Gene silencing

Abstract

An engineered transcriptional modulator (ETM) comprising: (a) at least one epigenetic effector domain; operably linked to (b) an endonuclease.

Description

GENE SILENCINGCROSS REFERENCE

[0001] This application claims the benefit under 35 U. S. C. § 119(e) of U. S.Provisional Application No. 63 / 711,591, filed October 24, 2024, entitled “GENE SILENCING,” which is incorporated herein by reference in its entirety.REFE ENCE TO AN ELECTRONIC SEQUENCE LISTING

[0002] The contents of the electronic sequence listing (C169870059WO00-SEQ-AXW.xml; Size: 4,222,010 bytes; and Date of Creation: October 24, 2025) are herein incorporated by reference in their entirety.FIELD OF THE INVENTION

[0003] The present invention relates to engineered transcriptional modulators (ETM), for example engineered transcriptional repressors (ETRs), for gene editing and epigenetic modification. More specifically, the present invention relates to ETMs (e.g., ETRs) for use in multiplexing methods for modifying the expression of at least two target genes, wherein the expression of a first target gene is modified by gene editing and the expression of second target gene is modified by epigenetic modification, including during gene therapy applications.BACKGROUND TO THE INVENTION

[0004] Adoptive immunotherapy using engineered T cells has emerged as a powerful approach to treat cancer. These cells can be prepared from the patient's own blood (autologous) or derived from a different donor (allogeneic) and are redirected against cancer cells by ectopic expression of a transgenic T Cell Receptor (TCR) or a Chimeric Antigen Receptor (CAR) recognizing tumor-related antigens. TCRs and CARs may be introduced into ex vivo expanded T cells by different means, including lentiviral and retroviral vectors. These vectors, however, tend to integrate semi-randomly in the genome of T cells, posing safety concerns related to 1#14533290vltranscriptional deregulation of tumor-promoting genes. To avoid this risk, genome editing with artificial nucleases, such as CRISPR / Cas9, has been used to drive insertion of the CAR sequence into the endogenous TCR locus (J. Eyquem etal., Nature 2017 Mar 2;543(7643):113-117), an approach that also enhances T-cell potency.

[0005] Genome editing has been further used to improve efficiency and reduce toxicity of T cell therapy via the knockout of additional key genes. In this regard, the most common targets are the TCR genes (encoded by TRAC and TRBC, with the latter present in two copies in cis on the same chromosome), the β-2 microglobulin (B2M) gene, and the programmed cell death 1 (PDCD1, also referred to as PD1) gene. Inactivation of TRAC and B2M '\s believed to reduce graft-versus-host reactions, whereas inactivation of PDCD1 is used to desensitize transplanted T cells to immune dampening signals originating from the cancer cells / microenvironment.SUMMARY OF THE INVENTION

[0006] While promising, these multiplexing gene editing approaches (i.e., disruption of multiple genes per cell) come with two related issues:(i) Induction of multiple DNA breaks per cell may over-activate cellular DNA damage responses, ultimately leading to apoptosis or poor performance / fitness of the transplanted cells. In this regard, triple editing has been posed as the upper limit for multiplexing, above which significant cell toxicity can be observed.(ii) Chromosomal translocations may occur between or among multiple DNA breaks (including on- and off-target sites of the nucleases and spontaneous breaks, the latter occurring at a relatively high rate in cultured T cells), further jeopardizing safety of the approach. Clinical and preclinical studies of multiplexing in CAR-T cell products have reported alarming levels of genomic translocations (up to 5%), even when dual-gene editing approaches were used (L. Poirot etal., Cancer Res. 2015 Sep 15;75(18):3853-64; W. Qasim etal., Sci Transl Med; 2017 Jan 25;9(374); E. Stadtaumer etal., Science 2020 Feb 28;367(6481)).

[0007] Targeted epigenetic modification (such as epi-silencing) may represent a safer alternative to gene editing approaches for multiplexing in T cells. Epi-silencing exploits epigenetics, rather than DNA breaks, to inactivate its intended target gene,2#14533290vlfor example through DNA methylation at CpG sites (A. Amabile etal., Cell. 2016 Sep 22;167(1 ):219-232).

[0008] Epi-silencing may be achieved by the transient delivery of Engineered Transcriptional Repressors (ETRs), proteins comprising, for example, a catalytically disabled Cas9 (dCas9) or a transcription activator-like effector (TALE) or a Zinc-finger protein (ZFP) fused to epigenetic domains from naturally occurring epigenetic effector proteins (such as KRAB, DNMT3L and DNMT3A). The application of ETRs in silencing individual as well as multi-copy genes in cell lines and in primary T lymphocytes was reported by A. Amabile supra and T. Mlambo etal., Nucleic Acids Res. 2018 May 18;46(9):4456-4468. However, the activity of ETRs appears to preferably occur at genes that possess a CpG island (CGI), thus excluding several potentially relevant targets (e.g., TCR genes and PD1 amongst others).

[0009] Accordingly, there remains a need for the development of technologies capable of modifying multiple genes within the same cell. Technologies which reduce the number of multiple DNA breaks per cell, compared to multiplexing gene editing strategies, may be a safer approach and may avoid cellular DNA damage responses and undesired chromosomal translocations.

[0010] The present invention relates to the development of a combined gene and epigenetic editing strategy to modify multiple genes within the same cell. In particular, it exploits an engineered transcriptional modulator (ETM), for example an engineered transcriptional repressor (ETR), which comprises an epigenetic effector domain operably linked to an endonuclease (such as a catalytically active Cas9) and guide ribonucleic acids (gRNAs) of different lengths to promote permanent epigenetic editing (e.g., silencing) of one or more genes and genetic editing (e.g., inactivation) of another gene.

[0011] This orthogonal approach overcomes the genotoxic risks associated with the use of nuclease-mediated genome editing technologies to inactivate multiple genes per cell. Advantageously, the present invention enables targeting of genes that may be more challenging to achieve with targeted epigenetic modification, enabling targeting of both genes having a CpG island (CGI) and genes which do not have a CGI in one multiplexing strategy.3#14533290vl

[0012] Thus, the present invention provides a combined strategy of gene editing coupled to epigenetic modification, such as epigenetic silencing. This combination will:(i) reduce the burden of genomic translocations compared to multiplexing gene editing methods. The target selected for gene editing will typically lack a CGI. This gene may be also used as a target site for insertion of exogenous expression cassettes encoding, for example, tumour restricted TCRs or CARs introduced with homologous recombination; and(ii) utilise epigenetics to modify, e.g., silence, one or more CGI-containing genes.(iii) allow the use of the same construct (an ETM) to achieve silencing in two different modalities, thus reducing the amount of gene editor-encoding RNA that needs to be added to the cell for correct silencing. An advantage of the present invention is to reduce the number of constructs required for multiplex modification, thus improving efficiency and decreasing manufacturing costs.

[0013] Suitably, gene editing may be limited to one gene (which lacks CGI) and at least one gene (such as at least two, or at least three or more genes) comprising a CGI may be modified epigenetically.

[0014] Overall, development of such a combined strategy will result in safer and more efficient T cell products for adoptive immunotherapy of cancer.

[0015] In an aspect, the present invention provides an engineered transcriptional modulator (ETM) comprising: a) at least one epigenetic effector domain; operably linked to b) an endonuclease.

[0016] In certain embodiments, the ETM is an engineered transcriptional repressor (ETR). In some embodiments, the ETM is an engineered transcriptional activator (ETA).

[0017] In some embodiments, the ETM (e.g., ETR) comprises one, two or three epigenetic effector domains. In some embodiments, the ETM (e.g., ETR) comprises one epigenetic effector domain. In some embodiments, the ETM (e.g., ETR) comprises two epigenetic effector domains. In some embodiments, the ETM (e.g., ETR) comprises three epigenetic effector domains.4#14533290vl

[0018] In some embodiments, the at least one epigenetic effector domain comprises a Kruppel-associated box (KRAB) domain, a DNA methyltransferase (DNMT) domain, a DNMT-like domain, and / or a histone methyltransferase (HMT) domain. In some embodiments, the epigenetic effector domain is a transcriptional repressor domain (e.g., a Kruppel-associated box (KRAB) domain).

[0019] In some embodiments, the at least one epigenetic effector domain is selected from the group consisting of: DNMT1, DNMT3A, DNMT3B, DNMT3L and SETDB1.

[0020] In some embodiments, the ETM (e.g., ETR) comprises a first epigenetic effector domain comprising a KRAB domain and a second epigenetic effector domain comprising a DNMT domain. In some embodiments, the ETM (e.g., ETR) comprises a first epigenetic effector domain comprising a KRAB domain and a second epigenetic effector domain comprising a DNMT-like domain. In some embodiments, the ETM (e.g., ETR) comprises a first epigenetic effector domain comprising a KRAB domain, a second epigenetic effector domain comprising a DNMT domain, and a third epigenetic effector domain comprising a DNMT-like domain. In certain embodiments, the ETM may comprise as epigenetic effector domains KRAB and DNMT3A; KRAB and DNMT3L; or KRAB, DNMT3A, and DNMT3L. In some embodiments, the ETM (e.g., ETR) comprises a transcriptional repressor domain (e.g., a Kruppel-associated box (KRAB) domain) and a DNMT3L domain. In some embodiments, the ETM (e.g., ETR) comprises a transcriptional repressor domain (e.g., a Kruppel-associated box (KRAB) domain), a DNMT3A domain and a DNMT3L domain.

[0021] In some embodiments, the endonuclease comprises an RNA binding domain.

[0022] In some embodiments, the endonuclease is a Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) / Cas system.

[0023] In some embodiments, the endonuclease is a Cas endonuclease.

[0024] In certain embodiments, the endonuclease is a Cas9 endonuclease. In certain embodiments, the endonuclease is a SpCas9 endonuclease.

[0025] In some embodiments, the ETM (e.g., ETR) comprises or consists of a Cas9-KRAB, Cas9-DNMT3A or Cas9-DNMT3L fusion protein, which can be used together.5#14533290vl

[0026] In some embodiments, the ETM (e.g., ETR) is a bi- or tri-partite fusion protein.

[0027] In another aspect, the present invention provides a gRNA which comprises a spacer sequence which comprises or consists of the sequence of any one of SEQ ID NOs: 23-46, 562-1076, 2778-4478, or 4553-4565 or a homologue or fragment thereof. In another aspect, the present invention provides a gRNA which comprises a spacer sequence which comprises or consists of the sequence of any one of SEQ ID NOs: 23-46, 562-1076, 2778-4478, and 4553-4565 or a homologue or fragment thereof.

[0028] In another aspect, the spacer sequence consists of a fragment of any one of SEQ ID NOs: 23-46, 562-1076 or 2778-4478, such as a 10, 11, 12,13, 14, 15,16, 17, 18, 19 or 20 nucleotide fragment of any one of SEQ ID NOs: 23-46, 562-1076, 2778-4478 or 4553-4565. In another aspect, the spacer sequence consists of a fragment of any one of SEQ ID NOs: 23-46, 562-1076, 2778-4478, and 4553-4565, such as a 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 nucleotide fragment of any one of SEQ ID NOs: 23-46, 562-1076, 2778-4478, and 4553-4565. The fragment may be a truncation of the sequence from the 5’ end.

[0029] In another aspect, the spacer sequence consists of a fragment of any one of SEQ ID NOs: 23-46, 562-1076 or 2778-4478, such as at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19 or at least 20 continuous nucleotides of any one of SEQ ID NOs: 23-46, 562-1076 or 2778-4478. In another aspect, the spacer sequence consists of a fragment of any one of SEQ ID NOs: 23-46, 562-1076, 2778-4478, and 4553-4565, such as at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19 or at least 20 continuous nucleotides of any one of SEQ ID NOs: 23-46, 562-1076, 2778-4478, and 4553-4565.

[0030] In another aspect, the present invention provides a combination (e.g., a system) comprising an ETM (e.g., ETR) according to the present invention, and at least one gRNA. The gRNA(s) may target the ETM (e.g., ETR) to one or more target gene(s). In another aspect, the present invention provides a combination (e.g., a system) comprising an ETM (e.g., ETR) according to the present invention, or polynucleotide(s) encoding therefor, and at least one gRNA, or polynucleotides coding therefor. The combination may comprise one or more ETMs (e.g., ETRs)6#14533290vlaccording to the present invention, such as one, two or three ETMs (e.g., ETRs), or polynucleotides encoding therefor.

[0031] In some embodiments, each ETM is a fusion protein comprising a catalytically active CRISPR / Cas endonuclease domain.

[0032] In another aspect, the present invention provides a combination for modifying transcription, expression and / or activity of one or more (e.g. two or more) gene in a cell, the combination comprising: (A) one or more fusion proteins each comprising a catalytically active CRISPR / Cas endonuclease domain, wherein the one or more fusion proteins collectively comprise a transcriptional repressor domain and a DNMT3L domain, or polynucleotide(s) encoding the one or more fusion proteins; (B) one or more guide RNAs (gRNAs) having a spacer sequence with a length that allows epigenetic editing and not gene editing of a first gene in the cell, wherein the first gene comprises a CpG island (CGI), or polynucleotide(s) coding for the one or more gRNAs; and (C) one or more gRNAs having a spacer sequence with a length that allows gene editing of a second gene in the cell, or polynucleotide(s) coding for the one or more gRNAs.

[0033] In some embodiments, at least one epigenetic effector domain is a transcriptional repressor domain e.g. a Kruppel-associated box (KRAB) domain), and / or at least one epigenetic effector domain is a DNMT3L domain. In some embodiments, at least one epigenetic effector domain is a transcriptional repressor domain (e.g. a Kruppel-associated box (KRAB) domain), at least one epigenetic effector domain is a DNMT3A domain, and / or at least one epigenetic effector domain is a DNMT3L domain.

