, or ex vivo. Cells suitable for modification can be obtained from a patient or a healthy donor.

[0208] In some embodiments, the cell is an immune cell. Immune cells can include T cells, B cells, natural killer (NK) cells, dendritic cells, and monocytes / macrophages. In some embodiments, the cell is an αβ T cell. In some embodiments, the cell is a γδ T cell. In some embodiments, the cell is a cytotoxic T cell, such as a CD8+ cytotoxic T cell. In some embodiments, the cell is a helper T cell, such as a CD4+ helper T cell. In some embodiments, the cell is a regulatory T cell. In some embodiments, the cell is an NK cell. In some embodiments, the cell is a dendritic cell. In some embodiments, the cell is a macrophage.

[0209] In some embodiments, the cell is a stem cell. A "stem cell" refers to an undifferentiated cell that has the ability to indefinitely generate a large number of stem cells of the same type, from which other specialized cells can arise by differentiation. Typically, adult stem cells are multipotent (able to differentiate within a specific range), and induced or embryonic stem cells are pluripotent (able to differentiate into any cell type).

[0210] In some embodiments, the cell is a progenitor cell. A "progenitor cell" is a cell that can differentiate to form one or more types of cells in vitro and in vivo, but has limited self-renewal ability.

[0211] In certain embodiments, the cells can differentiate into the immune cells described above. The cells may be, for example, embryonic stem cells (ESCs), hematopoietic stem cells (HSCs), hematopoietic progenitor cells (HPCs), or hematopoietic stem and progenitor cells (HSPCs). "Hematopoietic stem and progenitor cells" or "HSPCs" refer to cells that express the antigen marker CD34 (CD34+). In certain embodiments, the term "HSPC" refers to cells identified by the presence of the antigen marker CD34 (CD34+) and the absence of lineage (lin) markers. The cell population that is CD34+ and / or Lin- includes hematopoietic stem cells and hematopoietic progenitor cells.

[0212] In some embodiments, the cells are induced pluripotent stem cells (iPSCs) reprogrammed from somatic cells such as T cells.

[0213] In some embodiments, the cells are obtained from the umbilical cord blood of a healthy donor. In some embodiments, the cells are obtained from adult peripheral blood or collected from the bone marrow of a healthy donor.

[0214] In some embodiments, cells as described above are modified by a method comprising transfecting the cells with (a) one or more epigenetic editors described herein, or (b) a system comprising one or more nucleic acid molecules encoding said epigenetic editor. In certain embodiments, the modified cells are T cells. In some embodiments, the modified T cells express one or more epigenetic editors capable of selectively reducing or silencing the expression of one or more target genes within the cell. In certain embodiments, the target gene is B2M. In some embodiments, the T cells are modified ex vivo. The modified T cells may, in some embodiments, further express a modified TCR or CAR against at least one antigen expressed on the surface of a target cell (e.g., a malignant cell or an infected cell). In some embodiments, the modified T cells do not express at least one gene encoding an endogenous TCR component. In certain embodiments, the modified T cells are non-alloreactive. In certain embodiments, the modified T cells are particularly suitable for allotransplantation.

[0215] VIII. Pharmaceutical Compositions In one aspect, the present disclosure provides a pharmaceutical composition comprising, as an active ingredient (or the active ingredient alone), one or more epigenetic editors described herein or components thereof (e.g., fusion proteins and / or guide polynucleotides), or a nucleic acid molecule encoding said epigenetic editor or a component thereof. For example, the pharmaceutical composition may comprise a nucleic acid molecule encoding a fusion protein (and, where applicable, a guide polynucleotide) of an epigenetic editor described herein. In some embodiments, separate pharmaceutical compositions comprise the fusion protein and the guide polynucleotide. The pharmaceutical composition may also comprise cells that have undergone an epigenetic modification mediated or induced by an epigenetic editor provided herein.

[0216] In one aspect, the present disclosure provides a pharmaceutical composition comprising, as an active ingredient (or the only active ingredient), a cell that has undergone an epigenetic modification mediated or induced by one or more epigenetic editors provided herein. For example, a nucleic acid molecule encoding the epigenetic editor is administered ex vivo to the cell.

[0217] Generally, the epigenetic editors or components thereof described herein in the present disclosure, nucleic acid molecules encoding the epigenetic editors or components thereof, or cells modified by epigenetic editors are suitable for administration as a formulation, for example, together with one or more pharmaceutically acceptable excipients described below.

[0218] The term "excipient" is used herein to describe any component other than the compounds of the present disclosure. The choice of excipient will depend largely on factors such as the specific mode of administration, the effect of the excipient on solubility and stability, and the nature of the dosage form. As used herein, "pharmaceutically acceptable excipients" include any and all physiologically compatible solvents, dispersion media, coating agents, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like. Some examples of pharmaceutically acceptable excipients are water, physiological saline, phosphate buffered saline, dextrose, glycerol, ethanol, and the like, as well as combinations thereof. In many cases, it will be preferable to include in the composition an isotonic agent, such as a sugar, a polyhydric alcohol, such as mannitol, sorbitol, or sodium chloride. Additional examples of pharmaceutically acceptable substances are wetting agents or minor auxiliary substances, such as wetting agents or emulsifying agents, preservatives, or buffering agents, which enhance the shelf life or effectiveness of the antibody.

[0219] Formulations of pharmaceutical compositions suitable for parenteral administration typically include an active ingredient combined with a pharmaceutically acceptable carrier, such as sterile water or sterile isotonic saline. Such formulations can be prepared, packaged, or sold in a form suitable for bolus administration or continuous administration. The pharmaceutical compositions of the present disclosure can be administered, for example, subcutaneously, intradermally, intratumorally, intranodally, intramuscularly, intravenously, intralymphatically, or intraperitoneally. In certain embodiments, the pharmaceutical compositions of the present disclosure are administered intravenously to a subject.

[0220] IX. Delivery Methods In some embodiments, an epigenetic editor or a component thereof is introduced into a target cell in the form of an epigenetic editor or a nucleic acid molecule encoding the component thereof, and thus the pharmaceutical compositions herein include a nucleic acid molecule. Such nucleic acid molecules can be, for example, DNA, RNA, or mRNA, and / or modified nucleic acid sequences (e.g., having chemical modifications, 5' caps, or one or more 3' modifications). In some embodiments, the nucleic acid molecule may be delivered as naked DNA or RNA using, for example, transfection or electroporation, or may be conjugated to a molecule that facilitates uptake by the target cell (e.g., N-acetylgalactosamine). In some embodiments, the nucleic acid molecule may be included in a nucleic acid expression vector, which can include expression control sequences, such as promoters, enhancers, transcriptional signal sequences, transcriptional termination sequences, introns, polyadenylation signals, Kozak consensus sequences, internal ribosome entry sites (IRES), and the like. Such expression control sequences are well known in the art. The vector may also include a sequence encoding a signal peptide (e.g., for nuclear localization, nucleolar localization, or mitochondrial localization) associated with (e.g., inserted or fused to) the sequence encoding the protein.

[0221] Examples of vectors include, but are not limited to, plasmid vectors, vaccinia virus, poliovirus, adenovirus, adeno-associated virus, SV40, herpes simplex virus, human immunodeficiency virus, retroviruses (e.g., vectors derived from retroviruses such as murine leukemia virus or spleen necrosis virus, Rous sarcoma virus, Harvey sarcoma virus, avian leukemia virus, lentivirus, human immunodeficiency virus, myeloproliferative sarcoma virus, and mammary tumor virus), and other recombinant vectors. In certain embodiments, the vector is a plasmid or viral vector. Virus particles or virus-like particles (VLPs) can also be used to deliver nucleic acid molecules encoding the epigenetic editors or components thereof described herein. For example, "empty" virus particles can be assembled to contain any suitable cargo. Viral vectors and virus particles can also be engineered to incorporate targeting ligands to alter target tissue specificity.

[0222] In certain embodiments, the epigenetic editors or components thereof described herein are encoded by nucleic acid sequences present in one or more viral vectors or the appropriate capsid proteins of any viral vector. Examples of viral vectors include adeno-associated virus vectors (e.g., derived from AAV3, AAV3b, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAVrh8, AAV10, and / or variants thereof), retroviral vectors (e.g., Moloney murine leukemia virus, MML-V), adenoviral vectors (e.g., AD100), lentiviral vectors (e.g., vectors based on HIV and FIV), and herpesvirus vectors (e.g., HSV-2).