[0034] In some embodiments, the one or more ETMs collectively comprise a transcriptional repressor domain (e.g. a Kruppel-associated box (KRAB) domain) and a DNMT3L domain. In some embodiments, the one or more ETMs collectively comprise a transcriptional repressor domain (e.g. a Kruppel-associated box (KRAB) domain), a DNMT3A domain and a DNMT3L domain.

[0035] In some embodiments, the spacer sequence is less than or equal to 16 nucleotides in length. In some embodiments, the spacer sequence is 11 to 16 nucleotides in length, such as 12 to 16, 13 to 16, 14 to 16 or 15 to 16 nucleotides in length.7#14533290vl

[0036] In some embodiments, the spacer sequence is 17 or more nucleotides in length, such as 18 or more, 19 or more, or 20 or more nucleotides in length. In some embodiments, the spacer sequence is 17 to 30 nucleotides in length, such as 18 to 30, 19 to 30 or 20 to 30 nucleotides in length. In some embodiments, the spacer sequence is 17 to 25 nucleotides in length, such as 18 to 25, 19 to 25 or 20 to 25 nucleotides in length. In some embodiments, the spacer sequence is 17 to 20 nucleotides in length, such as 18 to 20 or 19 to 20 nucleotides in length.

[0037] In some embodiments, the spacer sequence is less than or equal to 17 nucleotides in length. In some embodiments, the spacer sequence is 11 to 17 nucleotides in length, such as 12 to 17, 13 to 17, 14 to 17, 15 to 17, 16 to 17, 12 to 16, 13 to 16, 14 to 16, or 15 nucleotides in length. In some embodiments, the one or more gRNAs in (B) has a spacer sequence of less than or equal to 17 nucleotides. In some embodiments, the one or more gRNAs in (B) has a spacer sequence of 11 to 17 nucleotides, such as 12 to 17, 13 to 17, 14 to 17, 15 to 17, 16 to 17, 12 to 16, 13 to 16, 14 to 16, or 15 nucleotides.

[0038] In some embodiments, the spacer sequence is 18 or more nucleotides in length, such as 19 or more, or 20 or more nucleotides in length. In some embodiments, the spacer sequence is 18 to 30 nucleotides in length, such as 19 to 30 or 20 to 30 nucleotides in length. In some embodiments, the spacer sequence is 18 to 25 nucleotides in length, such as 19 to 25 or 20 to 25 nucleotides in length. In some embodiments, the spacer sequence is 18 to 21 nucleotides in length, such as 19 to 21 or 20 to 21 nucleotides in length. In some embodiments, the spacer sequence is 18 to 20 nucleotides in length, such as 19 to 20 nucleotides in length. In some embodiments, the one or more gRNAs in (C) has a spacer sequence of 18 or more nucleotides, such as 19 or more, or 20 or more nucleotides. In some embodiments, the one or more gRNAs in (C) has a spacer sequence of 18 to 30 nucleotides, such as 19 to 30 or 20 to 30 nucleotides. In some embodiments, the one or more gRNAs in (C) has a spacer sequence of 18 to 25 nucleotides, such as 19 to 25 or 20 to 25 nucleotides. In some embodiments, the one or more gRNAs in (C) has a spacer sequence of 18 to 21 nucleotides, such as 19 to 21 or 20 to 21 nucleotides. In some embodiments, the one or more gRNAs in (C) has a spacer sequence of 18 to 20 nucleotides, such as 19 to 20 nucleotides.8#14533290vl

[0039] In certain embodiments, the combination comprises at least two gRNAs. Suitably, the combination may comprise two gRNAs. Suitably, the combination may comprise three, four, five, six, seven or eight gRNAs.

[0040] The at least two gRNAs may target the ETM (e.g., ETR) to different target genes. For example, a first gRNA may target the ETM (e.g., ETR) to a first target gene and a second gRNA may target the ETM (e.g., ETR) to a second target gene. A third gRNA may, for example, target the ETM (e.g., ETR) to a third target gene. Additional gRNAs may target the ETM (e.g., ETR) to additional target genes.

[0041] In some embodiments, one target gene may be targeted with two or more gRNAs. For example, it may be beneficial to target the same gene with several gRNAs for optimal epigenetic modification e.g., epigenetic silencing. A second target gene may be targeted with another gRNA.

[0042] In particular embodiments, the at least two gRNAs comprise spacer sequences of different lengths.

[0043] In some embodiments, at least one gRNA (e.g., one, two, three or more gRNAs) may have a spacer sequence with a length that allows epigenetic editing of a target gene by the ETM and / or at least one gRNA may have a spacer sequence with a length that allows gene editing of a target gene by the ETM.

[0044] In some embodiments, a first gRNA may have a spacer sequence with a length that allows epigenetic editing of a first target gene by the ETM and a second gRNA may have a spacer sequence with a length that allows gene editing of a second target gene by the ETM.

[0045] In some embodiments, at least one gRNA (e.g., one, two, three or more gRNAs) may have a spacer sequence with a length that allows epigenetic editing and not gene editing of a target gene by the ETM and / or at least one gRNA may have a spacer sequence with a length that allows gene editing of another target gene by the ETM.

[0046] In some embodiments, a first gRNA may have a spacer sequence with a length that allows epigenetic editing and not gene editing of a first target gene by the ETM and a second gRNA may have a spacer sequence with a length that allows gene editing of a second target gene by the ETM.

[0047] Suitably, at least one gRNA(s) may comprise a spacer sequence which is 15, 16, 17, 18, 19 or 20 nucleotides in length.9#14533290vl

[0048] Suitably, one of the at least two gRNAs may comprise a spacer sequence which is less than or equal to 17 (e.g., less than or equal to 16) nucleotides in length.

[0049] In some embodiments, the combination comprises:(a) a first gRNA comprises a spacer sequence which is less than or equal to 16 nucleotides in length, such as less than or equal to 15, less than or equal to 14, less than or equal to 13 or less than or equal to 12 nucleotides in length; and / or(b) a second gRNA comprises a spacer sequence which is 17 or more nucleotides in length, such as 18 or more, 19 or more, or 20 or more nucleotides in length.

[0050] In some embodiments, the combination comprises:(a) a first gRNA comprises a spacer sequence which is 11 to 16 nucleotides in length, such as 12 to 16, 13 to 16, 14 to 16 or 15 to 16 nucleotides in length; and / or(b) a second gRNA comprises a spacer sequence which is 17 to 30 nucleotides in length, such as 18 to 30, 19 to 30, 20 to 30, 17 to 25, 18 to 25, 19 to 25, 20 to 25, 17 to 20, 18 to 20 or 19 to 20 nucleotides in length.

[0051] In some embodiments, the combination comprises:(a) a first gRNA comprises a spacer sequence which is less than or equal to 17 nucleotides in length, such as less than or equal to 16, less than or equal to 15, less than or equal to 14, less than or equal to 13, less than or equal to 12 nucleotides, or equal to 11 nucleotides in length; and / or(b) a second gRNA comprises a spacer sequence which is 18 or more nucleotides in length, such as 19 or more, or 20 or more nucleotides in length.

[0052] In some embodiments, the combination comprises:10#14533290vl(a) a first gRNA comprises a spacer sequence which is 11 to 17 nucleotides in length, such as 12 to 17 (e.g., 12 or 16), 13 to 17 (e.g., 13 to 16), 14 to 17 (e.g., 14 to 16), 15 to 17 (e.g., 16), or 17 nucleotides in length; and / or(b) a second gRNA comprises a spacer sequence which is 18 to 30 nucleotides in length, such as 19 to 30, 20 to 30, 18 to 25, 19 to 25, 20 to 25, 18 to 20, or 19 to 20 nucleotides in length.

[0053] In some embodiments, the one or more guide RNAs (gRNAs) having a spacer sequence with a length that allows epigenetic editing and not gene editing of a first gene in the cell has a spacer sequence of:(a) less than or equal to 17 nucleotides (e.g., less than or equal to 16 nucleotides), such as less than or equal to 15, less than or equal to 14, less than or equal to 13, less than or equal to 12 nucleotides, or equal to 11 nucleotides; or(b) 11 to 17 nucleotides (e.g., 11 to 16 nucleotides), such as 12 to 17 (e.g., 12 or 16), 13 to 17 (e.g., 13 to 16), 14 to 17 (e.g., 14 to 16), 15 to 17 (e.g., 16), or 17 nucleotides.

[0054] In some embodiments, the one or more gRNAs having a spacer sequence with a length that allows gene editing of a second gene in the cell has a spacer sequence of:(a) 17 or more nucleotides (e.g., 18 or more nucleotides), such as 19 or more, or 20 or more nucleotides; or(b) 17 to 30 nucleotides, such as 18 to 30, 19 to 30, 20 to 30, 18 to 25, 19 to 25, 20 to 25, 18 to 20, or 19 to 20 nucleotides, optionally 18 to 25 nucleotides (e.g., 18 to 21 nucleotides).

[0055] In some embodiments, the at least one target gene is selected from: genes without CpG Islands (CGI), such as: TRAC; TRBC; PDCD1; TIM-3; TIGIT; LAG3; CTLA4; AAVS1; RFX5; CD3D; CD3G and CCR5; and / or genes having CGI, such as: B2M; TET2; TGFBR2; A2AR; CISH; PTPN11; PTPN6; PTPA; PTPN2; JUNB; TOX; TOX2; NR4A1; NR4A2; NR4A3; MAP4K1; REL; IRF4; DGKA; PIK3CD; HLA-A;USP16; DCK; CD3Z; CIITA; ADORA2A; RFXAPand FAS. For example, the target genes may comprise one or more of B2M, TRAC, TET2, and TGFBR2. In some 11#14533290vlembodiments, the target genes may comprise, e.g., B2M and TRAC. In some embodiments, the target genes may comprise, e.g., B2M, TRAC, TET2, and TGFBR2. In some embodiments, the target genes may comprise a combination of B2M, TET2, and TRAC; a combination of B2M, TET2, and TGFBR2; a combination of B2M, TGFBR2 and TRAC; or a combination of TET2, TGFBR2, and TRAC.

[0056] In some embodiments, the first gene is selected from B2M, TET2, TGFBR2, A2AR, CISH, PTPN11, PTPN6, PTPA, PTPN2, JUNB, TOX, TOX2, NR4A1, NR4A2, NR4A3, MAP4K1, REL, IRF4, DGKA, PIK3CD, HLA-A, USP16, DCK, CD3Z; CIITA; ADORA2A; RFXAP and FAS; and / or the second gene is selected from TRAC, TRBC, PDCD1, TIM-3, TIGIT, LAG3, CTLA4, AAVS1, RFX5, CD3D, CD3G and CCR5.

[0057] In some embodiments, the second gene is a TRAC gene, optionally wherein the one or more gRNAs targeting the TRAC gene comprise a spacer having the sequence of one of SEQ ID NOs: 562-611, optionally SEQ ID NO: 604.

[0058] In some embodiments, the first gene is a B2M gene, optionally wherein the one or more gRNAs targeting the B2M gene each comprise a spacer having the sequence of one of SEQ ID NOs: 28-33 and 39-44; or the sequence of one of SEQ ID NOs: 2778-2878 with a 3 to 9 nucleotide truncation at the 5’ end, optionally one of SEQ ID NOs: 2778, 2780, 2801, and 2863 with a 3 to 9 nucleotide truncation at the 5’ end, selected from SEQ ID NOs: 4486-4492, 4497-4503, 4508-4514, and 4519-4525.

[0059] In some embodiments, the first gene is a TGFBR2 gene, optionally wherein the one or more gRNAs targeting the TGFBR2 gene each comprise a spacer having the sequence of one of SEQ ID NOs: 2929-2978 and 4553-4559 with a 3 to 9 nucleotide truncation at the 5’ end.

[0060] In some embodiments, the first gene is a TET2 gene, optionally wherein the one or more gRNAs targeting the TET2 gene each comprise a spacer having the sequence of one of SEQ ID NOs: 4429-4478 and 4560-4565 with a 3 to 9 nucleotide truncation at the 5’ end.

[0061] In some embodiments, the combination is for modifying transcription, expression and / or activity of one or more e.g. two or more) gene in a cell, wherein the cell is a mammalian cell, optionally a human cell, optionally wherein the cell is a human immune cell or human T cell.12#14533290vl

[0062] In some embodiments, the combination further comprises a donor DNA (e.g., a donor template nucleic acid) comprising 5’ and 3’ arms that are homologous to sequences in the second gene.

[0063] In some embodiments, the combination further comprises an agent:i) which promotes the survival, proliferation and / or activity of a cell, such as a cell which comprises the combination or a cell which does not comprise the combination; and / orii) which is detrimental to the survival, proliferation, activity, chemoresistance and / or chemotaxis of a cell, such as a cell which comprises the combination or a cell which does not comprise the combination; and / oriii) which enables selection of a cell, such as a cell which comprises the combination or a cell which does not comprise the combination. In some embodiments, the agent is a CAR or transgenic TCR. In some embodiments, the agent is FIX.

[0064] In another aspect the invention provides a combination for regulating one or more gene in a human cell, optionally an immune cell or a T cell, the combination comprising:one or more (e.g. one to three) fusion proteins each comprising a catalytically inactive Cas9, optionally SpCas9, endonuclease domain, wherein the one or more e.g. one to three) fusion proteins collectively comprise a transcriptional repressor and a DNMT3L domain, or polynucleotide(s) encoding the one ore more (e.g. one to three) fusion proteins, wherein the gene comprises a CpG island (CGI) and is(i) a B2M gene and the combination further comprises two or more gRNAs each comprising a spacer having the sequence of one of SEQ ID NOs: 2778-2878 optionally with a 1 to 9 nucleotide truncation at the 5’ end, or comprises polynucleotide(s) coding for the gRNAs;(ii) a TGFBR2gene and the combination further comprises a gRNA that comprises a spacer having the sequence of any one of SEQ ID NOs: 2929-2978 and 4553-4559 optionally with a 1 to 9 nucleotide truncation at the 5’ end, or comprises polynucleotide(s) coding for the gRNA; or13#14533290vl(iii) a TET2 gene and the combination further comprises a gRNA that comprises a spacer having the sequence of any one of SEQ ID NOs: 4429-4478 and 4560-4565 optionally with a 1 to 9 nucleotide truncation at the 5’ end, or comprises polynucleotide(s) coding for the gRNA.