[0223] In some embodiments, delivery comprises an adeno-associated virus (AAV) vector. AAV vector delivery can be particularly useful when the DNA-binding domain of the epigenetic editor fusion protein is a zinc finger array. Without wishing to be bound by any theory, the relatively small size of the zinc finger array compared to large DNA-binding domains, such as Cas protein domains, may allow such fusion proteins to be conveniently packaged into viral vectors, such as AAV vectors.

[0224] Any AAV serotype, such as a human AAV serotype, can be used in the AAV vectors described herein, including, but not limited to, AAV serotype 1 (AAV1), AAV serotype 2 (AAV2), AAV serotype 3 (AAV3), AAV serotype 4 (AAV4), AAV serotype 5 (AAV5), AAV serotype 6 (AAV6), AAV serotype 7 (AAV7), AAV serotype 8 (AAV8), AAV serotype 9 (AAV9), AAV serotype 10 (AAV10), and AAV serotype 11 (AAV11), as well as variants thereof. In some embodiments, the AAV variant has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% amino acid sequence identity to wild-type AAV. In certain embodiments, the AAV variant may be engineered such that its capsid protein has reduced immunogenicity or enhanced transduction ability in humans. In some cases, one or more regions of at least two different AAV serotype viruses are shuffled and reassembled to generate a chimeric variant. For example, a chimeric AAV can contain inverted terminal repeat sequences (ITRs) from a serotype that is heterologous compared to the serotype of the capsid. The resulting chimeric AAV may have different antigen reactivity or recognition compared to its parental serotype. In some embodiments, the chimeric variant of AAV contains amino acid sequences derived from two, three, four, five, or more different AAV serotypes.

[0225] Non-viral systems are also contemplated for delivery as described herein. Non-viral systems include, but are not limited to, electroporation, sonoporation, calcium phosphate transfection, microinjection, DNA gene gun, lipid-mediated transfection, transfection through heat shock, small DNA-mediated transfection, lipofection, cationic agent-mediated transfection, and nucleic acid transfection methods including transfection with liposomes, immunoliposomes, exosomes, or cationic facial amphiphiles (CFA). In certain embodiments, one or more mRNAs encoding the epigenetic editor fusion proteins described herein can be co-electroporated with one or more guide polynucleotides (e.g., gRNA) described herein. One important category of non-viral nucleic acid vectors are nanoparticles, which can be organic (e.g., lipid) or inorganic (e.g., gold). For example, organic (e.g., lipid and / or polymer) nanoparticles can be suitable for use as delivery vehicles in certain embodiments of the present disclosure.

[0226] In some embodiments, delivery is accomplished using lipid nanoparticles (LNP). LNP compositions are typically on the micrometer scale or smaller in size and can include a lipid bilayer. In some embodiments, LNP refers to any particle having a diameter of less than 1000 nm, less than 500 nm, less than 250 nm, less than 200 nm, less than 150 nm, less than 100 nm, less than 75 nm, less than 50 nm, or less than 25 nm. In some embodiments, the nanoparticles can range in size from 1 - 1000 nm, 1 - 500 nm, 1 - 250 nm, 25 - 200 nm, 25 - 100 nm, 35 - 75 nm, or 25 - 60 nm. Nanoparticle compositions include lipid nanoparticles (LNP), liposomes (e.g., lipid vesicles), and lipoplexes.

[0227] The LNPs described herein can be made from cationic, anionic, or neutral lipids. In some embodiments, the LNP can include a neutral lipid, such as a fusogenic phospholipid 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE) or cholesterol which is a membrane component, as a helper lipid to enhance transfection activity and nanoparticle stability. In some embodiments, the LNP can include hydrophobic lipids, hydrophilic lipids, or both hydrophobic and hydrophilic lipids. Any lipid or combination of lipids known in the art can be used to produce the LNP. The lipids can be combined in any molar ratio for producing the LNP. In some embodiments, the LNP is a T cell-targeting (e.g., preferentially or specifically targeting T cells) LNP.

[0228] X. Use of Epigenetic Editors and Modified Cells in Therapy The present disclosure also provides a method for treating or preventing a disease in a subject, comprising administering to the subject a) one or more epigenetic editors described herein, b) one or more nucleic acid molecules encoding the epigenetic editor, c) a cell modified by the epigenetic editor, or d) a pharmaceutical composition comprising any of a) to c).

[0229] In one aspect, the epigenetic editor effects an epigenetic modification of a target polynucleotide sequence within a target gene associated with a disease, disorder, or condition in a subject, thereby regulating the expression of the target gene to treat or prevent the disease, disorder, or condition. In some embodiments, the epigenetic editor reduces the expression of the target gene to an extent sufficient to achieve a desired effect, such as a therapeutically appropriate effect, for example, prevention or treatment of a disease, disorder, or condition.

[0230] In some embodiments, cells (e.g., allogeneic cells) modified by one or more of the epigenetic editors of the present disclosure are administered as a drug to a subject having a disease, disorder, or impairment, whereby the disease, disorder, or impairment can be treated. In some embodiments, the subject receives administration of allogeneic T cells epigenetically modified as described herein, for example, to reduce or silence B2M expression. In some embodiments, the modified T cells further express a modified TCR or CAR against at least one antigen expressed on the surface of target cells (e.g., malignant cells or infected cells). In some embodiments, the modified T cells do not express at least one gene encoding an endogenous TCR component.

[0231] In some embodiments, the subject can be a mammal, such as a human. In some embodiments, the subject is selected from non-human primates such as chimpanzees, cynomolgus monkeys, or macaques, as well as other ape and monkey species.

[0232] XI. Definitions As used herein, the term "nucleic acid" refers to any oligonucleotide or polynucleotide that contains nucleotides (e.g., deoxyribonucleotides or ribonucleotides) in either single-stranded or double-stranded form, including DNA and RNA. A "nucleotide" contains a sugar deoxyribose (DNA) or ribose (RNA), a base, and a phosphate group, and is linked through the phosphate groups. "Bases" include purines and pyrimidines, such as natural compounds, e.g., adenine, thymine, guanine, cytosine, uracil, inosine, and natural analogs, as well as synthetic derivatives of purines and pyrimidines with new reactive groups, e.g., amines, alcohols, thiols, carboxylic acids, alkyl halides, etc., but are not limited thereto. Nucleic acids can contain known nucleotide analogs and / or modified backbone residues or linkages, which may be synthetic, naturally occurring, or non-naturally occurring. Such nucleotide analogs, modified residues, and modified linkages are well known in the art and can result in nucleic acid molecules having enhanced cellular uptake, reduced immunogenicity, and / or increased stability in the presence of nucleases.

[0233] As used herein, an "isolated" or "purified" nucleic acid molecule is a nucleic acid molecule that exists separate from its native environment. For example, an "isolated" or "purified" nucleic acid molecule is (1) separated from the nucleic acids of genomic DNA or cellular RNA from which it originated, and / or (2) does not exist in nature. In some embodiments, an "isolated" or "purified" nucleic acid molecule is a recombinant nucleic acid molecule.

[0234] In addition to the specific proteins and nucleic acid molecules referred to herein, it will be understood that the present disclosure also contemplates the use of their variants, derivatives, homologs, and fragments. A variant of any given sequence can have a specific sequence of residues (amino acid residues or nucleic acid residues) that are modified in such a way that the polypeptide or polynucleotide in question substantially retains at least one of its endogenous functions. Variant sequences can be obtained by addition, deletion, substitution, modification, replacement, and / or variation of at least one residue (in some embodiments, one or fewer, two or fewer, three or fewer, four or fewer, five or fewer, six or fewer, seven or fewer, eight or fewer, nine or fewer, ten or fewer, fifteen or fewer, or twenty or fewer residues) present in the naturally occurring sequence. For the specific proteins described herein (e.g., the KRAB, dCas9, DNMT3A, and DNMT3L proteins described herein), the present disclosure also contemplates either the naturally occurring form of the protein or a variant or homolog that retains at least one of its endogenous functions (e.g., at least 50%, 60%, 70%, 80%, 90%, 85%, 96%, 97%, 98%, or 99% of its function as compared to the specific protein described).