[0065] In some embodiments, the combination comprises at least one gRNA according to the present invention. In some embodiments, the combination comprises one or more gRNAs comprising one or more gRNA sequences shown in Table 8. In some embodiments, the present disclosure provides a combination for regulating a gene comprising one or more gRNAs comprising one or more gRNA sequences shown in Table 8.

[0066] In some embodiments, the gene comprising a CGI is a B2M gene and the gRNAs targeting it are two or three gRNAs each independently comprising a spacer having the sequence of: C8 (SEQ ID NO: 35), F4 (SEQ ID NO: 24), H8 (SEQ ID NO: 2780), H10 (SEQ ID NO: 2863), H11 (SEQ ID NO: 2778), or H12 (SEQ ID NO:2801 ), optionally with a 1 to 9, optionally 3 to 9, nucleotide truncation at the 5’ end.

[0067] In some embodiments, the B2M-targeting gRNAs comprise a gRNA comprising a spacer having the sequence of F4 optionally with a 1 to 9, optionally 3 to 9, nucleotide truncation at the 5’ end, a gRNA comprising a spacer having the sequence of H8 optionally with a 1 to 9, optionally 3 to 9, nucleotide truncation at the 5’ end, and a gRNA comprising a spacer having the sequence of H10 optionally with a 1 to 9, optionally 3 to 9, nucleotide truncation at the 5’ end.

[0068] In some embodiments, the B2M-targeting gRNAs comprise a gRNA comprising a spacer having the sequence of C8 optionally with a 1 to 9, optionally 3 to 9, nucleotide truncation at the 5’ end, a gRNA comprising a spacer having the sequence of H8 optionally with a 1 to 9, optionally 3 to 9, nucleotide truncation at the 5’ end, and a gRNA comprising a spacer having the sequence of H10 optionally with a 1 to 9, optionally 3 to 9, nucleotide truncation at the 5’ end.

[0069] In some embodiments, the B2M-targeting gRNAs comprise a gRNA comprising a spacer having the sequence of F4 optionally with a 1 to 9, optionally 3 to 9, nucleotide truncation at the 5’ end, and a gRNA comprising a spacer having the sequence of H8 optionally with a 1 to 9, optionally 3 to 9, nucleotide truncation at the 5’ end.14#14533290vl

[0070] In some embodiments, the B2M-targeting gRNAs comprise a gRNA comprising a spacer having the sequence of F4 optionally with a 1 to 9, optionally 3 to 9, nucleotide truncation at the 5’ end, and a gRNA comprising a spacer having the sequence of H10 optionally with a 1 to 9, optionally 3 to 9, nucleotide truncation at the 5’ end.

[0071] In some embodiments, the B2M-targeting gRNAs comprise a gRNA comprising a spacer having the sequence of H8 optionally with a 1 to 9, optionally 3 to 9, nucleotide truncation at the 5’ end, and a gRNA comprising a spacer having the sequence of H10 optionally with a 1 to 9, optionally 3 to 9, nucleotide truncation at the 5’ end.

[0072] In some embodiments, the gene comprising a CGI is a TGFBR2 gene and the combination comprises one or more gRNAs targeting it, or coding sequences of the one or more gRNAs, the one or more gRNAs each independently comprising a spacer having the sequence ofTG1 (SEQ ID NO: 4553),TG2 (SEQ ID NO: 4554),TG3 (SEQ ID NO: 4555),TG4 (SEQ ID NO: 4556),TG5 (SEQ ID NO: 4557),TG6 (SEQ ID NO: 2940),TG7 (SEQ ID NO: 2937),TG8 (SEQ ID NO: 2930),TG9 (SEQ ID NO: 2955),TG10 (SEQ ID NO: 4558),TG11 (SEQ ID NO: 2957),TG12 (SEQ ID NO: 2929),TG13 (SEQ ID NO: 4559),TG14 (SEQ ID NO: 2945),TG15 (SEQ ID NO: 2931),TG16 (SEQ ID NO: 2942),TG17 (SEQ ID NO: 2939),TG18 (SEQ ID NO: 2935),TG19 (SEQ ID NO: 2938), or15#14533290vlTG20 (SEQ ID NO: 2932),optionally with a 1 to 9, optionally 3 to 9, nucleotide truncation at the 5’ end.

[0073] In some embodiments, the TGFBR2-targeting gRNAs comprise(i) a gRNA comprising a spacer having the sequence of TG7 optionally with a 1 to 9, optionally 3 to 9, nucleotide truncation at the 5’ end, anda gRNA comprising a spacer having the sequence of TG8 optionally with a 1 to 9, optionally 3 to 9, nucleotide truncation at the 5’ end; or(ii) a gRNA comprising a spacer having the sequence of TG19 optionally with a 1 to 9, optionally 3 to 9, nucleotide truncation at the 5’ end, anda gRNA comprising a spacer having the sequence of TG20 optionally with a 1 to 9, optionally 3 to 9, nucleotide truncation at the 5’ end.

[0074] In some embodiments, the gene comprising a CGI is a TET2 gene and the combination comprises one or more gRNAs targeting it, or coding sequences of the one or more gRNAs, the one or more gRNAs each independently comprising a spacer having the sequence ofTE1 (SEQ ID NO: 4560),TE2 (SEQ ID NO: 4561),TE3 (SEQ ID NO: 4562),TE4 (SEQ ID NO: 4563),TE5 (SEQ ID NO: 4443),TE6 (SEQ ID NO: 4434),TE7 (SEQ ID NO: 4466),TE8 (SEQ ID NO: 4438),TE9 (SEQ ID NO: 4429),TE10 (SEQ ID NO: 4469),TE11 (SEQ ID NO: 4564),TE12 (SEQ ID NO: 4449),TE13 (SEQ ID NO: 4433),TE14 (SEQ ID NO: 4442),TE15 (SEQ ID NO: 4430),TE16 (SEQ ID NO: 4431),TE17 (SEQ ID NO: 4474),TE18 (SEQ ID NO: 4432),16#14533290vlTE19 (SEQ ID NO: 4565), orTE20 (SEQ ID NO: 4478),optionally with a 1 to 9, optionally 3 to 9, nucleotide truncation at the 5’ end.

[0075] In some embodiments, the TET2-targeting gRNAs comprise(i) a gRNA comprising a spacer having the sequence of TE13 optionally with a 1 to 9, optionally 3 to 9, nucleotide truncation at the 5’ end, anda gRNA comprising a spacer having the sequence of TE14 optionally with a 1 to 9, optionally 3 to 9, nucleotide truncation at the 5’ end; or(ii) a gRNA comprising a spacer having the sequence of TE19 optionally with a 1 to 9, optionally 3 to 9, nucleotide truncation at the 5’ end, anda gRNA comprising a spacer having the sequence of TE20 optionally with a 1 to 9, optionally 3 to 9, nucleotide truncation at the 5’ end.

[0076] In some embodiments, the ETM(s) (e.g., one or more fusion proteins) collectively further comprise a DNMT1, DNMT3A, DNMT3B, or SETDB1 domain, optionally DNMT3A.

[0077] In some embodiments, the combination comprises: (i) a first fusion protein comprising a transcriptional repressor domain and a Gas endonuclease domain, and a second fusion protein comprising a DNMT3L domain and a Gas endonuclease domain, or (ii) a fusion protein comprising, optionally from N-terminus to C-terminus, a transcriptional repressor domain, a Gas endonuclease domain, and a DNMT3L domain.

[0078] In some embodiments, the combination comprises (i) a first fusion protein comprising a transcriptional repressor domain and a Gas endonuclease domain, a second fusion protein comprising a DNMT3L domain and a Gas endonuclease domain, and a third fusion protein comprising a DNMT3A domain and a Gas endonuclease domain, or (ii) a fusion protein comprising a transcriptional repressor domain, a Gas endonuclease domain, a DNMT3L domain, and a DNMT3A domain.

[0079] In some embodiments, the epigenetic effector domain (e.g. transcriptional repressor domain) is a Kruppel-associated box (KRAB) domain, optionally derived from human Kox1 or ZIM3.

[0080] In some embodiments, the combination comprises a fusion protein comprising, optionally from N terminus to C terminus, a KRAB domain derived from17#14533290vlZIM3, a catalytically active Cas9 domain, and a DNMT3L domain, optionally comprising an amino acid sequence of SEQ ID NO: 4482.

[0081] In some embodiments, the combination further comprises gRNAs for targeting one or more additional genes in the cell, optionally wherein the combination comprises gRNAs targeting the following genes, or comprises polynucleotides coding for the gRNAs: (i) B2M and TRAC, (ii) B2M, TRAC, and TGFBR2, (iii) B2M, TRAC, and TET2, (iv) B2M, TGFBR2, and TET2, or (v) B2M, TGFBR2, TET2, and TRAC.

[0082] In some embodiments, the gRNA(s) are chemically modified, optionally wherein the chemically modified gRNA(s) comprise phosphorothioate internucleoside linkages at the 5’ and / or 3’ ends, and / or 2’-O-methyl nucleotides.

[0083] In a further aspect, the present invention provides a polynucleotide encoding at least one ETM (e.g., ETR) according to the present invention.

[0084] In another aspect, the present invention provides a nucleic acid construct comprising a nucleic acid sequence encoding at least one ETM (e.g., ETR) according to the present invention.

[0085] In some embodiments, the nucleic acid construct further comprises a nucleic acid sequence:i) which promotes the survival, proliferation and / or activity of a cell, such as a cell which expresses said nucleic acid construct or a cell which does not express said nucleic acid construct; and / orii) which is detrimental to the survival, proliferation, activity, chemoresistance and / or chemotaxis of a cell, such as a cell which expresses said nucleic acid construct or a cell which does not express said nucleic acid construct; and / oriii) which enables selection of a cell, such as a cell which comprises the nucleic acid construct or a cell which does not comprise the construct.

[0086] In an aspect, the present invention provides a vector comprising a polynucleotide according to the present invention or a nucleic acid construct according to the present invention.18#14533290vl

[0087] In another aspect, the present invention provides a kit of polynucleotides comprising:a) at least one polynucleotide encoding at least one ETM (e.g., ETR) according to the present invention; andb) a polynucleotide providing at least one gRNA disclosed herein; and optionally,c) a further polynucleotide comprising a nucleic acid sequence which encodes an agent:i) which promotes the survival, proliferation and / or activity of a cell, such as a cell which comprises the polynucleotides or a cell which does not comprise the polynucleotides; and / orii) which is detrimental to the survival, proliferation, activity, chemoresistance and / or chemotaxis of a cell, such as a cell which comprises said polynucleotides or a cell which does not comprise said polynucleotides; and / oriii) which enables selection of a cell, such as a cell which comprises the polynucleotides or a cell which does not comprise the polynucleotides.

[0088] In another aspect, the present invention provides a cell (such as an engineered cell) comprising an ETM (e.g., ETR) according to the present invention, at least one gRNA according to the present invention, a combination according to the present invention, a polynucleotide according to the present invention, a nucleic acid construct according to the present invention, a vector according to the present invention or a kit of polynucleotides according to the present invention. In another aspect, the invention provides a progeny of the cell.

[0089] In another aspect, the invention provides a cell obtained by the use or method of the invention, or a progeny thereof.

[0090] In some embodiments, the cell is a human T cell, optionally engineered to express a recombinant antigen receptor, optionally selected from a recombinant T cell receptor (TCR) or a chimeric antigen receptor (CAR).19#14533290vl

[0091] In a further aspect, the present invention provides a composition comprising an ETM (e.g., ETR) according to the present invention, at least one gRNA according to the present invention, a combination according to the present invention, a polynucleotide according to the present invention, a nucleic acid construct according to the present invention, a vector according to the present invention, a kit of polynucleotides according to the present invention or a cell according to the present invention.

[0092] In another aspect, the present invention provides a pharmaceutical composition comprising an ETM (e.g., ETR) according to the present invention, at least one gRNA according to the present invention, a combination according to the present invention, a polynucleotide according to the present invention, a nucleic acid construct according to the present invention, a vector according to the present invention, a kit of polynucleotides according to the present invention or a cell according to the present invention.

[0093] In a further aspect, the present invention provides the use of an ETM (e.g., ETR) according to the present invention, at least one gRNA according to the present invention, a combination according to the present invention, a polynucleotide according to the present invention, a nucleic acid construct according to the present invention, a vector according to the present invention, a kit of polynucleotides according to the present invention or a cell according to the present invention for modifying the transcription, expression and / or activity at least one target gene. The use may, for example, be in vitro or ex vivo use.

[0094] In another aspect, the present invention provides a method of modifying the transcription, expression and / or activity of at least one target gene in a cell comprising the step of administering an ETM (e.g., ETR) according to the present invention, at least one gRNA according to the present invention, a combination according to the present invention, a polynucleotide according to the present invention, a nucleic acid construct according to the present invention, a vector according to the present invention or a kit of polynucleotides according to the present invention to a cell. The cell may be, for example, a T cell.

[0095] In some embodiments, the modifying the transcription, expression and / or activity is repressing transcription, expression and / or activity, e.g., silencing.20#14533290vl

[0096] In some embodiments, the method comprises repressing the transcription and / or expression of at least two different target genes in a cell.

[0097] In some embodiments, the method comprises silencing at least two different target genes in a cell.