[0235] As used herein, a homolog of any polypeptide or nucleic acid sequence contemplated herein includes a sequence having a certain homology with the wild-type amino acid and nucleic acid sequences. The homologous sequences can include sequences that are at least 50%, 55%, 65%, 75%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the target sequence, for example, amino acid sequences. The term "percent identical" in the context of an amino acid or nucleotide sequence refers to the percentage of residues that are the same in two sequences when aligned for maximum correspondence. In some embodiments, the length of the reference sequence aligned for comparison purposes is at least 30% (e.g., at least 40, 50, 60, 70, 80, or 90%, or 100%) of the reference sequence. Sequence identity can be measured using sequence analysis software (e.g., the Sequence Analysis Software Package of the Genetics Computer Group, University of Wisconsin Biotechnology Center, 1710 University Avenue, Madison, Wis. 53705, BLAST, BESTFIT, GAP, or PILEUP / PRETTYBOX programs). Such software correlates identical or similar sequences by assigning a degree of homology to various substitutions, deletions, and / or other modifications. In an exemplary approach for determining the degree of identity, the BLAST program can be used, and probability scores of e-3 to e-100 indicate closely related sequences.

[0236] The percent identity of two nucleotide or polypeptide sequences is determined, for example, using BLAST® with its default parameters (available on the website of the U.S. National Library of Medicine's National Center for Biotechnology Information). In some embodiments, the length of the reference sequence aligned for comparison purposes is at least 30% of the reference sequence (e.g., at least 40, 50, 60, 70, 80, or 90%).

[0237] It will be understood that the numbering of specific positions or residues in a polypeptide sequence may depend on the particular protein and numbering scheme used. The numbering may, for example, differ between the precursor of a mature protein and the mature protein itself, and sequence differences between species may affect the numbering. One of ordinary skill in the art will be able to identify the respective residues in any homologous protein and its encoding nucleic acid by methods well known in the art, such as sequence alignment and determination of homologous residues.

[0238] The terms "modulate" or "alter" refer to a change in the amount, degree, or extent of a function. For example, the epigenetic editors described herein can modulate the activity of a promoter sequence by binding to a motif within the promoter, thereby inducing, enhancing, or repressing the transcription of a gene operably linked to the promoter sequence. As another example, the epigenetic editors described herein can block the ability of RNA polymerase to transcribe a gene or inhibit the translation of an mRNA transcript. The terms "inhibit," "suppress," "repress," "silencing," etc., when used with reference to the epigenetic editors or components thereof described herein, refer to a decrease or prevention of the activity of a nucleic acid sequence (e.g., a target gene) or protein (e.g., transcription) as compared to the activity of the nucleic acid sequence or protein in the absence of the epigenetic editor or its components. This term can include partially or completely blocking the activity or preventing or delaying the activity. The inhibited activity can be, for example, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% lower than that of a control, or can be, for example, at least one-fifteenth, one-half, one-third, one-fourth, one-fifth, or one-tenth of that of a control.

[0239] The term "about" or "approximately" means within an acceptable error range for a particular value as determined by one of ordinary skill in the art, which will depend in part on how the value is measured or determined, e.g., the limitations of the measurement system. For example, "about" can mean within one or more standard deviations of a given value according to the convention in the art. When a particular value is recited in the present application and claims, the term "about" should be assumed to mean within the acceptable error range of that particular value, unless otherwise indicated. The ranges provided in this specification are understood to be all truncated forms of values within the range. For example, a range of 1 to 50 includes any number, combination of numbers, or sub-range from the group consisting of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50, as well as all intervening fractional values between the aforementioned integers, such as 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, and 1.9. With respect to sub-ranges, "nested sub-ranges" extended from either endpoint of the range are specifically contemplated. For example, nested sub-ranges of the exemplary range of 1 to 50 can include, in one direction, 1 to 10, 1 to 20, 1 to 30, and 1 to 40, or in the other direction, 50 to 40, 50 to 30, 50 to 20, and 50 to 10.

[0240] Unless otherwise defined herein, scientific and technical terms used in connection with the present disclosure shall have the meanings commonly understood by one of ordinary skill in the art. Although exemplary methods and materials are described below, methods and materials similar or equivalent to those described herein can also be used in the practice or testing of the present disclosure. In case of conflict, the present specification, including definitions, will control. Further, unless the context requires otherwise, singular terms shall include pluralities and plural terms shall include singulars. Throughout this specification and the embodiments, the terms "have" and "comprise" or variations such as "has", "having", "comprises", or "comprising" are meant to imply the inclusion of the stated integer or group of integers, but not the exclusion of any other integer or group of integers. Unless otherwise indicated, the description of a list of elements herein shall include any one of the elements alone or in any combination. The description of embodiments herein includes that embodiment as a single embodiment or in combination with any other embodiment herein. All publications, patents, patent applications, and other references cited herein are hereby incorporated by reference in their entirety. To the extent that the references incorporated by reference conflict with the present disclosure contained herein, the present specification is intended to be prior and / or superior to any such conflicting material. Although several documents are cited herein, this citation does not constitute an admission that any of these documents forms part of the common general knowledge in the art.

[0241] According to the present disclosure, a backward reference in a dependent claim means a shorthand for the direct and explicit disclosure of the combination of each individual claim indicated by the backward reference. Further, the headings herein are for ease of construction and are in no way intended to limit the scope of the invention described in the claims.

[0242] To better understand the present disclosure, the following examples are described. These examples are for illustrative purposes only and should in no way be construed as limiting the scope of the present disclosure.

Examples

[0243] [Example 1] Design and synthesis of fusion proteins A fusion protein (“CRISPR-off”) containing dCas9, DNMT3A, DNMT3L, and KOX1 KRAB was created. This protein had the following functional domains and linkers from the N-terminus to the C-terminus: huDNMT3A-linker-huDNMT3L-XTEN80-NLS-dSpCas9-NLS-XTEN16-huKOX1 KRAB (SEQ ID NO: 658). The CRISPR-off plasmid construct is described in Nunez et al., Cell (2021) 184(9):2503-19.

[0244] A ZF fusion protein (“ZF-off”) containing DNMT3A, 3L, and KOX1 KRAB was also created. This fusion protein had the following general structure: huDNMT3A-linker-huDNMT3L-XTEN80-NLS-ZFP domain-NLS-XTEN16-huKOX1 Krab (SEQ ID NO: 659).

[0245] [Example 2] Selection of the B2M region for gRNA targeting Using the Benchling gRNA platform for humans (GRCh38), gRNAs targeting genomic regions within 1 kb from the TSS of the human B2M gene were computationally designed. gRNAs containing the polyTTTT sequence were initially excluded. gRNA off-target analysis was performed using CasOFFinder (Bae et al., Bioinformatics (2014) 30(10):1473-5). gRNAs that matched multiple locations within the target genome were excluded.

[0246] A final set of 258 gRNA sequences was selected for primary screening in GripTite™ HEK 293 cells. DNA plasmids containing the coding sequences of the gRNAs under the control of the U6 promoter were obtained from a vendor.

[0247] [Example 3] Selection of ZFP Target Sites and Design of ZFPs Using a library of two-finger ZFPs (2F units) that each recognize a 6-bp DNA site, a large six-finger ZFP array targeting an 18-bp DNA binding site was designed. The source of the 2F units was a set of three-finger Zn finger proteins selected to bind to specific target sites using a bacterial two-hybrid (B2H) selection system (Hurt et al., PNAS (2003) 100:12271-6; Maeder et al., Mol Cell (2008) 31(2):294-301). All possible three-combinations of the 6-bp binding sites shown in the library were generated, allowing either 0 or 1 bp between the 6-bp target sites to create a list of targetable DNA sites. To identify ZF target sites within human B2M, sequences within 1 kb from the TSS (human (GRCh38)) were compared to this list.

[0248] For each identified ZF target site, multiple ZF proteins could be designed. The design of the six recognition helices used to generate the complete protein was made by selecting 2F units, considering factors such as the known binding ability of Zn finger proteins, the frequency with which amino acids at positions -1, 2, 3, and 6 are selected to bind to the desired target bases in the B2H selection system, the avoidance of amino acids at positions -1, 2, 3, 6 selected to bind to multiple different bases in B2H, and the maintenance of context-dependence by matching adjacent bases if possible. The entire ZF sequence was derived from the naturally occurring Zif268 protein, and the selected recognition helices were maintained in the sequence configuration selected by B2H (either finger 1-2 or finger 2-3 of Zif268).