[0098] Suitably, transcription and / or expression of at least one of the at least two target genes may be epigenetically repressed (e.g., silenced) and at least one of the at least two target genes may be repressed (e.g., silenced) by gene editing, wherein at least one ETM (e.g., ETR) and at least two gRNAs are administered to said cell simultaneously, sequentially, or separately.

[0099] In an aspect, an ETM (e.g., ETR) according to the present invention, at least one gRNA according to the present invention, a combination according to the present invention, a polynucleotide according to the present invention, a nucleic acid construct according to the present invention, a vector according to the present invention, a kit of polynucleotides according to the present invention, a cell according to the present invention or a pharmaceutical composition according to the present invention may be for use in therapy.

[0100] In another aspect the invention provides use of an ETM (e.g., ETR) according to the present invention, at least one gRNA according to the present invention, a combination according to the present invention, a polynucleotide according to the present invention, a nucleic acid construct according to the present invention, a vector according to the present invention, a kit of polynucleotides according to the present invention, a cell according to the present invention or a pharmaceutical composition according to the present invention in the manufacture of medicament for treating a human in need thereof.

[0101] Suitably, at least one ETM (e.g., ETR) and at least two gRNAs may be administered to a subject simultaneously, sequentially, or separately.

[0102] In another aspect, the present invention provides a method for treating and / or preventing a disease, which comprises the step of administering an ETM (e.g., ETR) according to the present invention, at least one gRNA according to the present invention, a combination according to the present invention, a polynucleotide according to the present invention, a nucleic acid construct according to the present invention, a vector according to the present invention, a kit of polynucleotides according to the present invention, a cell according to the present invention or a 21#14533290vlpharmaceutical composition according to the present invention to a subject in need thereof.

[0103] Suitably, at least one ETM (e.g., ETR) and at least two gRNAs may be administered to a subject simultaneously, sequentially, or separately.

[0104] In an aspect, the present invention provides a method of gene therapy which comprises the steps:(i) isolation of a cell containing sample;(ii) introduction of an ETM (e.g. ETR) according to the present invention, at least one gRNA according to the present invention, a polynucleotide according the present invention, a nucleic acid construct according to the present invention, a vector according to the present invention or a kit of polynucleotides according to the present invention to the cell(s); and(iii) administering the cell(s) from step (ii) to a subject.

[0105] The polynucleotide, nucleic acid construct or vector may, for example, be introduced by transduction or transfection.

[0106] In some embodiments, the cell is autologous. In some embodiments, the cell is allogeneic.

[0107] Some aspects of this disclosure provide a combination for (1 ) regulating one or more genes, and (2) editing the sequence of a gene, in a human cell, the combination comprising: (A) a fusion protein, wherein the fusion protein comprises a catalytically active Cas9 endonuclease domain, a transcriptional repressor, and a DNMT domain, or a polynucleotide encoding the fusion protein, and (B) a guide RNA; wherein each of the one or more genes of (1) is characterized by the presence of a CpG island, and wherein the gene of (2) is a safe harbor genomic locus. In some embodiments, the human cell is a human lymphocyte, optionally, wherein the human lymphocyte is in vivo. In some embodiments, the human lymphocyte is a cytotoxic lymphocyte. In some embodiments, the human lymphocyte is a cytotoxic T-cell. In some embodiments, the human lymphocyte is an NK cell. In some embodiments, the human lymphocyte is a macrophage. In some embodiments, the one or more genes of (1 ) is B2M, TET2, TGFBR2, A2AR, CISH, PTPN11, PTPN6,22#14533290vlPTPA, PTPN2, JUNB, TOX, TOX2, NR4A1, NR4A2, NR4A3, MAP4K1, REL, IRF4, DGKA, PIK3CD, HLA-A, USP16, DCK, CD3z, CIITA, or FAS, or a combination of two or more of these. In some embodiments, the gene of (2) is TRAC, TRBC, PDCD1, TIM-3, TIGIT, LAG3, CTLA4, AAVS1, hRosa26, or CCR5. In some embodiments, the one or more genes of (1) comprise a TGFBR2 gene, wherein the combination further comprises a gRNA that comprises a spacer having the sequence of any one of SEQ ID NOs: 2929-2978 and 4553-4559 optionally with a 1 to 9 nucleotide truncation at the 5’ end, or comprises polynucleotide(s) coding for the gRNA. In some embodiments, the one or more genes of (1) comprise a TET2 gene and the combination further comprises a gRNA that comprises a spacer having the sequence of any one of SEQ ID NOs: 4429-4478 and 4560-4565 optionally with a 1 to 9 nucleotide truncation at the 5’ end, or comprises polynucleotide(s) coding for the gRNA. In some embodiments, the gene of (2) is a TRAC gene and the combination further comprises a gRNA comprising a spacer having the sequence of one of SEQ ID NOs: 562-611 or 4575, or comprises polynucleotide(s) coding for the gRNA. In some embodiments, the one or more genes of (1) comprise (i) a TGFBR2 gene, wherein the combination further comprises a gRNA that comprises a spacer having the sequence of any one of SEQ ID NOs: 2929-2978 and 4553-4559 with 1 to 9 nucleotide truncation at the 5’ end, optionally with a 3 to 9 nucleotide truncation at the 5’ end, or comprises polynucleotide(s) coding for the gRNA; and (ii) a TET2 gene and the combination further comprises a gRNA that comprises a spacer having the sequence of any one of SEQ ID NOs: 4429-4478 and 4560-4565 with a 1 to 9 nucleotide truncation at the 5’ end, optionally with a 3 to 9 nucleotide truncation at the 5’ end, or comprises polynucleotide(s) coding for the gRNA; and the gene of (2) is a TRAC gene and the combination further comprises a gRNA comprising a spacer having the sequence of one of SEQ ID NOs: 562-611, or comprises polynucleotide(s) coding for the gRNA. In some embodiments, the one or more genes of (1) comprise (i) a TGFBR2 gene, wherein the combination further comprises a gRNA comprising a spacer having the sequence of SEQ ID NO: 4645, and / or a gRNA comprising a spacer having the sequence of SEQ ID NO 4646; each individually with a 3 to 9 nucleotide truncation at 5’ end, e.g., comprising a fragment of 11, 12, 13, 14, 15, 16, or 17 nucleotides of said sequence, or comprises polynucleotide(s) coding for the gRNA; and (ii) a TET2 gene and the combination 23#14533290vlfurther comprises a gRNA that comprises a spacer having the sequence of SEQ ID NO: 4643, and / or a gRNA that comprises a spacer having the sequence of SEQ ID NO: 4644; each individually with a 3 to 9 nucleotide truncation at the 5’ end, e.g., comprising a fragment of 11, 12, 13, 14, 15, 16, or 17 nucleotides of said sequence, or comprises polynucleotide(s) coding for the gRNA. In some embodiments, the gene of (2) is a TRAC gene, wherein the combination further comprises a gRNA comprising a spacer having the sequence of SEQ ID NO: 4575. In some embodiments, the one or more genes of (1) comprise (i) a TGFBR2 gene, wherein the combination further comprises a gRNA comprising a spacer having the sequence of SEQ ID NO: 4645, and / or a gRNA comprising a spacer having the sequence of SEQ ID NO 4646; each individually with a 3 to 9 nucleotide truncation at 5’ end, or comprises polynucleotide(s) coding for the gRNA; and (ii) a TET2 gene and the combination further comprises a gRNA that comprises a spacer having the sequence of SEQ ID NO: 4643, and / or a gRNA that comprises a spacer having the sequence of SEQ ID NO: 4644; each individually with a 3 to 9 nucleotide truncation at the 5’ end, or comprises polynucleotide(s) coding for the gRNA; and the gene of (2) is a TRAC gene, wherein the combination further comprises a gRNA comprising a spacer having the sequence of SEQ ID NO: 4575. In some embodiments, the fusion protein further comprises a DNMT1, DNMT3A, DNMT3B, or SETDB1 domain, optionally a DNMT3A domain. In some embodiments, the transcriptional repressor domain is a Kruppel-associated box (KRAB) domain, optionally derived from human Kox1 or ZIM3. In some embodiments, the combination comprises a fusion protein comprising, optionally from N terminus to C terminus, a KRAB domain derived from ZIM3, a dCas9 domain, and a DNMT3L domain. In some embodiments, the combination comprises a fusion protein comprising, optionally from C terminus to N terminus, a KRAB domain derived from ZIM3, a dCas9 domain, and a DNMT3L domain. In some embodiments, the combination comprises a fusion protein comprising, optionally from N terminus to C terminus, a KRAB domain derived from ZIM3, a catalytically active Cas9 domain, and a DNMT3L domain. In some embodiments, the combination comprises a fusion protein comprising, optionally from C terminus to N terminus, a KRAB domain derived from ZIM3, a catalytically active Cas9 domain, and a DNMT3L domain. In some embodiments, the combination comprises a fusion protein comprising an amino acid sequence of SEQ 24#14533290vlID NO: 4481. In some embodiments, the combination comprises a fusion protein comprising an amino acid sequence of SEQ ID NO: 4482. In some embodiments, the gRNA(s) are chemically modified, optionally wherein the chemically modified gRNA(s) comprise phosphorothioate internucleoside linkages at the 5’ and / or 3’ ends, and / or 2’-O-methyl nucleotides. In some embodiments, the combination further comprises a donor template nucleic acid. In some embodiments, the donor template nucleic acid comprises, from 5’ to 3’, a 5’ homology arm, a transgene, and a 3’ homology arm, optionally wherein each homology arm, individually, comprises a sequence of 30-300 nucleotides that are homologous to a sequence of the gene of (2). In some embodiments, the donor template nucleic acid comprises, from 5’ to 3’, a 5’ TRAC homology arm, a transgene, and a 3’ TRAC homology arm, wherein each homology arm, individually, comprises a sequence of 30-300 nucleotides that are homologous to a sequence of a TRAC gene. In some embodiments, the donor template nucleic acid comprises a 5’ TRAC homology arm and a 3’ TRAC homology arm, wherein each homology arm, individually, comprises a sequence of 30-300 nucleotides that are homologous to a sequence of a TRAC gene immediately adjacent to the sequence of SEQ ID NO: 4575 comprised in the TRAC gene. In some embodiments, the transgene comprised in the donor sequence comprises a nucleic acid sequence encoding a CAR. In some embodiments, the transgene comprised in the donor sequence comprises a nucleic acid sequence encoding a CAR targeting CD19. In some embodiments, the combination is formulated in an LNP. In some embodiments, the combination is formulated in a T-cell targeting LNP. In some embodiments, the combination is formulated in a VLP. In some embodiments, the combination is formulated in a lymphocyte targeting VLP. Some aspects of this disclosure provide pharmaceutical compositions comprising a combination as disclosed herein. Some aspects of this disclosure provide cells comprising a combination as disclosed herein. In some embodiments, the cell or the progeny thereof is characterized by expressing the transgene and by a reduction of the expression of TGFBR2 and TET2 as compared to a cell of the same cell type but not comprising the combination. Some aspects of this disclosure provide methods and modalities for modifying transcription, expression and / or activity of one or more gene in a human cell, for example, in a human lymphocyte, such as a human T cell, NK-cell, or macrophage, and, in some such embodiments, the method or modality 25#14533290vlcomprises introducing a combination as disclosed herein into the cell in vitro or ex vivo. In other embodiments, the method or modality comprises introducing a combination as disclosed herein into the cell in vivo. In some embodiments, the method further comprises expressing a transgene, e.g., a transgene provided herein, in the human cell. Some aspects of this disclosure provide cells obtained by the methods and modalities provided herein, or progeny (daughter cells) of cells so obtained. In some embodiments, the cells are human lymphocytes, e.g., cytotoxic human lymphocytes, T cells, NK cells, or macrophages, that are engineered via the method and modalities disclosed herein to express a recombinant antigen receptor, optionally selected from a recombinant T cell receptor (TCR) or a chimeric antigen receptor (CAR). In some embodiments, the engineered cells provided herein are for use in treating a human in need thereof. In some embodiments, the present disclosure provides a use of cells engineered according to the present disclosure in the manufacture of a medicament for treating a human in need thereof. Some aspects of this disclosure provide methods and modalities for treating a subject, for example a mammalian subject, or a human subject, in need thereof, wherein such methods and modalities comprise, in some embodiments, administering to the subject an engineered cell according to the present disclosure.

[0108] Some aspects of this disclosure provide methods that comprise administering to a subject in need thereof a pharmaceutical composition, for example, a pharmaceutical composition encapsulated in a delivery vehicle, e.g., an LNP, VLP, or other suitable delivery vehicle, wherein the delivery vehicle targets human lymphocytes, e.g., human T cells, NK cells, and / or macrophages, and wherein the pharmaceutical composition comprises (A) a fusion protein, wherein the fusion protein comprises a catalytically active Cas9 endonuclease domain, a transcriptional repressor, and a DNMT domain, or a polynucleotide encoding the fusion protein, (B) one or more guide RNAs each individually having a spacer sequence that is identical to a target sequence of a gene characterized by the presence of a CGI, or a polynucleotide encoding the guide RNA; (C) one or more guide RNAs having a spacer sequence that is identical to a target sequence of a gene that is a safe harbor genomic locus; and (D) a donor template nucleic acid that comprises, from 5’ to 3’, a 5’-homology arm, a transgene, and a 3’ homology arm, wherein the 5’ and the 3’ homology arms comprise a sequence of at least 3026#14533290vlnucleotides that is identical with a sequence immediately adjacent to the target sequence of the safe harbor genomic locus. In some embodiments, the gene characterized by the presence of a CGI is B2M, TET2, TGFBR2, A2AR, CISH, PTPN11, PTPN6, PTPA, PTPN2, JUNB, TOX, TOX2, NR4A1, NR4A2, NR4A3, MAP4K1, REL, IRF4, DGKA, PIK3CD, HLA-A, USP16, DCK, CD3z, CIITA, or FAS, or a combination of two or more of these. In some embodiments, the gene that is a safe harbor genomic locus is TRAC, TRBC, PDCD1, TIM-3, TIGIT, LAG3, CTLA4, AAVS1, hRosa26, or CCR5. In some embodiments, the gene characterized by the presence of a CGI comprises a TGFBR2 gene, wherein the combination further comprises a gRNA that comprises a spacer having the sequence of any one of SEQ ID NOs: 2929-2978 and 4553-4559 optionally with a 1 to 9 nucleotide truncation at the 5’ end, or comprises polynucleotide(s) coding for the gRNA. In some embodiments, the gene characterized by the presence of a CGI comprises a TET2 gene and the combination further comprises a gRNA that comprises a spacer having the sequence of any one of SEQ ID NOs: 4429-4478 and 4560-4565 optionally with a 1 to 9 nucleotide truncation at the 5’ end, or comprises polynucleotide(s) coding for the gRNA. In some embodiments, the gene that is a safe harbor genomic locus is a TRAC gene and the combination further comprises a gRNA comprising a spacer having the sequence of one of SEQ ID NOs: 562-611 or 4575, or comprises polynucleotide(s) coding for the gRNA. In some embodiments, the gene characterized by the presence of a CGI comprises (i) a TGFBR2 gene, wherein the combination further comprises a gRNA that comprises a spacer having the sequence of any one of SEQ ID NOs: 2929-2978 and 4553-4559 with 1 to 9 nucleotide truncation at the 5’ end, optionally with a 3 to 9 nucleotide truncation at the 5’ end, or comprises polynucleotide(s) coding for the gRNA; and (ii) a TET2 gene and the combination further comprises a gRNA that comprises a spacer having the sequence of any one of SEQ ID NOs: 4429-4478 and 4560-4565 with a 1 to 9 nucleotide truncation at the 5’ end, optionally with a 3 to 9 nucleotide truncation at the 5’ end, or comprises polynucleotide(s) coding for the gRNA; and the gene that is a safe harbor genomic locus is a TRAC gene and the combination further comprises a gRNA comprising a spacer having the sequence of one of SEQ ID NOs: 562-611, or comprises polynucleotide(s) coding for the gRNA. In some embodiments, the gene characterized by the presence of a CGI comprises (i) a TGFBR2 gene, wherein the 27#14533290vlcombination further comprises a gRNA comprising a spacer having the sequence of SEQ ID NO: 4645, and / or a gRNA comprising a spacer having the sequence of SEQ ID NO 4646; each individually with a 3 to 9 nucleotide truncation at 5’ end, e.g., comprising a fragment of 11, 12, 13, 14, 15, 16, or 17 nucleotides of said sequence, or comprises polynucleotide(s) coding for the gRNA; and (ii) a TET2 gene and the combination further comprises a gRNA that comprises a spacer having the sequence of SEQ ID NO: 4643, and / or a gRNA that comprises a spacer having the sequence of SEQ ID NO: 4644; each individually with a 3 to 9 nucleotide truncation at the 5’ end, e.g., comprising a fragment of 11, 12, 13, 14, 15, 16, or 17 nucleotides of said sequence, or comprises polynucleotide(s) coding for the gRNA. In some embodiments, the gene that is a safe harbor genomic locus is a TRAC gene, wherein the combination further comprises a gRNA comprising a spacer having the sequence of SEQ ID NO: 4575. In some embodiments, the gene characterized by the presence of a CGI comprises (i) a TGFBR2 gene, wherein the combination further comprises a gRNA comprising a spacer having the sequence of SEQ ID NO: 4645, and / or a gRNA comprising a spacer having the sequence of SEQ ID NO 4646; each individually with a 3 to 9 nucleotide truncation at 5’ end, or comprises polynucleotide(s) coding for the gRNA; and (ii) a TET2 gene and the combination further comprises a gRNA that comprises a spacer having the sequence of SEQ ID NO: 4643, and / or a gRNA that comprises a spacer having the sequence of SEQ ID NO: 4644; each individually with a 3 to 9 nucleotide truncation at the 5’ end, or comprises polynucleotide(s) coding for the gRNA; and the gene that is a safe harbor genomic locus is a TRAC gene, wherein the combination further comprises a gRNA comprising a spacer having the sequence of SEQ ID NO: 4575. In some embodiments, the fusion protein further comprises a DNMT1, DNMT3A, DNMT3B, or SETDB1 domain, optionally a DNMT3A domain. In some embodiments, the transcriptional repressor domain is a Kruppel-associated box (KRAB) domain, optionally derived from human Kox1 or ZIM3. In some embodiments, the combination comprises a fusion protein comprising, optionally from N terminus to C terminus, a KRAB domain derived from ZIM3, a dCas9 domain, and a DNMT3L domain. In some embodiments, the combination comprises a fusion protein comprising, optionally from C terminus to N terminus, a KRAB domain derived from ZIM3, a dCas9 domain, and a DNMT3L domain. In some embodiments, the28#14533290vlcombination comprises a fusion protein comprising, optionally from N terminus to C terminus, a KRAB domain derived from ZIM3, a catalytically active Cas9 domain, and a DNMT3L domain. In some embodiments, the combination comprises a fusion protein comprising, optionally from C terminus to N terminus, a KRAB domain derived from ZIM3, a catalytically active Cas9 domain, and a DNMT3L domain. In some embodiments, the combination comprises a fusion protein comprising an amino acid sequence of SEQ ID NO: 4481. In some embodiments, the combination comprises a fusion protein comprising an amino acid sequence of SEQ ID NO: 4482. In some embodiments, the gRNA(s) are chemically modified, optionally wherein the chemically modified gRNA(s) comprise phosphorothioate internucleoside linkages at the 5’ and / or 3’ ends, and / or 2’-O-methyl nucleotides. In some embodiments, each homology arm of the donor template nucleic acid, individually, comprises a sequence of 30-300 nucleotides. In some embodiments, the gene that is a safe harbor genomic locus is a TRAC gene, and wherein the donor template nucleic acid comprises, from 5’ to 3’, a 5’ TRAC homology arm, a transgene, and a 3’ TRAC homology arm. In some embodiments, the donor template nucleic acid comprises a 5’ TRAC homology arm and a 3’ TRAC homology arm, wherein each homology arm, individually, comprises a sequence of 30-300 nucleotides that are homologous to a sequence of a TRAC gene immediately adjacent to the sequence of SEQ ID NO: 4575 comprised in the TRAC gene. In some embodiments, the transgene comprised in the donor template nucleic acid sequence comprises a nucleic acid sequence encoding a CAR. In some embodiments, the transgene comprised in the donor template nucleic acid sequence comprises a nucleic acid sequence encoding a CAR targeting CD19. In some embodiments, the subject has been diagnosed with a malignancy. In some embodiments, the subject has been diagnosed with a hematological malignancy. In some embodiments, the subject has been diagnosed with a B-cell leukemia or lymphoma. In some embodiments, the pharmaceutical composition encapsulated in the LNP is administered to the subject.

[0109] It is understood that an ETM (e.g., ETR) according to the present invention, at least one gRNA according to the present invention, a combination according to the present invention, a polynucleotide according to the present invention, a nucleic acid construct according to the present invention, a vector according to the present invention, a kit of polynucleotides according to the present invention, a cell according 29#14533290vlto the present invention or a pharmaceutical composition according to the present invention may be used in a method of treatment described herein, may be for use in a treatment described herein, or may be used in the manufacture of a medicament for a treatment described herein.

[0110] Other features, objects, and advantages of the invention are apparent in the detailed description that follows. It should be understood, however, that the detailed description, while indicating embodiments and aspects of the invention, is given by way of illustration only, not limitation. Various changes and modification within the scope of the invention will become apparent to those skilled in the art from the detailed description.DESCRIPTION OF THE DRAWINGS

[0111] FIG. 1 shows (A) the sequence (SEQ ID NOs: 21 and 22 for the sense and antisense strands, respectively) within the B2M gene which may be targeted by Cas9 or dCas9-ETRs and which is targeted in the Examples herein (the protospacer adjacent motif (PAM) sequence is underlined), and (B) the sequences of spacers which may be used in gRNAs to target B2M and which are used in the Examples herein (SEQ ID NOs: 23-34, in order of appearance).

[0112] FIG. 2 shows a histogram illustrating the percentage of mutated B2M alleles in cells transfected with Cas9 and the indicated gRNAs. Data are represented as % of non-homologous end-joining (NHEJ) at B2M (n=3; mean±s.d.). UT: untreated.

[0113] FIG. 3 shows a histogram illustrating the percentage of tdTomato-negative cells 44 days upon transfection with the triple combination of dCas9-based ETRs and the indicated gRNAs (n=3; mean±s.d.).

[0114] FIG. 4 shows histograms illustrating the percentage of tdTomato-negative cells 20 days upon transfection with the triple combination of dCas9-based ETRs (left panel) or Cas9 (right panel) and the indicated gRNAs H8, C8, and H10, which were either full length or truncated as indicated. The full-length sequences of H8, C8, and H10 are SEQ ID NOs: 2780, 35, and 2863, respectively. The truncated versions (19, 18, 17, 16, 15, 14, 13, 12, 11, or 10 nucleotide versions) are truncated at the 5’ end of the full-length sequence by 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 nucleotides, respectively.30#14533290vl

[0115] FIG. 5 shows a histogram illustrating the percentage of mutated TRAC alleles in cells transfected with Cas9 and the T AC gRNA. Data are represented as % of NHEJ at TRAC (n=3; mean±s.d.).

[0116] FIG. 6 shows a histogram illustrating the percentage of B2M-negative cells (B2IVT cells) 25 days after transfection with the indicated Cas9 constructs (i.e., Cas9, dCas9-ETRs, Cas9-ETRs (namely ETM)) and gRNA combinations (n=3; mean±s.d.).

[0117] FIG. 7 shows representative flow cytometry dot plots analyses of the cells treated with the 16 nt B2MgRNA and either Cas9-ETRs (namely ETM) or dCas9-ETRs. Analysis was performed at day 25 post-treatment.

[0118] FIG. 8 shows time-course flow cytometric analysis of cells treated as indicated. Data are shown as % of B2M-negative cells normalized to Untreated (UT) cells. Analysis was performed at day 25 post-treatment (n=3; mean±s.d.).

[0119] FIG. 9 shows a histogram illustrating the percentage of gene editing at the B2Mor TRAC gene for the indicated treatment conditions (n=3; mean±s.d.).

[0120] FIG. 10 shows polymerase chain reaction (PCR) analysis of the indicated treatment conditions for reciprocal chromosomal translocations between the B2M and the TRAC locus. Top: it shows a schematic diagram of the PCR strategy indicating the primers used (arrows) for the analysis. Bottom: it shows a picture of the agarose-stained gel loaded with the PCR products from the indicated treatment conditions (each in triplicate). Translocations were detected only in samples treated with Cas9 or Cas9-ETR (namely ETM) in combination with the 20nt B2MgRNA. MW: molecular weight.

[0121] FIG. 11 is a diagram of the B2M gene showing the CpG island (CGI) and the distribution of gRNAs H8 (SEQ ID NO: 2780), C8 (SEQ ID NO: 35), F4 (SEQ ID NO: 2878), H10 (SEQ ID NO: 2863), H11 (SEQ ID NO: 2778), and H12 (SEQ ID NO: 2801).

[0122] FIG. 12 shows the percentage of B2M silencing by the triple combination of dCas9-based ETRs at days 12 and 25 post-treatment with the indicated gRNAs, either alone (first row of each table) or in combinations (second and third row of each table). Data are shown as heatmap.

[0123] FIG. 13 shows representative flow cytometry analyses of T cells treated with the indicated gRNA combinations (namely C8+F4, C8+H8 or H8+F4) and the triple combination of dCas9-based ETRs at days 12 and 25 post-treatment. The fold 31#14533290vlincrease in terms of efficiency of B2Mepi-silencing between the C8+F4 and H8+F4 conditions is indicated.

[0124] FIG. 14 shows a time-course flow cytometry analysis of T cells treated with the triple combination of dCas9-based ETRs and the indicated gRNAs combinations. Data are shown as % of B2M-negative cells. UT: untreated T cells. Vertical dashed red lines indicate the days at which T cells were restimulated.

[0125] FIG. 15 shows a histogram illustrating the fold change in the percentage of B2M negative T cells between day 25 and 12 post-treatment, calculated based on the data shown in FIG. 14. Data are represented as fold decrease in B2M negative cells.

[0126] FIG. 16 shows a time-course flow cytometry analysis of T cells treated with the triple combination of dCas9-based ETRs and the indicated gRNAs combinations. Data are shown as % of B2M-negative cells. UT: untreated T cells. Vertical dashed red lines indicate the days at which T cells were restimulated.

[0127] FIG. 17 shows a histogram illustrating the fold change in the percentage of B2M negative T cells between day 25 and 12 post-treatment, calculated based on the data shown in FIG. 16. Data are represented as fold decrease in B2M negative cells.

[0128] FIG. 18A shows a time-course flow cytometry analysis of T cells treated with the indicated ETR combinations and the gRNA combination C8+F4. Data are shown as % of B2M-negative cells. UT: untreated T cells. K+3A+3L: standard triple ETR combination; K: KRAB-based ETR alone: 3A+3L: double ETR combination containing DNMT3A and DNMT3L; K+3A: double ETR combination containing KRAB and DNMT3A; K+3L: double ETR combination containing KRAB and DNMT3L; triple Vertical dashed red lines indicate the days at which T cells were restimulated.

[0129] FIG. 18B shows representative flow cytometry analyses of T cells from FIG. 18A and treated with the indicated ETR combinations and the gRNA combination C8+F4. K+3A+3L: standard triple ETR combination; K: KRAB-based ETR alone: 3A+3L: double ETR combination containing DNMT3A and DNMT3L; K+3A: double ETR combination containing KRAB and DNMT3A; K+3L: double ETR combination containing KRAB and DNMT3L.