[0249] The 2F units were linked with linker TGSQKP (SEQ ID NO: 651) when the 6bp binding sites were adjacent, and with linker TGGGGSQKP (SEQ ID NO: 652) when the 6bp binding sites were separated by 1bp. Additionally, a final set of 280 ZFPs targeting 41 different binding sites within 1 kb from the B2M TSS (chr15:44711517) with no exact match to the genome (GRCh38) were selected for the primary screening (Table 1).

[0250] [Example 4] Guide RNA Screening in GripTite (trademark) HEK293 MSR Cells This example describes a study screening the effectiveness of gRNAs targeting B2M in HEK293 cells (human fetal kidney cells).

[0251] Introduction of gRNA + CRISPR-Off into HEK293 Cells Six 96-well plates (Sigma-Aldrich) were seeded with 20,000 GripTite (trademark) 293 MSR cells (Thermo Fisher, Cat No. R79507) per well in appropriate cell culture medium. These cells are derived from human embryonic kidney cells (HEK293). After plating, the cells were grown for 24 hours in an incubator at 5% CO2 and 37 °C. 25 ng of the gRNA-encoding DNA fragment and 50 ng of the CRISPR-off-encoding plasmid were resuspended in DPBS buffer (Thermo Fisher, Cat. No. 14190144). Additionally, 10 ng of the EF1a: puromycin resistance plasmid (PLA015) was also added to the transfection mixture to obtain a total payload of 85 ng of DNA.

[0252] The transfection mixture was prepared by adding the resuspended components to Mirus TransIT®-LT1 Transfection Reagent (Mirus, Cat. No. MIR2300). The transfection mixture was added in duplicate to a total of six screening plates. Wild-type (WT) CRISPR Cas9 with two different TSS-adjacent gRNAs (positive control), CRISPR-off without gRNA (negative control), CRISPR-off with a gRNA targeting a non-B2M locus (negative control), and empty vector only (negative control) were also part of this experiment. The cells were passaged twice a week by treating them with trypsin and EDTA and then splitting them into fresh medium in a new culture plate.

[0253] B2M Flow Cytometry On the 6th, 13th, and 20th days after transfection, the transfected GripTite (trademark) 293 MSR cells were treated with trypsin and EDTA and washed with PBS containing 2% FBS. Then, the cells were stained with a 1:300 dilution of PE-labeled anti-human B2M antibody (BioLegend, Cat. No. 395704) and a 1:1000 dilution of Zombie Violet Fixable Viability Dye (BioLegend, Cat. No. 423113) in PBS containing 2% FBS, which was prepared in advance according to the manufacturer's recommendations, for 20 minutes at 4°C. The stained cells were washed and incubated in Fixation Buffer (BioLegend, Cat. No. 420801) for 20 minutes. Next, after washing the cells, data were acquired using an Agilent Novocyte Penteon flow cytometer, and a maximum of 20,000 live cell events per well could be collected. The percentage of silencing was evaluated by comparing the screening situation with the expression level of the negative control (without gRNA).

[0254] Results The relative B2M expression levels of cells transfected with one of the 258 gRNAs tested are shown in Figure 1 and Table 8. The B2M gRNA showing the most excellent effect is indicated as "Yes" selection in Figure 1 in combination with the quantification of B2M expression in the control experiment without gRNA. The smoothed fitting of the entire screening shows a pattern of effective gRNA silencing centered on the TSS of B2M, as shown in Figure 1.

[0255] After treatment with a large number of gRNA candidates, strong silencing of the B2M gene, which causes a decrease in B2M expression, was observed, and only 30 - 40% of B2M-positive cells were observed.

[0256] [Table 8] TIFF2025524456000037.tif245170TIFF2025524456000038.tif248170TIFF2025524456000039.tif248170TIFF2025524456000040.tif248170TIFF2025524456000041.tif250170TIFF2025524456000042.tif194170

[0257] 172 gRNAs that showed the most excellent effects from the above primary screening (i.e., gRNAs with the highest B2M protein knockdown efficiency) were ordered in the mRNA / sgRNA format for further follow-up research as single guide RNAs (sgRNAs).

[0258] [Example 5] Confirmation of gRNA Screening in Primary T Cells In this example, a study on screening gRNAs in human primary T cells will be described.

[0259] T cells are isolated from human leukapheresis products (StemCell Technologies, Cat. No. 70500) using the EasySep™ Human T cell Isolation Kit (StemCell Technologies, Cat. No. 17951). The T cells are thawed and activated. Prior to nucleofection, the T cells are thawed, washed, and stimulated in complete T cell medium (X-VIVO15 medium; Lonza, Cat. No. BEBP04-744Q) supplemented with 5% human AB serum (Gemini Bio-Product, Cat. No. 100-512), 2 mM L-alanyl-L-glutamine, 5 ng / mL IL-7, and 5 ng / mL IL-15 using Dynabeads Human T-Activator CD3 / CD28 for T Cell Expansion and Activation (Thermo Fisher, Cat. 11131D) at a bead:cell ratio of 3:1 at 37 °C, 5% CO2 for approximately 48 hours. Subsequently, the beads are removed from the culture by magnetic force and the T cells are cultured in fresh complete T cell medium for approximately 24 hours. Next, 2 × 10 5 cells / well of 2.5 μg CRISPR-off mRNA (TriLink) + 2.5 μg sgRNA (IDT) are introduced into the T cells by nucleofection using the P3 Primary Cell 96 Well Nucleofector Kit (Lonza, Cat. No. V4SP-3960) and Amaxa 4D Nucleofector® (Lonza).

[0260] After nucleofection, the T cells are resuspended in complete T cell medium and maintained by medium change and passaging twice a week as needed. On day 13 after nucleofection, the cells are restimulated with ImmunoCult™ Human CD3 / CD28 T Cell Activator (StemCell Technologies, Cat. No. 10991).

[0261] Cell surface B2M protein expression of live T cells is evaluated by flow cytometry on days 6, 13, and 20 after nucleofection. Controls of no mRNA, CRISPR-off mRNA + non-B2M-targeting sgRNA, CRISPR-off mRNA without gRNA, WT Cas9 mRNA + exon-targeting sgRNA, staining only (no mRNA or gRNA), isotype (no mRNA or gRNA), and no staining (no mRNA or gRNA) are also run on each screening plate.

[0262] The B2M flow cytometry assay is performed as described in Example 5. The percentage of silencing is evaluated by comparing the expression level of the test sample to that of the negative control (CRISPR-off mRNA without sgRNA).

[0263] [Example 6] ZF Screening in Primary T Cells In this example, a study is described in which zinc finger protein (ZFP) domains targeting various genomic regions of the B2M gene are screened in human primary T cells.

[0264] T cells were isolated from human leukapheresis products and cryopreserved. Before nucleofection, the T cells were thawed and stimulated with CD3 / CD28 beads in complete T cell medium at 37 °C and 5% CO2 for approximately 48 hours. Subsequently, the beads were removed by magnetic force and the T cells were cultured in fresh complete T cell medium. ZF-off mRNA was introduced into the T cells by nucleofection using the Lonza Amaxa 4D Nucleofector. After nucleofection, the T cells were resuspended in complete T cell medium and maintained twice a week by medium change and cell splitting as needed. On day 13 after nucleofection, the cells were restimulated with soluble CD3 / CD28 T Cell Activator. On days 6, 13, and 20 after nucleofection, the cell surface B2M protein expression of viable T cells was evaluated by flow cytometry. Controls without mRNA, non-B2M-targeted ZF-off mRNA, WT Cas9 mRNA + exon-targeted gRNA, staining only, isotype, and no staining were also run on each screening plate.