[0130] FIG. 19 shows a time-course flow cytometry analysis of T cells treated with the double ETR combination containing KRAB and DNMT3L, plus the indicated 32#14533290vlgRNAs combinations. Data are shown as % of B2M-negative cells. UT: untreated T cells. Vertical dashed red lines indicate the days at which T cells were restimulated.

[0131] FIG. 20A shows on the left a schematic of the ZIM3:dCas9:3L fusion ETR and on the right a time-course flow cytometry analysis of T cells co-treated with ether the double ETR combination containing DNMT3A and DNMT3L or ZIM3:dCas9:3L, plus the indicated gRNAs combinations. Data are shown as % of B2M-negative cells. UT: untreated T cells. Vertical dashed red lines indicate the days at which T cells were restimulated.

[0132] FIG. 20B shows representative flow cytometry analyses of T cells from FIG. 20A and treated with the indicated ETRs and gRNA combinations. Indicated is also the fold change increase in the efficiency of epi-silencing between sample treated with the double ETR combination and the ETR fusion.

[0133] FIG. 21 shows representative flow cytometry analyses of T cells treated with decreasing doses (in micrograms) of the mRNA encoding for ZIM3:dCas9:3L fusion ETR and the indicated gRNA combination.

[0134] FIG. 22 shows representative flow cytometry analyses of T cells treated or not with Cas9, a gRNA against TRAC (see FIG. 5) and transduced with an AAV6 for targeted integration into TRAC of the NY-ESO engineered TOR. Upper left quadrant shows wild-type, un-edited cells. Bottom left quadrant shows cells with genetically disrupted TOR. Upper right quadrant shows T cells with targeted integration of the NY-ESO TOR.

[0135] FIG. 23 shows on the left a schematic representation of the double ETM combination containing the catalytically active Cas9 and the KRAB and DNMT3L effectors, while, on the right, it shows representative flow cytometry analyses of T cells treated with these ETMs and the indicated truncated gRNA against B2M, plus the full-length gRNA against TRAC and the AAV6 for targeted integration of the NY-ESO TOR into TRAC. The flow cytometry dot plot on the left reports the expression levels of B2M. The flow cytometry dot plot on the middle reports the expression levels of the endogenous TOR and the targeted NY-ESO. The flow cytometry dot plot on the right reports the expression level of NY-ESO and B2M. SSCH: side scatter height.

[0136] FIG. 24 shows on the left a schematic representation of the ETM containing the catalytically active Cas9 and the ZIM3 and DNMT3L effectors (namely33#14533290vlZIM3: Cas9:3L), while, on the right, it shows representative flow cytometry analyses of T cells treated with this ETM and the indicated truncated gRNA against B2M, plus the full-length gRNA against TRAC and the AAV6 for targeted integration of the NY-ESO TCR into TRAC. The flow cytometry dot plot on the top left reports the expression levels of B2M. The flow cytometry dot plot on the top middle reports the expression levels of the endogenous TCR and the targeted NY-ESO. The flow cytometry dot plot on the bottom reports the expression level of NY-ESO and B2M. The flow cytometry dot plot on the top right shows, within the NY-ESO positive cells, the expression levels of B2M. The flow cytometry dot plot on the bottom right shows, within the endogenous TCR negative cells, the expression levels of B2M.

[0137] FIG. 25 shows a polymerase chain reaction (PCR) analysis of the indicated treatment conditions for reciprocal chromosomal translocations between the B2M and TRAC. Top: it shows a schematic diagram of the PCR strategy indicating the primers used (arrows) for the analysis. Bottom: it shows a picture of the agarose-stained gel loaded with the PCR products from the indicated treatment conditions. Expected position of the B2M-TRAC translocation band is shown by the asterisks. MW: molecular weight. Translocations were detected only in samples treated with the ETM in combination with the 20nt gRNAs for B2M and TRAC.

[0138] FIG. 26 shows schematics of the TGFBR2 (top) and TET2 (bottom) genes, in which are indicated the relative positions of each gRNA and their pairing (P). The CpG Island (CGI) of each gene are also indicated.

[0139] FIG. 27 shows the percentages of TGFBR2 epi-silencing for the indicated combinations of gRNA pairs. Percentages are reported in the boxes. Unlabeled boxes indicate combinations that were already present in the matrix, np: not performed.

[0140] FIG. 28 shows the percentages of TFT epi-silencing for the indicated combinations of gRNA pairs. Percentages are reported in the boxes. Unlabeled boxes indicate combinations that were already present in the matrix. Negative data indicate upregulation of TET2. np: not performed.

[0141] FIG. 29 shows histograms illustrating the percentages of epi-silencing of TGFBR2 (left) and TET2 (right) in T cells treated with the triple ETR combination and the indicated pairs (P) of gRNAs, either alone or in combination. The pairs used in these studies correspond to those described in FIG.s 27 and 28.34#14533290vl

[0142] FIG. 30 shows a histogram illustrating the percentage of epigenetic silencing of the indicated genes as measured by ddPCR.

[0143] FIG. 31 shows on the left representative flow cytometry analyses for B2M (left plot) and TRAC (right plot) expression by T cells treated as indicated and on the right a histogram illustrating the percentage of epigenetic silencing of TGFBR2.

[0144] FIG. 32 shows on the left representative flow cytometry analyses for B2M (left plot) and TRAC (right plot) expression by T cells treated as indicated and on the right a histogram illustrating the percentage of epigenetic silencing of TGFBR2.

[0145] FIG. 33 shows polymerase chain reaction (PCR) analyses of the indicated treatment conditions for reciprocal chromosomal translocations among B2M, TGFBR2 and TRAC. Top: a schematic diagram of the PCR strategy for two hypothetical genes (X and Y), where arrows indicate the primers used for analysis. Bottom: pictures of the agarose-stained gels loaded with the PCR products from the indicated treatment conditions. Expected positions of translocations bands are indicated by the asterisks. MW: molecular weight. Translocations were detected only in samples treated with the ETM in combination with the 20nt gRNAs for B2M, TGFBR2 and TRAC.

[0146] FIG. 34 shows on the left representative flow cytometry analyses for B2M (left plot) and TRAC (right plot) expression by T cells treated as indicated and on the right a histogram illustrating the percentage of epigenetic silencing of TET2.

[0147] FIG. 35 shows on the left representative flow cytometry analyses for B2M (left plot) and TRAC (right plot) expression by T cells treated as indicated and on the right a histogram illustrating the percentage of epigenetic silencing of TET2.

[0148] FIG. 36 shows polymerase chain reaction (PCR) analyses of the indicated treatment conditions for reciprocal chromosomal translocations among B2M, TET2 and TRAC. Top: a schematic diagram of the PCR strategy for two hypothetical genes (X and Y), where arrows indicate the primers used for analysis. Bottom: pictures of the agarose-stained gels loaded with the PCR products from the indicated treatment conditions. Expected positions of translocations bands are indicated by the asterisks. MW: molecular weight. Translocations were detected only in samples treated with the ETM in combination with the 20nt gRNAs for B2M, TET2 and TRAC.

[0149] FIG. 37 shows on the left representative flow cytometry analyses for B2M (left plot) and TRAC (right plot) expression by T cells treated as indicated and on the 35#14533290vlright a histogram illustrating the percentage of epigenetic silencing of TGFBR2 and TET2.

[0150] FIG. 38 shows on the left representative flow cytometry analyses for B2M (left plot) and TRAC (right plot) expression by T cells treated as indicated and on the right a histogram illustrating the percentage of epigenetic silencing of TGFBR2 and TET2.

[0151] FIG. 39 shows polymerase chain reaction (PCR) analyses of the indicated treatment conditions for reciprocal chromosomal translocations among B2M, TGFBR2, TET2 and TRAC. Top: a schematic diagram of the PCR strategy for two hypothetical genes (X and Y), where arrows indicate the primers used for analysis. Bottom: pictures of the agarose-stained gels loaded with the PCR products from the indicated treatment conditions. Expected positions of translocations bands are indicated by the asterisks. MW: molecular weight. Translocations were detected only in samples treated with the ETM in combination with the 20nt gRNAs for B2M, TGFBR2, TET2 and TRAC.

[0152] FIG. 40 shows a schematic of an approach to simultaneously editing TRAC with cutting and editing both B2M and TGFBR2wW epigenetic silencing.

[0153] FIG. 41 shows a schematic of an approach to simultaneously editing TRAC with cutting and editing both TET2 and TGFBR2 with epigenetic silencing.

[0154] FIG. 42 shows representative flow cytometry analyses of T cells treated according to the schematic of FIG. 40 indicating, analyzing for NY-ESO1 and TCR at 26 (left), and a histogram of ddPCR analyses for % silencing of B2 / W and TGFBR2at Day 26.

[0155] FIG. 43 shows representative flow cytometry analyses of T cells treated according to the schematic of FIG. 41 indicating, analyzing for NY-ESO1 and TCR at 20 (left), and a histogram of ddPCR analyses for % silencing of TET2 and TGFBR2 at Day 22.DETAILED DESCRIPTION OF THE INVENTIONEngineered transcriptional modulator (ETM)

[0156] In an aspect, the present invention provides an engineered transcriptional modulator (ETM), for example an engineered transcriptional repressor (ETR),36#14533290vlcomprising: a) at least one epigenetic effector domain; operably linked to b) an endonuclease.

[0157] The ETMs of the invention may be ETRs. ETRs may repress transcription and / or expression of target gene(s).

[0158] The ETMs (e.g., ETRs) of the invention are agents that may enable multiplexing of gene editing and epigenetic editing of different target genes. For example, the ETMs (e.g., ETRs) according to the present invention may enable repression of transcription and / or expression (e.g., silencing) of multiple different target genes, wherein one gene is repressed (e.g., silenced) by genetic editing and at least one gene is repressed (e.g., silenced) by epigenetic repression (e.g., silencing). An advantage of this poly-functional editing system is that there is no reciprocal translocation between the simultaneously edited genes, thus greatly improving the safety of multiplex gene editing. Furthermore, application of such a poly-functional editing approach allows performance of orthogonal edits in one step, without the need for sequential engineering procedures, thus greatly facilitating product manufacturing and reducing associated costs and cell toxicity. The target gene selected for gene editing also may be used as a target site for insertion of exogenous expression cassettes.

[0159] The ETMs may be referred to as programmable multi-editors (ProMEs). For example, the composition of the editor and selection of endonuclease(s), as well as the design of gRNAs, may allow an ETM to be programmed to modify the sequence or modify transcription, expression and / or activity of multiple targets in the same cell. The ETMs (e.g., ETRs) may be chimeric or fusion proteins that are comprised of at least one (such as one) endonuclease operably linked to at least one effector domain (e.g., a KRAB domain, a SETDB1 domain, a DNMT3A, DNMT3B or DNMT1 domain or a DNMT3L domain, or homologues thereof; wherein the domains may be full-length proteins or functional fragments thereof and may be referred to herein as “KRAB,” “SETDB1,” “DNMT3A,” “DNMT3B,” “DNMT1,” or “DNMT3L,” respectively). The endonuclease may enable cleavage of specific DNA sequence(s), and may be chosen or engineered to bind to nucleic acid sequence(s) of choice. The endonuclease may comprise endonucleases from multiple species, e.g. S. pyogenes and S. aureus, and enable cleavage of multiple specific DNA sequence(s) in the same cell. The epigenetic effector domain may harbor a catalytic activity which 37#14533290vlenables modification (such as repression) of transcription of a target gene or target genes. Alternatively, or additionally, the effector domain may recruit additional agents within a cell to a target gene or target genes, which results in the modification (such as repression) of transcription of the target gene. The present invention also envisages ETMs that are engineered transcription activators (ETAs). ETAs may increase transcription and / or expression of target gene(s).

[0160] By “operably linked”, it is to be understood that the individual components are linked together in a manner which enables them to carry out their function (e.g., cleavage of DNA, binding to DNA, catalyzing a reaction or recruiting additional agents from within a cell) substantially unhindered. For example, an endonuclease may be conjugated to an epigenetic effector domain, for example to form a fusion protein. Methods for conjugating polypeptides are known in the art, for example through the provision of a linker amino acid sequence connecting the polypeptides (e.g., a linker comprising glycine and / or serine residues). Alternative methods of conjugating polypeptides known in the art include chemical and light-induced conjugation methods (e.g., using chemical cross-linking agents). In an example, the endonuclease and epigenetic effector domain (e.g., KRAB domain, DNMT3A, DNMT3B or DNMT1 domain and / or DNMT3L domain, or homologue thereof) of the ETM form a fusion protein.

[0161] In some aspects, the ETM (e.g., ETR) comprises an RNA binding domain. The RNA binding domain may bind to a gRNA which is complementary to a genomic target site. Thus, the RNA binding domain may direct the ETM (e.g., ETR) to a target gene.

[0162] In some aspects, the ETM (e.g., ETR) is a fusion protein comprising a) at least one epigenetic effector domain; and b) an endonuclease.

[0163] In some aspects, the ETM (e.g., ETR) is a bi-partite fusion protein. For example, the ETM (e.g., ETR) may comprise two effector domains fused to the same endonuclease.

[0164] In some aspects, the ETM (e.g., ETR) is a tri-partite fusion. For example, the ETM (e.g., ETR) may comprise three effector domains fused to the same endonuclease.

[0165] In some aspects, the ETM (e.g., ETR) may comprise four or five or six or more effector domains fused to the same endonuclease.38#14533290vl

[0166] Suitably, where the ETM (e.g., ETR) comprises multiple effector domains, the effector domains may be different. Suitably, where the ETM (e.g., ETR) comprises multiple effector domains, the effector domains may be the same.

[0167] In some aspects, an ETM (e.g., ETR) according to the present invention comprises or consists of a Cas9-KRAB, Cas9-DNMT3A or Cas9-DNMT3L fusion protein.

[0168] Suitably, an ETM (e.g., ETR) according to the present invention may be a fusion protein comprising or consisting of endonuclease, KRAB and DNMT3A domains. Suitably, an ETM (e.g., ETR) according to the present invention may be a fusion protein comprising or consisting of endonuclease, DNMT3L and DNMT3A domains. Suitably, an ETM (e.g., ETR) according to the present invention may be a fusion protein comprising or consisting of endonuclease, DNMT3L and KRAB domains. Suitably, an ETM (e.g., ETR) according to the present invention may be a fusion protein comprising or consisting of endonuclease, DNMT3L, KRAB and DNMT3A domains.

[0169] Suitably, an ETM (e.g., ETR) according to the present invention may be a fusion protein comprising or consisting of Gas (e.g., Cas9), KRAB, and DNMT3A domains. Suitably, an ETM (e.g., ETR) according to the present invention may be a fusion protein comprising or consisting of Gas (e.g., Cas9), DNMT3L and DNMT3A domains. Suitably, an ETM (e.g., ETR) according to the present invention may be a fusion protein comprising or consisting of Gas (e.g., Cas9), DNMT3L and KRAB domains. Suitably, an ETM (e.g., ETR) according to the present invention may be a fusion protein comprising or consisting of Gas (e.g., Cas9), DNMT3L, KRAB and DNMT3A domains.

[0170] In some aspects, the ETM (e.g., ETR) comprises or consists of an endonuclease-KRAB fusion protein such as a Cas-KRAB, e.g., Cas9-KRAB domain fusion protein.

[0171] An exemplary sequence of an ETM according to the present invention comprising a KRAB domain (ETM-KRAB) is set forth below in SEQ ID NO: 18: M P DVPD AS\PRRRRRV\EASDKKYSIGLDIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHS IKKNLIGALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEES FL VEEDKKHERHP IF GN I VDE VA YHEKYP TI YHLRKKL VD S TDKADLRL I YLALAHMIKFR G HFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRLENLIAQ LPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQIGDQYADLF39#14533290vlLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVRQQLPEKYKEIFF DQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLRKQRTFDNGSIPHQI HLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARGNSRFAWMTRKSEETITPW NFEEWDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEYFTVYNELTKVKYVTEGMRKPA FLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFDSVEISGVEDRFNASLGTYHDLLKI IKDKDFLDNEENED PLED I VL TL TLFEDREMIEERLKTYAHLFDDKVMKQLKRRR YTGWGRL SRKLINGIRDKQSGKTILDFLKSDGFANRNFMQLIHDDSLTFKEDIQKAQVSGQGDSLHEHI AN LA GSPA IKKGIL Q TVKWDEL VKVMGRHKPENI VIEMARENQTTQKGQKNSRERMKR I EE GIKELGSQILKEHPVENTQLQNEKLYLYYLQNGRDMYVDQELDINRLSDYDVDHIVPQSFLK DDSIDNKVL TRSDKNRGKSDNVPSEEWKKMKNYWRQLLNAKLITQRKFDNL TKAERGGLSE LDKAGFIKRQLVETRQITKHVAQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQF YKVREINNYHHAHDAYLNAWGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATA KYFFYSNIMNFFKTEITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVK KTEVQTGGFSKESILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLK SVKELLGITIMERSSFEKNPIDFLEAKGYKEVKKDLI IKLPKYSLFELENGRKRMLASAGEL QKGNELALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVIL ADANLDKVLSAYNKHRDKPIREQAENI IHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVLD ATLIHQSITGLYETRIDLSQLGGDSRAIKKKRK. VGSGGGALSPQHSAVTQGSIIKNKEGMD AKSLTAWSRTLVTFKDVFVDFTREEWKLLDTAQQIVYRNVMLENYKNLVSLGYQLTKPDVIL RLEKGEEPWLVEREIHQETHPDSETAFEIKSSV (SEQ ID NO: 18)

[0172] In the above sequence, the Cas9 domain is shown in italics, a haemagglutinin (HA) tag is shown in bold, a linker domain is shown in bold and double-underlined, and the KRAB domain is in italics and underlined. Nuclear localization signal (NLS) sequences are boxed.

[0173] It will be appreciated that alternatives to the HA tag and glycine-serine linker shown in these exemplary ETMs may be used in ETMs according to the present invention, or they may be absent.

[0174] In some aspects, the ETM e.g., ETR) comprises or consists of an endonuclease-DNMT3A fusion protein such as a Cas-DNMT3A, e.g., a Cas9- DNMT3A domain fusion protein.

[0175] An exemplary sequence of an ETM according to the present invention comprising a DNMT3A domain (ETM-D3A) is set forth below in SEQ ID NO: 19: M P DVPD AS^KKKRKNEASDKKYSIGLDIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHS IKKNLIGALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEES FL VEEDKKHERHP IF GN I VDE VA YHEKYP TI YHLRKKL VD S TDKADLRL I YLALAHMIKFR G HFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRLENLIAQ LPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQIGDQYADLF LAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVRQQLPEKYKEIFF DQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLRKQRTFDNGSIPHQI HLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARGNSRFAWMTRKSEETITPW NFEEWDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEYFTVYNELTKVKYVTEGMRKPA FLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFDSVEISGVEDRFNASLGTYHDLLKI40#14533290vlIKDKDFLDNEENED I LED I VL TL TLFEDREMIEERLKTYAHLFDDKVMKQLKRRR YTGWGRL SRKLINGIRDKQSGKTILDFLKSDGFANRNFMQLIHDDSLTFKEDIQKAQVSGQGDSLHEHI AN LA GSR A IKKGIL Q TVKWDEL VKVMGRHKPENI VIEMARENQTTQKGQKNSRERMKR TEE GIKELGSQILKEHPVENTQLQNEKLYLYYLQNGRDMYVDQELDINRLSDYDVDHIVPQSFLK DDSIDNKVL TRSDKNRGKSDNVPSEE KKMKNYWRQLLNAKLITQRKFDNL TKAERGGLSE LDKAGFIKRQLVETRQITKHVAQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQF YKVREINNYHHAHDAYLNAWGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATA KYFFYSNIMNFFKTEITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVK KTEVQTGGFSKESILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLK SVKELLGITIMERSSFEKNPIDFLEAKGYKEVKKDLI IKLPKYSLFELENGRKRMLASAGEL QKGNELALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVIL ADANLDKVLSAYNKHRDKPIREQAENI IHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVLD ATLIHQSITGLYETRIDLSQLGGDSRAD^KKKRKV\GSGG TYGLLRRREDWPSRLQMFFANN HDQEFDPPKVYPPVPAEKRKPIRVLSLFDGIATGLLVLKDLGIQVDRYIASEVCEDSITVGM VRHQGKIMYVGDVRSVTQKHIQEWGPFDLVIGGSPCNDLSIVNPARKGLYEGTGRLFFEFYR LLHDARPKEGDDRPFFWLFENWAMGVSDKRDISRFLESNPVMIDAKEVSAAHRARYFWGNL PGMNRPLASTVNDKLELQECLEHGRIAKFSKVRTITTRSNSIKQGKDQHFPVFMNEKEDILW CTEMERVFGFPVHYTDVSNMSRLARQRLLGRSWSVPVIRHLFAPLKEYFACV (SEQ ID NO: 19)

[0176] In the above sequence, the Cas9 domain is shown in italics, an HA tag is shown in bold, a linker domain is shown in bold and double-underlined, and the DNMT3A domain is in italics and underlined. NLS sequences are boxed.

[0177] In some aspects, the ETM (e.g., ETR) comprises or consists of an endonuclease-DNMT3L fusion protein such as a Cas-DNMT3L, e.g., a Cas9-DNMT3L domain fusion protein.

[0178] An exemplary sequence of an ETM according to the present invention comprising a DNMT3L domain (ETM-D3L) is set forth below in SEQ ID NO: 20:M P DVPD AS^KKKRKNEASDKKYSIGLDIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHS IKKNLIGALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEES FL VEEDKKHERHP IF GN I VDE VA YHEKYP TI YHLRKKL VD S TDKADLRL I YLALAHMIKFR G HFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRLENLIAQ LPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQIGDQYADLF LAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVRQQLPEKYKEIFF DQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLRKQRTFDNGSIPHQI HLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARGNSRFAWMTRKSEETITPW NFEEWDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEYFTVYNELTKVKYVTEGMRKPA FLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFDSVEISGVEDRFNASLGTYHDLLKI IKDKDFLDNEENED I LED I VL TL TLFEDREMIEERLKTYAHLFDDKVMKQLKRRR YTGWGRL SRKLINGIRDKQSGKTILDFLKSDGFANRNFMQLIHDDSLTFKEDIQKAQVSGQGDSLHEHI AN LA GSPA IKKGIL Q TVKWDEL VKVMGRHKPENI VIEMARENQTTQKGQKNSRERMKR TEE GIKELGSQILKEHPVENTQLQNEKLYLYYLQNGRDMYVDQELDINRLSDYDVDHIVPQSFLK DDSIDNKVL TRSDKNRGKSDNVPSEEWKKMKNYWRQLLNAKLITQRKFDNL TKAERGGLSE LDKAGFIKRQLVETRQITKHVAQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQF YKVREINNYHHAHDAYLNAWGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATA41#14533290vlKYFFYSNIMNFFKTEITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVK KTEVQTGGFSKESILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLK SVKELLGITIMERSSFEKNPIDFLEAKGYKEVKKDLI IKLPKYSLFELENGRKRMLASAGEL QKGNELALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVIL ADANLDKVLSAYNKHRDKPIREQAENI IHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVLD ATLIHQSITGLYETRIDLSQLGGDSRAIKKKRK. VGSGGGMAAIPALDPEAEPSMDVILVGS SELSSSVSPGTGRDLIAYEVKANQRNIEDICICCGSLQVHTQHPLFEGGICAPCKDKFLDAL FLYDDDGYQSYCSICCSGETLLICGNPDCTRCYCFECVDSLVGPGTSGKVHAMSNWVCYLCL PSSRSGLLQRRRKWRSQLKAFYDRESENPLEMFETVPVWRRQPVRVLSLFEDIKKELTSLGF LESGSDPGQLKHVVDVTDTVRKDVEEWGPFDLVYGATPPLGHTCDRPPSWYLFQFHRLLQYA RPKP GSPRPFFWMFVDNL VLNKEDLD VA SRFLEMEP VT I PD VHGGSL QNA VR VWSNIPA IR S RHWALVSEEELSLLAQNKQSSKLAAKWPTKLVKNCFLPLREYFKYFSTELTSSL (SEQ ID NO: 20)

[0179] The Cas9 domain is shown in italics, an HA tag is shown in bold, a linker domain is shown in bold and double-underlined, and the DNMT3L domain is in italics and underlined. NLS sequences are boxed.

[0180] A fusion protein may, for example, comprise an amino acid sequence that has at least 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identity to SEQ ID NO: 18, 19, 20, 4481 or 4482, e.g., wherein the amino acid sequence substantially retains the natural function of the protein represented by SEQ ID NO: 18, 19, 20, 4481 or 4482.

[0181] A fusion protein may, for example, be encoded by a polynucleotide comprising a nucleic acid sequence which encodes the protein of SEQ ID NO: 18, 19, 20, 4481 or 4482, or a protein that has at least 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99% or 100% amino acid identity to SEQ ID NO: 18, 19, 20, 4481 or 4482, e.g., wherein the amino acid sequence substantially retains the natural function of the protein represented by SEQ ID NO: 18, 19, 20, 4481 or 4482, respectively. The coding sequence may be codon-optimized for optimal expression in human cells.

[0182] In some aspects, the present disclosure provides ETMs that comprises a fusion protein comprising a DNA methyltransferase (DNMT) domain (e.g., a DNMT3L and / or a DNMT3A domain), an endonuclease domain (e.g., a catalytically active polynucleotide guided DNA-binding domain, such as, for example, a CRISPR / Cas domain, preferably a Cas9 domain). In particular embodiments, the repressor domain is a KRAB domain, e.g., a Kox1 KRAB domain or a Zim3 KRAB domain, a Cas9 endonuclease domain), and a transcriptional repressor domain (e.g., a KRAB domain). Such ETMs comprising DNMT domain, endonuclease domain,42#14533290vland transcriptional repressor domain are also sometimes referred to as all-in-one ETMs.

[0183] In some embodiments, the present disclosure provides an ETM that comprises a fusion protein comprising a combination of an endonuclease domain with one or more DNMT domains and / or a KRAB domain in one of the following configurations (domains listed in order from N- to C terminus of the fusion protein:N-[DNMT domain]-[endonuclease domain]-[repressor domain]-C,N-[repressor domain]-[endonuclease domain]-[DNMT domain]-C,N-[DNMT domain]-[repressor domain]- [endonuclease domain]-C,orN-[repressor domain]-[DNMT domain]- [endonuclease domain]-C.

[0184] In some embodiments, a connecting structure “]-[“in any one of the ETMr structures is a linker, e.g., a peptide linker; a detectable tag; a peptide bond; a nuclear localization signal; and / or a promoter or regulatory sequence. In an ETM comprising more than one connecting structure, the multiple connecting structures “]-[“ may be the same or may each be of a different sequence and / or type e.g., linker, tag, NLS, or peptide bond). In particular embodiments, the DNMT domain comprises DNMT3A, DNMT3L, or both.