[0265] The B2M flow cytometry assay was performed as described in Example 5. The screening situation was compared with the expression level of the negative (non-B2M-targeted ZF) control, and the percentage of silencing was evaluated. The following ZF constructs were tested:

[0266] TIFF2025524456000043.tif175161TIFF2025524456000044.tif252161TIFF2025524456000045.tif252161TIFF2025524456000046.tif254160TIFF2025524456000047.tif254161TIFF2025524456000048.tif253161TIFF2025524456000049.tif255162TIFF2025524456000050.tif252162TIFF2025524456000051.tif253162TIFF2025524456000052.tif252162TIFF2025524456000053.tif253162TIFF2025524456000054.tif254162TIFF2025524456000055.tif254163TIFF2025524456000056.tif254163TIFF2025524456000057.tif252163TIFF2025524456000058.tif253163TIFF2025524456000059.tif254163TIFF2025524456000060.tif253163TIFF2025524456000061.tif253163TIFF2025524456000062.tif255162TIFF2025524456000063.tif252163TIFF2025524456000064.tif52163

[0267] [Example 7] Full specificity screening of constructs in primary human T cells Test the specificity of CRISPR-off and ZF-off constructs for silencing B2M in primary human T cells. The readouts for assessing specificity are RNAseq, methylation arrays, and whole-genome bisulfite sequencing analysis assays. Genome-wide expression and methylation changes after epigenetic editing compared to negative controls are profiled.

[0268] [Example 8] CpG methylation pattern Examine the CpG methylation pattern in primary human T cells treated with CRISPR-off or ZF-off. Perform a hybrid capture assay on bisulfite-treated DNA to examine the methylation pattern of CpG sites induced by CRISPR-off or ZF-off in a 1 kb region near the B2M TSS.

[0269] [Example 9] Screening follow-up and hit validation The top hits obtained from the gRNA and ZF-off screening are reconfirmed by repeating the screening experimental conditions and appropriately adjusting the dose of CRISPR-off mRNA + gRNA or ZF-off mRNA up and down by about several times (about 3.2) times to establish a dose-response profile. Select the gRNA and ZF-off mRNA showing the highest potency and long-term persistence profile for downstream candidate development. 0.5 (about 3.2) times up and down by adjusting the dose of CRISPR-off mRNA + gRNA or ZF-off mRNA up and down by about several times (about 3.2) times to establish a dose-response profile. Select the gRNA and ZF-off mRNA showing the highest potency and long-term persistence profile for downstream candidate development.

[0270] [Example 10] Allogeneic function assay in primary T cells The response of allogeneic healthy donor CD8+ T cells to mock-modified T cells or B2M-silenced T cells is evaluated by a mixed lymphocyte co-culture assay and / or a cytotoxicity assay.

[0271] The proliferation and / or activation of allogeneic healthy donor CD8+ T cells are evaluated by measuring cell dye dilution and cell surface expression of activation markers by flow cytometry, respectively, after co-culture with mock-modified T cells or B2M-silencing T cells. A decrease in the response to B2M-silencing cells compared to the response to mock-modified cells is expected, which indicates a decrease in the proliferation and activation of allogeneic healthy donor CD8+ T cells. Furthermore, the death of modified T cells after co-incubation with allogeneic healthy donor CD8+ T cells is evaluated by flow cytometry using a viability dye stain or by image analysis of cell viability. B2M-silencing T cells are expected to survive preferentially over mock-modified T cells in the presence of healthy donor CD8+ T cells.

[0272] [Example 11] Screening of guide RNAs in primary T cells using CRISPR-off constructs Using 172 guide RNAs (shown in Table 9 below) and mRNA encoding fusion protein construct 15, re-screening of B2M single guide RNAs in primary T cells was performed. The annotation of the amino acid sequence of fusion protein Configuration 15 is shown below. The results are shown in Table 9 below.

[0273] Ten guides showed silencing exceeding 20%, and 18 guides showed silencing exceeding 10%. RNA988 showed 40% silencing.

[0274] TIFF2025524456000065.tif104163

[0275] Table 9. The percentage of normalized B2M+ cells in a primary T cell population treated with a CRISPR-off epigenetic repressor using various gRNAs targeting B2M in primary human T cells was measured on day 6 after application. Data obtained from two replicate plates ("Plate 1" and "Plate 2") are shown together with the weighted average of the two replicate plates. Also provided is the starting position of each gRNA on chromosome 15 (GRCh38).

[0276]

Table 9

[0277] [Example 12] B2M Dual Guide Screening To improve the robustness and persistence of silencing, an assay was performed in which two guides were applied to the same cell. In this example, a study targeting gRNA pairs in human primary T cells is described.

[0278] T cells were isolated from human leukapheresis products (StemCell Technologies, Cat. No. 70500) using the EasySep™ Human T cell Isolation Kit (StemCell Technologies, Cat. No. 17951). The T cells were thawed and activated. Before nucleofection, the T cells were thawed, washed, and stimulated in complete T cell medium (X-VIVO15 medium; Lonza, Cat. No. BEBP04-744Q) containing 5% human AB serum (Gemini Bio-Products, Cat. No. 100-512), 2 mM L-alanyl-L-glutamine, 5 ng / mL IL-7, and 5 ng / mL IL-15 with Dynabeads Human T-Activator CD3 / CD28 for T Cell Expansion and Activation (Thermo Fisher, Cat. 11131D) at a bead:cell ratio of 3:1 at 37 °C, 5% CO2 for approximately 48 h. Subsequently, the beads were removed from the culture by magnetic force, and the T cells were cultured in fresh complete T cell medium for approximately 24 h. Next, 2 × 10 5 cells / well of 2.5 μg CRISPR-off mRNA (TriLink) + 2.5 μg sgRNA (IDT) were introduced into the T cells by nucleofection using the P3 Primary Cell 96 Well Nucleofector Kit (Lonza, Cat. No. V4SP-3960) and Amaxa 4D Nucleofector (Lonza) with the pulse code EO115.

[0279] After nucleofection, the T cells were resuspended in complete T cell medium and maintained by medium change and passage twice a week as needed. On day 13 after nucleofection, the cells were restimulated with ImmunoCult™ Human CD3 / CD28 T Cell Activator (StemCell Technologies, Cat. No. 10991).

[0280] Cell surface B2M protein expression of live T cells was evaluated by flow cytometry on days 6, 13, and 20 after nucleofection. Controls without mRNA, CRISPR-off mRNA + non-B2M-targeting sgRNA, CRISPR-off mRNA without gRNA, WT Cas9 mRNA + exon-targeting sgRNA, staining only (without mRNA or gRNA), isotype (without mRNA or gRNA), and without staining (without mRNA or gRNA) were also run on each screening plate.

[0281] The B2M flow cytometry assay was performed as described in Example 5. The gating strategy is shown in Figure 2A (without gRNA) and Figure 2B (RNA102 and RNA964). Test samples were compared to the expression level of the negative (CRISPR-off mRNA without sgRNA) control to evaluate the silencing percentage. The results are shown in Figure 2C.

[0282] Figures 3A - 3B show the percentage of B2M+ positive cells observed after applying different guide RNA pairs and the distance from the guide RNA binding site to the B2M TSS. Figure 4 shows B2M silencing by six guide RNA pairs measured on days 6, 13, and 20. All six guide RNA pairs decreased B2M expression compared to the gRNA-free control at each time point.

[0283] [Example 13] B2M CpG methylation pattern The CpG methylation pattern in primary human T cells treated with CRISPR-off was examined. A hybrid capture assay was performed on bisulfite-treated DNA to examine the methylation pattern of CpG sites induced by CRISPR-off in a 1 kb region near the B2M TSS.

[0284] B2M was silenced with a combination of two pairs of double guides (RNA138 / 949 and RNA104 / 988). Samples were sorted on day 14 after nucleofection, and pure B2M-negative (B2M−) and B2M-positive (B2M+) cell populations were sent for methylation analysis. More than 99% of the sorted B2M+ cells were B2M positive, and less than 1% of the B2M− cells were B2M positive. After sorting, B2M− samples were either restimulated with PMA / ionomycin or left in standard medium; after incubating these samples to observe silencing, the restimulated and control samples were also sent for hybrid capture methylation analysis.

[0285] An overview of the experimental procedures for each sample is shown in FIG. 6A. FIG. 6B shows the methylation patterns under each condition near the B2M locus. As shown in FIGS. 7A-7B, robust B2M CpG methylation was observed in the sorted B2M-negative population. As shown in FIG. 8, extensive B2M CpG methylation was achieved with the combination of RNA138 / 949 and RNA104 / 988.

[0286] [Example 14] B2M Silencing Under Multiple Effector / Guide Conditions Under Both Fresh and Frozen Conditions Fresh primary human T cells were transfected with various combinations of effector (FP13 or FP11a) and / or RNA (guide 1, guide 2, Milan TRACR, or US TRACR), or WT Cas9 (FIG. 9A). Six days after transfection, the percentage of T cells expressing B2M was measured. Silencing was achieved when the effector and guide were combined, but not when the effector or guide was used alone. B2M expression in the transfected T cells was also measured on day 14 after transfection (FIG. 9B). When the effector and guide were combined, silencing was maintained for a long period.

[0287] The same transfection was repeated in cryopreserved primary human T cells (Figure 10A). Similarly in this case, silencing was achieved when the effector and guide were combined. Six days after transfection, a comparison of B2M silencing in primary human T cells from two different donors (DON23, Figure 10B and DON24, Figure 10C) was also performed (transfection was carried out as above). The effectiveness of B2M silencing when the effector and guide were combined varied between donors, and DON24 (Figure 10C) showed stronger B2M silencing with all effector / guide combinations. Transfections were performed using fusion protein 13 and fusion protein 11a, as well as gRNA, WT Cas9, or a control without gRNA, and B2M expression was evaluated at 6, 12, 20, 28, and 35 days after transfection. Silencing was achieved when the effector and guide were combined, and cryopreserved T cells retained greater B2M silencing over a long period of time.

[0288] [Example 15] B2M Silencing at Multiple Serum Concentrations B2M silencing in primary human T cells under different serum conditions (comparing 5% human serum and 10% human serum) was measured over time after transfection with B2M silencing gRNA, WT Cas9, or a control without gRNA. A representative gating strategy for B2M expression measurement is shown in Figure 11A. No differences in B2M silencing in different media were observed at any time point after transfection (Figure 11B).

[0289] [Example 16] B2M Silencing under Multitarget Multiplex Conditions at Multiple Introduction Timings Transducing chimeric antigen receptors (CARs) into T cells treated with silencing gRNAs may affect the efficiency of gRNA silencing, CAR expression, or both. To determine whether this is the case for the gRNAs described above, primary human T cells obtained from donors DON001, DON006, DON020, and DON023 were nucleofected on the second or third day after thawing. The T cells were also transduced with a B cell maturation antigen (BCMA) CAR on the first, second, or third day after thawing. The T cells were transduced with a combination of 2.5 μg of fusion protein 11a and pairs created from six different gRNAs. When nucleofection with gRNA was performed on the third day after thawing, more robust B2M silencing was achieved, as indicated by a decrease in the expression of B2M, HLA-DR, and CD3, when combined with transduction of the BCMA CAR. Furthermore, when the BCMA CAR was transduced on the first or second day after thawing, the expression of B2M, HLA-DR, and CD3 remained decreased compared to the third day. Different pairs of gRNAs showed diverse B2M silencing capabilities (Figure 12A).

[0290] The transduction efficiency of the BCMA CAR differed depending on the day of transduction. Transducing the BMCA CAR into T cells on the first or third day after thawing resulted in higher CAR expression than transduction on the second day after thawing (Figure 12B). B2M silencing by gRNA was more effective in CAR− cells, but B2M silencing was equally effective in CAR+ cells, as evaluated by the expression of B2M, HLA-DR, and CD3 (Figure 12C), indicating that human T cells with silenced B2M express the further transduced CAR.

[0291] [Example 17] B2M Silencing with gRNAs from Multiple Manufacturing Batches To determine whether the variability per nucleofection was due to the quality of the gRNA, primary human T cells from donors DON006 and DON023 were transfected with different batches of gRNA. Three batches of two B2M silencing gRNAs were pairwise tested in combination with 2.5 μg of effector fusion protein 11a. A representative gating strategy for nucleofected T cells is shown in Fig. 13A. All gRNA pairs resulted in significant suppression of B2M expression, but there was some batch-dependent variation in the silencing efficiency of the gRNA on day 7 after nucleofection (Fig. 13B).

[0292] [Example 18] Dosage response assay of B2M dual guide The dosage responses of 12 guide pairs were assayed at two time points. 2.5 μg of fusion protein 11a was used, and similarly, the starting dosage of each sgRNA was also 2.5 μg. The responses were observed on day 6 (Fig. 14A) and day 13 (Fig. 14B).

[0293] [Example 19] Allogeneic function assay in primary T cells The responses of allogeneic healthy donor CD8+ T cells to mock-modified T cells or B2M-silencing T cells were evaluated by a mixed lymphocyte co-culture assay.

[0294] The proliferation and / or activation of allogeneic healthy donor CD8+ T cells were evaluated by measuring the cell dye dilution and the cell surface expression of activation markers by flow cytometry, respectively, after co-culture with mock-modified T cells or B2M-silencing T cells. The decrease in the response of allogeneic T cells to B2M-silencing cells resulted in a decrease in the proliferation (measured by CellTrace Violet dilution over a 7-day assay) and activation (measured by cell surface staining for CD25 expression) of CD8+ and CD4+ T cells, and such a decrease in the response of allogeneic T cells to B2M-silencing cells was observed compared to the response to unmodified cells. The results are shown in Figs. 15A - 15B.

[0295] T cells were isolated from human leukapheresis products (StemCell Technologies, Cat. No. 70500) using the EasySep™ Human T cell Isolation Kit (StemCell Technologies, Cat. No. 17951) and cryopreserved in CryoStor® CS10 cryopreservation medium (Biolife Solutions, Cat. No. 210502). Prior to nucleofection, the T cells were thawed, washed, and stimulated in complete T cell medium (ImmunoCult™-XF T Cell Expansion Medium; StemCell Technologies, Cat. No. 10981) containing 5% heat-inactivated human AB serum (Gemini Bio-Product, Cat. No. 100-512), 2 mM L-alanyl-L-glutamine, 5 ng / mL IL-7, and 5 ng / mL IL-15 at 37 °C, 5% CO2 for approximately 72 h using Dynabeads Human T-Activator CD3 / CD28 for T Cell Expansion and Activation (Thermo Fisher, Cat. 11131D) at a bead:cell ratio of 1:1. The beads were then removed from the culture by magnetic force, and then 2.5 μg CRISPR-off mRNA + 2.5 μg sgRNA (IDT) was introduced into the T cells by nucleofection at 2 × 10 5 cells / well using the P3 Primary Cell 96 Well Nucleofector Kit (Lonza, Cat. No. V4SP-3960) and Amaxa 4D Nucleofector (Lonza) with pulse code EO115.

[0296] After nucleofection, the T cells were resuspended in complete T cell medium and maintained by medium change and subculture twice a week as needed. On day 8 after nucleofection, B2M-silencing cells were sorted and the culture was restarted and continued until the assay day. On the assay day, unedited T cells and B2M-silencing T cells were treated with 50 μg / ml mitomycin C at 37 °C for 30 minutes, then washed, and then stained with 0.5 μM CFSE in PBS at room temperature for 3 minutes, and then washed. Allogeneic PBMCs were thawed and stained with CTV by incubating in 10 mM CellTrace Violet (CTV) in PBS at 37 °C for 10 minutes, then washed. T cells and PBMCs were co-incubated for 7 days at a T cell:PBMC ratio of 1:1 in T cell medium without cytokine addition. At the end point of the assay, the cell surface expression of CD3, CD4, CD8, and CD25 was evaluated by flow cytometry of the co-culture samples. The proliferation of CD8+ T cells and CD4+ T cells in allogeneic PBMCs was evaluated by analyzing the CFSE-CD3+CD8+ or CFSE-CD3+CD4+ cell population and quantifying the number of CTV dilutions. The activation of CD8+ T cells and CD4+ T cells in allogeneic PBMCs was evaluated by analyzing the CFSE-CD3+CD8+ or CFSE-CD3+CD4+ cell population and quantifying the frequency of cell surface expression of CD25.

[0297] [Example 27] B2M triple guide screening To improve the robustness and persistence of silencing, an assay was performed in which three guides were applied to the same cells. In this example, a study of screening a triple set of gRNAs in human primary T cells is described (FIGS. 16A-C).

[0298] T cells were isolated from human leukapheresis products (StemCell Technologies, Cat. No. 70500) using the EasySep™ Human T cell Isolation Kit (StemCell Technologies, Cat. No. 17951). The T cells were thawed and activated. Prior to nucleofection, the T cells were thawed, washed, and stimulated for approximately 48 hours at 37°C and 5% CO2 in complete T cell medium (X-VIVO15 medium; Lonza, Cat. No. BEBP04-744Q) supplemented with Dynabeads Human T-Activator CD3 / CD28 for T Cell Expansion and Activation (Thermo Fisher, Cat. 11131D) at a bead:cell ratio of 3:1, 5% human AB serum (Gemini Bio-Product, Cat. No. 100-512), 2 mM L-alanyl-L-glutamine, 5 ng / mL IL-7, and 5 ng / mL IL-15. Subsequently, the beads were removed from the culture by magnetic force, and the T cells were cultured in fresh complete T cell medium for approximately 24 hours. Next, 2 × 10 5 cells / well of 2.5 μg CRISPR-off mRNA (TriLink) + 2.5 μg sgRNA (IDT) were introduced into the T cells by nucleofection using the P3 Primary Cell 96 Well Nucleofector Kit (Lonza, Cat. No. V4SP-3960) and Amaxa 4D Nucleofector (Lonza) with pulse code EO115.

[0299] After nucleofection, the T cells were resuspended in complete T cell medium and maintained by medium change and passaging twice a week as needed. On day 13 after nucleofection, the cells were restimulated with ImmunoCult™ Human CD3 / CD28 T Cell Activator (StemCell Technologies, Cat. No. 10991).

[0300] The cell surface B2M protein expression of live T cells was evaluated by flow cytometry on days 6, 13, and 20 after nucleofection. Controls without mRNA, CRISPR-off mRNA + non-B2M-targeted sgRNA, CRISPR-off mRNA without gRNA, WT Cas9 mRNA + exon-targeted sgRNA, staining only (without mRNA or gRNA), isotype (without mRNA or gRNA), and without staining (without mRNA or gRNA) were also run on each screening plate.

[0301] The B2M flow cytometry assay was performed as described in Example 5. Test samples were evaluated for the percentage of silencing by comparing their expression levels to those of a negative (CRISPR-off mRNA without sgRNA) control. The results are shown in Figures 16A-16B.

[0302] Sequence Listing The sequence numbers (SEQ) for the nucleotide (nt) and amino acid (aa) sequences described in this disclosure are shown below.

[0303] TIFF2025524456000079.tif229168TIFF2025524456000080.tif255169TIFF2025524456000081.tif252170TIFF2025524456000082.tif255169TIFF2025524456000083.tif254167TIFF2025524456000084.tif253166TIFF2025524456000085.tif254166TIFF2025524456000086.tif252166TIFF2025524456000087.tif254166TIFF2025524456000088.tif252167TIFF2025524456000089.tif252167TIFF2025524456000090.tif254167TIFF2025524456000091.tif254167TIFF2025524456000092.tif254167TIFF2025524456000093.tif252167TIFF2025524456000094.tif254167TIFF2025524456000095.tif252167TIFF2025524456000096.tif252167TIFF2025524456000097.tif253167TIFF2025524456000098.tif251167TIFF2025524456000099.tif246166TIFF2025524456000100.tif240167TIFF2025524456000101.tif255165TIFF2025524456000102.tif255166TIFF2025524456000103.tif253166TIFF2025524456000104.tif254167TIFF2025524456000105.tif255167TIFF2025524456000106.tif255167TIFF2025524456000107.tif252167TIFF2025524456000108.tif255166TIFF2025524456000109.tif253167TIFF2025524456000110.tif251167TIFF2025524456000111.tif251167TIFF2025524456000112.tif253167TIFF2025524456000113.tif241166TIFF2025524456000114.tif244167TIFF2025524456000115.tif242167TIFF2025524456000116.tif242167TIFF2025524456000117.tif241166TIFF2025524456000118.tif254166TIFF2025524456000119.tif241166TIFF2025524456000120.tif241166TIFF2025524456000121.tif244166TIFF2025524456000122.tif253165TIFF2025524456000123.tif241166TIFF2025524456000124.tif241166TIFF2025524456000125.tif241166TIFF2025524456000126.tif253167TIFF2025524456000127.tif241167TIFF2025524456000128.tif249167TIFF2025524456000129.tif242168TIFF2025524456000130.tif247167TIFF2025524456000131.tif247166TIFF2025524456000132.tif251166TIFF2025524456000133.tif241166TIFF2025524456000134.tif240166TIFF2025524456000135.tif241166TIFF2025524456000136.tif251166TIFF2025524456000137.tif241166TIFF2025524456000138.tif255166TIFF2025524456000139.tif244166TIFF2025524456000140.tif254166TIFF2025524456000141.tif249166TIFF2025524456000142.tif254166TIFF2025524456000143.tif254166TIFF2025524456000144.tif249166TIFF2025524456000145.tif255166TIFF2025524456000146.tif254166TIFF2025524456000147.tif248166TIFF2025524456000148.tif254166TIFF2025524456000149.tif249166TIFF2025524456000150.tif254167TIFF2025524456000151.tif253167TIFF2025524456000152.tif254167TIFF2025524456000153.tif255166TIFF2025524456000154.tif254167TIFF2025524456000155.tif251167TIFF2025524456000156.tif254168TIFF2025524456000157.tif253168TIFF2025524456000158.tif254167TIFF2025524456000159.tif253167TIFF2025524456000160.tif255167TIFF2025524456000161.tif254167TIFF2025524456000162.tif255167TIFF2025524456000163.tif254167TIFF2025524456000164.tif254167TIFF2025524456000165.tif249167TIFF2025524456000166.tif244168TIFF2025524456000167.tif246168TIFF2025524456000168.tif245167TIFF2025524456000169.tif245167TIFF2025524456000170.tif245166TIFF2025524456000171.tif247166TIFF2025524456000172.tif248167TIFF2025524456000173.tif244168TIFF2025524456000174.tif249164TIFF2025524456000175.tif243164TIFF2025524456000176.tif247164TIFF2025524456000177.tif243164TIFF2025524456000178.tif246164TIFF2025524456000179.tif245163TIFF2025524456000180.tif245163TIFF2025524456000181.tif244163TIFF2025524456000182.tif246163TIFF2025524456000183.tif247163TIFF2025524456000184.tif247163TIFF2025524456000185.tif245163TIFF2025524456000186.tif245163TIFF2025524456000187.tif245163TIFF2025524456000188.tif246163TIFF2025524456000189.tif245163TIFF2025524456000190.tif246160TIFF2025524456000191.tif245163TIFF2025524456000192.tif245163TIFF2025524456000193.tif245164TIFF2025524456000194.tif245163TIFF2025524456000195.tif248163TIFF2025524456000196.tif246163TIFF2025524456000197.tif245163TIFF2025524456000198.tif245163TIFF2025524456000199.tif249163TIFF2025524456000200.tif246163TIFF2025524456000201.tif246163TIFF2025524456000202.tif246163TIFF2025524456000203.tif247162TIFF2025524456000204.tif246162TIFF2025524456000205.tif246162TIFF2025524456000206.tif243162TIFF2025524456000207.tif248162TIFF2025524456000208.tif247162TIFF2025524456000209.tif246162TIFF2025524456000210.tif246162TIFF2025524456000211.tif245163TIFF2025524456000212.tif248163TIFF2025524456000213.tif247163TIFF2025524456000214.tif245164TIFF2025524456000215.tif245164TIFF2025524456000216.tif126164.

Claims

1. A system for repressing the transcription of the human B2M gene in human cells, and possibly in human T lymphocytes or human NK cells, This system a) A fusion protein comprising a DNA methyltransferase (DNMT) domain and / or a domain that recruits DNMT, and a transcriptional repressor domain, Here, the DNMT domain and / or mobilization domain may include the DNMT3A domain and / or the DNMT3L domain, and the mobilized DNMT may be DNMT3A. Each domain is linked to a DNA-binding domain, and the DNA-binding domain is dead CRISPR Cass (dCas9) domain and sequence numbers 710, 741-744, 746, 747, 749-759, 770-780, 782-849, 851-854, 856-859, 861-863, 866-871, 873-883, 885-896, 888-915, 917-931, 933-1007, 1015, 1018-1020, 1023, 1024, 1028, 1029, 1031-1077, 1083-1093, 1096, 1098, 1101, 1104, 1105, 1110-1112, 1114-1116, 1120 The fusion protein comprising one or more guide RNAs including one of the following: 1122-1124, 1126, 1135, 1137-1148, 1150-1154, 1162-1167, 1169-1171, 1174-1185, 1193, 1194, 1196-1198, 1205, 1207, 1214-1218, 1220, 1222-1233, 1246-1252, 1254, 1256, 1258-1260, 1266, 1268-1270, 1274-1276, 1278-1282 and 1735-1737; or b) One or more nucleic acid molecules encoding the aforementioned fusion protein Includes, That system does not cause DNA breaks in the B2M gene. A system for suppressing the transcription of the aforementioned human B2M gene.

2. A system for repressing the transcription of the human B2M gene in human cells, and possibly in human T lymphocytes or human NK cells, This system a) DNMT3A domain, DNMT3L domain, DNA-binding domain, and Contains transcriptional repressor domains Fusion protein, or b) Nucleic acid molecule encoding the fusion protein Includes, That system does not cause DNA breaks in the B2M gene. The DNA-binding domain includes a dead CRISPR-Cas (dCas9) domain, and comprises (i) one or more guide RNAs containing one of sequence numbers 1012-1282, or (ii) nucleic acid molecules encoding the one or more guide DNAs mentioned above. A system for suppressing the transcription of the aforementioned human B2M gene.

3. a) The dCas domain may include a dCas9 sequence and a sequence having at least 90% identity with sequence number 12 or 13; b) The DNMT3A domain contains a sequence that is at least 90% identical to sequence number 574 or 575; c) The DNMT3L domain contains a sequence that has at least 90% identity with a sequence selected from sequence numbers 578-581; The DNMT3L domain contains a sequence that has at least 90% identity with a sequence selected from sequence numbers 582-599; or The DNMT domain contains a sequence that has at least 90% identity with a sequence selected from sequence numbers 601-603; and / or, d) The transcriptional repressor domain contains a sequence that has at least 90% identity with a sequence selected from SEQ ID NOs: 33-570. The system according to claim 1 or 2.

4. The system according to claim 1 or 2, wherein the transcriptional repressor domain comprises a KRAB domain derived from KOX1, ZIM3, ZFP28, or ZN627.

5. The system according to claim 4, wherein the KRAB domain comprises a sequence having at least 90% identity with a sequence selected from sequence numbers 89, 116, 245, and 255.

6. The system according to claim 1 or 2, wherein the transcriptional repressor domain comprises a fusion of the N-terminal and C-terminal regions of ZIM3 and KOX1 KRAB, and may also comprise the amino acid sequence of SEQ ID NO: 571 or 572.

7. The system according to claim 1 or 2, wherein the transcriptional repressor domain is derived from KAP1, MECP2, HP1a / CBX5, HP1b, CBX8, CDYL2, TOX, TOX3, TOX4, EED, EZH2, RBBP4, RCOR1, or SCML2.

8. The system a) A fusion protein comprising a DNMT3A domain, a DNMT3L domain, a transcriptional repressor domain, and a DNA-binding domain, or b) Nucleic acid molecule encoding the fusion protein Includes, Here, either or both of the DNMT3A domain and the DNMT3L domain may be human domains. The DNA-binding domain may be a dCas domain or a ZFP domain. The system according to claim 1 or 2.

9. The fusion protein moves from the N-terminus to the C-terminus. a) DNMT3A domain, first peptide linker, DNMT3L domain, second peptide linker, DNA binding domain, third peptide linker, and transcriptional repressor domain; b) DNMT3A domain, first peptide linker, DNMT3L domain, second peptide linker, first nuclear localization signal (NLS), DNA binding domain, second NLS, third peptide linker, and transcriptional repressor domain; c) First nuclear localization signal (NLS), DNMT3A domain, first peptide linker, DNMT3L domain, second peptide linker, DNA binding domain, third peptide linker, transcriptional repressor domain, and second NLS; or d) comprising the DNMT3A domain, the first peptide linker, the DNMT3L domain, the second peptide linker, the DNA binding domain, the third peptide linker, the transcriptional repressor domain, and the third and fourth NLS, The system according to claim 8.

10. The system according to claim 9, wherein one or both of the second and third peptide linkers are XTEN linkers, which may be selected from XTEN80 and XTEN16, and further optionally the second peptide linker is XTEN80 and the third peptide linker is XTEN16.

11. The fusion protein moves from the N-terminus to the C-terminus. a) Human DNMT3A domain, first peptide linker, human DNMT3L domain, XTEN80 peptide linker, first NLS, dCas domain including dead Streptococcus pyogenes Cas9 (dSpCas9) domain, second NLS, XTEN16 peptide linker, and human KOX1 KRAB domain; b) Human DNMT3A domain, first peptide linker, human DNMT3L domain, XTEN80 peptide linker, first NLS, ZFP domain, second NLS, XTEN16 peptide linker, and human KOX1 KRAB domain; c) The first and second NLSs, the human DNMT3A domain, the first peptide linker, the human DNMT3L domain, the XTEN80 peptide linker, the dSpCas9 domain, the XTEN16 peptide linker, the human KOX1 KRAB domain, and the third and fourth NLSs; d) The first and second NLSs, the human DNMT3A domain, the first peptide linker, the human DNMT3L domain, the XTEN80 peptide linker, the ZFP domain, the XTEN16 peptide linker, the human KOX1 KRAB domain, and the third and fourth NLSs; e) The first and second NLSs, the human DNMT3A domain, the first peptide linker, the human DNMT3L domain, the XTEN80 peptide linker, the dSpCas9 domain, the XTEN16 peptide linker, the human ZFP28 KRAB domain, and the third and fourth NLSs; f) The first and second NLSs, the human DNMT3A domain, the first peptide linker, the human DNMT3L domain, the XTEN80 peptide linker, the ZFP domain, the XTEN16 peptide linker, the human ZFP28 KRAB domain, and the third and fourth NLSs; g) The first and second NLSs, the human DNMT3A domain, the first peptide linker, the human DNMT3L domain, the XTEN80 peptide linker, the dSpCas9 domain, the XTEN16 peptide linker, the human ZN627 KRAB domain, and the third and fourth NLSs; h) The first and second NLSs, the human DNMT3A domain, the first peptide linker, the human DNMT3L domain, the XTEN80 peptide linker, the ZFP domain, the XTEN16 peptide linker, the human ZN627 KRAB domain, and the third and fourth NLSs; i) the first and second NLS, human DNMT3A domain, first peptide linker, human DNMT3L domain, XTEN80 peptide linker, dSpCas9 domain, XTEN16 peptide linker, human ZIM3 KRAB domain, and the third and fourth NLS; or j) comprising the first and second NLS, human DNMT3A domain, first peptide linker, human DNMT3L domain, XTEN80 peptide linker, ZFP domain, XTEN16 peptide linker, human ZIM3 KRAB domain, and third and fourth NLS, The system according to claim 8.

12. The system according to claim 8, wherein the fusion protein comprises sequence number 658 or a sequence at least 90% identical thereto; sequence number 659 or a sequence at least 90% identical thereto; sequence number 660 or a sequence at least 90% identical thereto; sequence number 661 or a sequence at least 90% identical thereto; sequence number 662 or a sequence at least 90% identical thereto; sequence number 663 or a sequence at least 90% identical thereto; or sequence number 667 or a sequence at least 90% identical thereto.

13. The system according to claim 8, comprising at least one SV40 NLS.

14. The system a) A first fusion protein comprising a first DNA-binding domain and further comprising or mobilizing a DNMT3A domain, A second fusion protein containing a second DNA-binding domain and further containing or mobilizing a DNMT3L domain, and A third fusion protein comprising a third DNA-binding domain and further comprising or mobilizing a transcriptional repressor domain; or b) One or more nucleic acid molecules representing the fusion protein including, The system according to claim 1 or 2.

15. Human cells, or offspring of such cells, comprising the system according to claim 1 or 2, wherein the cells may be T lymphocytes or NK cells.

16. Human cells modified by the system according to claim 1 or 2, or offspring of such cells, wherein the cells may be T lymphocytes or NK cells, and the cells may be ex vivo modified.

17. A pharmaceutical composition comprising the system according to claim 1 or 2, and a pharmaceutically acceptable excipient, wherein the composition may comprise lipid nanoparticles (LNPs) comprising the system, and / or the DNA-binding domain may be a dCas domain, and the LNPs may further comprise one or more gRNAs.

18. A pharmaceutical composition comprising human cells and a pharmaceutically acceptable excipient as described in claim 15.

19. The system according to claim 1 or 2 for use in treating a subject that requires it.

20. The system according to claim 1 or 2 for use in the treatment of a subject having cancer or an autoimmune disease.