[0185] In some embodiments, the present disclosure provides an ETM that comprises a fusion protein comprising a combination of an endonuclease domain with one or more DNMT domains and / or a KRAB domain in one of the following configurations (domains listed in order from N- to C terminus of the fusion protein:N-[DNMT3A-DNMT3L]-[endonuclease domain]-[KRAB]-CN-[KRAB]-[endonuclease domain]-[DNMT3A-DNMT3L]-CN-[KRAB]-[endonuclease domain]-[DNMT3A]-CN-[DNMT3A]-[endonuclease domain]-[KRAB]-CN-[KRAB]-[endonuclease domain]-[DNMT3A]-[DNMT3L]-CN-[DNMT3A]- [DNMT3L]- [endonuclease domain]-[KRAB]-CN-[DNMT3A]-[endonuclease domain]-CN-[endonuclease domain]-[DNMT3A]-CN-[DNMT3L]-[endonuclease domain]-CN-[endonuclease domain]-[DNMT3L]-C43#14533290vl

[0186] wherein [DNMT3A-DNMT3L] indicates that the DNMT3A and DNMT3L domains are directly fused via a peptide bond, and wherein the connecting structure ]-[ is any one of the linkers as described herein, a detectable tag, an affinity domain, a peptide bond, a nuclear localization signal, a promoter, and / or a regulatory sequence. In some embodiments, where an ETM comprises both a DNMT3L and a DNMT3A domain, the DNMT3L and DNMT3A domains are both derived from human proteins. In some embodiments, the DNMT3L and DNMT3A domains are derived from human and mouse proteins, respectively. In some embodiments, the DNMT3L and DNMT3A domains are derived from mouse and human proteins, respectively. In some embodiments, the DNMT3L and DNMT3A are both derived from mouse parental proteins. In some embodiments, the Cas9 is dSpCas9. In some embodiments, the KOX1 KRAB domain is derived from human KOX1. In some embodiments, the Zim3 KRAB domain is derived from human Zim3.

[0187] While some exemplary ETM configurations and some exemplary repressor domains, DNMT domains, and endonuclease domains are disclosed herein, additional suitable configurations and domains will be apparent to the skilled artisan based on the present disclosure and the knowledge in the art. Non-limiting examples of suitable configurations and domains include, for example, those disclosed in International (PCT) Publication Nos. WO / 2023 / 250183;WO / 2023 / 250148; WO / 2024 / 238700; WO / 2019 / 204766; and WO / 2024 / 173896, the entire contents of each of which are incorporated herein by reference. The skilled artisan will appreciate that where such configurations are disclosed in the art using a catalytically inactive Cas, e.g., a dCas9 domain, such configurations are useful in the context of the present disclosure using a catalytically (endonuclease) active Cas, e.g., Cas9.Epigenetic effector domains

[0188] As used herein, the term “epigenetic effector domain”, is to be understood as referring to the part of the ETM which provides for the epigenetic effect on a target gene, for example by catalysing a reaction on the DNA or chromatin e.g.,44#14533290vlmethylation of DNA), or by recruiting an additional agent from within a cell, e.g., resulting in the repression of the transcription of a gene.

[0189] “Domain” is to be understood in this context as referring to a part of the ETM that harbours a certain function. The domain may be an individual domain e.g., a catalytic domain) isolated from a natural protein or it may be an entire, full-length natural protein. Put another way, either the full-length protein or a functional fragment thereof can be used as an effector domain. Therefore, for example, “Kruppel-associated box (KRAB) domain” or “KRAB domain” refers to the part of the ETM that comprises an amino acid sequence with the function of a KRAB domain.

[0190] Chromatin remodeling enzymes that are known to be involved in the permanent epigenetic silencing of endogenous retroviruses (ERVs; Feschotte, C. et al. (2012) Nat. Rev. Genet. 13: 283-96; Leung, D. C. etal. (2012) Trends Biochem. Sci. 37: 127-33) may provide suitable effector domains for exploitation in the present invention.

[0191] In an aspect, the epigenetic effector domain is capable of repressing transcription and / or expression of at least one target gene. A factor capable of repressing transcription of a gene is also called a transcriptional repressor. In an aspect, the epigenetic effector domain is a repressor domain, e.g., a transcriptional repressor domain.

[0192] In an aspect, the epigenetic effector domain initiates chemical modification of chromatin and / or chromatin remodeling.

[0193] In an aspect, the epigenetic effector domain initiates DNA modification, such as DNA methylation. In an aspect, the epigenetic effector domain is a DNA methyltransferase and / or is capable of recruiting a DNA methyltransferase.

[0194] In an aspect, the epigenetic effector domain initiates histone modification, such as histone methylation or histone acetylation. In an aspect, the epigenetic effector domain is a histone methyltransferase or histone acetyltransferase.

[0195] In an aspect, the at least one epigenetic effector domain comprises a Kruppel-associated box (KRAB) domain, a DNA methyltransferase (DNMT) domain, a DNMT-like domain, or a histone methyltransferase (HMT) domain.

[0196] In an aspect, the at least one epigenetic effector domain is an antibody or derivative thereof, such as a nanobody, which binds an epigenetic regulator, such as45#14533290vla chromatin regulator which may chemically modify chromatin and / or remodel chromatin.

[0197] See, for example, Van et al., Nat Commun. 2021 Jan 22;12(1 )537, which describes nanobody-mediated control of gene expression and epigenetic memory. KRAB

[0198] In some aspects, the at least one epigenetic effector domain comprises a KRAB domain. The family of the Kruppel-associated box containing zinc finger proteins (KRAB-ZFP; Huntley, S. etal. (2006) Genome Res. 16: 669-77) plays an important role in the silencing of endogenous retroviruses. These transcription factors bind to specific ERV sequences through their ZFP DNA binding domain, while they recruit the KRAB Associated Protein 1 (KAP1) with their conserved KRAB domain. KAP1 in turn binds a large number of effectors that promote the local formation of repressive chromatin (Iyengar, S. etal. (2011) J. Biol. Chem. 286:26267-76).

[0199] An ETM of the present invention may, for example, comprise a KRAB domain. Various KRAB domains are known in the family of KRAB-ZFP proteins. For example, an ETM of the present invention may comprise the KRAB domain of human zinc finger protein 10 (ZNF10; Szulc, J. etal. (2006) Nat. Methods 3: 109-16):ALSPQHSAVTQGSIIKNKEGMDAKSLTAWSRTLVTFKDVFVDFTREEWKLLDTAQQI VYRNVMLENYKNLVSLGYQLTKPDVILRLEKGEEPWLVEREIHQETHPDSETAFEIKssv (SEQ ID NO: 1)

[0200] Further examples of suitable KRAB domains for use in the present invention include:MNNSQGRVTFEDVTVNFTQGEWQRLNPEQRNLYRDVMLENYSNLVSVGQGETTKPDV ILRLEQGKEPWLEEEEVLGSGRAEKNGDIGGQIWKPKDVKESL(the KRAB domain of the human ZIM3 protein; SEQ ID NO: 2) ITLEDVAVDFTWEEWQLLGAAQKDLYRDVMLENYSNLVAVGYQASKPDALFKLEQGE QLWTIEDGIHSGACS(the KRAB domain of the ZNF350 protein; SEQ ID NO: 3) VMFEEVSVCFTSEEWACLGPIQRALYWDVMLENYGNVTSLEWETMTENEEVTSKPSS SQRADSHKGTSKRLQG46#14533290vl(the KRAB domain of the ZNF197 protein; SEQ ID NO: 4) VSFKDVAVDFTQEEWQQLDPDEKITYRDVMLENYSHLVSVGYDTTKPNVI IKLEQGE EPWIMGGEFPCQHSP(the KRAB domain of the RBAK protein; SEQ ID NO: 5) VKIEDMAVSLILEEWGCQNLARRNLSRDNRQENYGSAFPQGGENRNENEESTSKAET SEDSASRGETTGRSQKE(the KRAB domain of the ZKSCAN1 protein; SEQ ID NO: 6) LTFKDVFVDFTLEEWQQLDSAQKNLYRDVMLENYSHLVSVGYLVAKPDVIFRLGPGE ESWMADGGTPVRTCA(the KRAB domain of the KRBOX4 protein; SEQ ID NO: 7) VTFEDVTLGFTPEEWGLLDLKQKSLYREVMLENYRNLVSVEHQLSKPDWSQLEEAE DFWPVERGIPQDTIP(the KRAB domain of the ZNF274 protein; SEQ ID NO: 8)

[0201] The above KRAB domains are illustrative only. Functional variants thereof are also contemplated herein. For example, the ZIM3 KRAB domain shown in SEQ ID NO: 4481 and 4482 (see Examples 3 and 4 below) may also be used. That ZIM3 KRAB domain has the following sequence:MGRVTFEDVTVNFTQGEWQRLNPEQRNLYRDVMLENYSNLVSVGQGETTKPDVILRL EQGKEPWLEEEEVLGSGRAEKNGDIGGQIWKPKDVKESL (SEQ ID NO: 4637) DNMT

[0202] In some aspects, the epigenetic effector domain comprises a DNA methyltransferase (DNMT) domain. DNMTs catalyse the transfer of a methyl group to DNA. Examples of DNMTs are DNMT1, DNMT3A and DNMT3B.

[0203] An ETM of the present invention may, for example, comprise a domain of human DNA methyltransferase 3A (DNMT3A; Law, J. A. etal. (2010) Nat. Rev.Genet. 11: 204-20), e.g., the catalytic domain. For example, an ETM of the present invention may comprise the sequence:TYGLLRRREDWPSRLQMFFANNHDQEFDPPKVYPPVPAEKRKP IRVLSLFDGIATGL LVLKDLGIQVDRYIASEVCEDSITVGMVRHQGKIMYVGDVRSVTQKHIQEWGPFDLV IGGSPCNDLSIVNPARKGLYEGTGRLFFEFYRLLHDARPKEGDDRPFFWLFENWAM GVSDKRDISRFLESNPVMIDAKEVSAAHRARYFWGNLPGMNRPLASTVNDKLELQEC47#14533290vlLEHGRIAKFSKVRTITTRSNSIKQGKDQHFPVFMNEKEDILWCTEMERVFGFPVHYT DVSNMSRLARQRLLGRSWSVPVIRHLFAPLKEYFACV(the catalytic domain of human DNMT3A; SEQ ID NO: 9)

[0204] DNA methyltransferases 3B and 1 (DNMT3B and DNMT1), similarly to DNMT3A, are also responsible for the deposition and maintenance of DNA methylation, and may also be used in an ETM of the present invention. For example, an ETM of the present invention may comprise any of the sequences:CHGVLRRRKDWNVRLQAFFTSDTGLEYEAPKLYPAIPAARRRP IRVLSLFDGIATGY LVLKELGIKVGKYVASEVCEESIAVGTVKHEGNIKYVNDVRNITKKNIEEWGPFDLV IGGSPCNDLSNVNPARKGLYEGTGRLFFEFYHLLNYSRPKEGDDRPFFWMFENWAM KVGDKRDISRFLECNPVMIDAIKVSAAHRARYFWGNLPGMNRPVIASKNDKLELQDC LEYNRIAKLKKVQTITTKSNSIKQGKNQLFPWMNGKEDVLWCTELERIFGFPVHYT DVSNMGRGARQKLLGRSWSVPVIRHLFAPLKDYFACE(the catalytic domain of human DNMT3B; SEQ ID NO: 10)MVAELISEEDLEFMKGDTRHLNGEEDAGGREDSILVNGACSDQSSDSPPILEAIRTP EIRGRRSSSRLSKREVSSLLSYTQDLTGDGDGEDGDGSDTPVMPKLFRETRTRSESP AVRTRNNNSVSSRERHRPSPRSTRGRQGRNHVDESPVEFPATRSLRRRATASAGTPW PSPPSSYLTIDLTDDTEDTHGTPQSSSTPYARLAQDSQQGGMESPQVEADSGDGDSS E YQD GKEF G I GD LVWGK I KGF S WWP AMWS WKAT S KRQAMS GMRWVQWF GD GKF S EV SADKLVALGLFSQHFNLATFNKLVSYRKAMYHALEKARVRAGKTFPSSPGDSLEDQL KPMLEWAHGGFKPTGIEGLKPNNTQPENKTRRRTADDSATSDYCPAPKRLKTNCYNN GKDRGDEDQSREQMASDVANNKSSLEDGCLSCGRKNPVSFHPLFEGGLCQTCRDRFL ELFYMYDDDGYQSYCTVCCEGRELLLCSNTSCCRCFCVECLEVLVGTGTAAEAKLQE PWSCYMCLPQRCHGVLRRRKDWNVRLQAFFTSDTGLEYEAPKLYPAIPAARRRP IRV LSLFDGIATGYLVLKELGIKVGKYVASEVCEESIAVGTVKHEGNIKYVNDVRNITKK NIEEWGPFDLVIGGSPCNDLSNVNPARKGLYEGTGRLFFEFYHLLNYSRPKEGDDRP FFWMFENWAMKVGDKRDISRFLECNPVMIDAIKVSAAHRARYFWGNLPGMNRPVIA SKNDKLELQDCLEYNRIAKLKKVQTITTKSNSIKQGKNQLFPWMNGKEDVLWCTEL ERIFGFPVHYTDVSNMGRGARQKLLGRSWSVPVIRHLFAPLKDYFACE(human DNMT3B: SEQ ID NO: 11)LRTLDVFSGCGGLSEGFHQAGISDTLWAIEMWDPAAQAFRLNNPGSTVFTEDCNILL KLVMAGETTNSRGQRLPQKGDVEMLCGGPPCQGFSGMNRFNSRTYSKFKNSLWSFL SYCDYYRPRFFLLENVRNFVSFKRSMVLKLTLRCLVRMGYQCTFGVLQAGQYGVAQT RRRAI ILAAAPGEKLPLFPEPLHVFAPRACQLSVWDDKKFVSNITRLSSGPFRTIT VRDTMSDLPEVRNGASALEISYNGEPQSWFQRQLRGAQYQP ILRDHICKDMSALVAA RMRHIPLAPGSDWRDLPNIEVRLSDGTMARKLRYTHHDRKNGRSSSGALRGVCSCVE AGKACDPAARQFNTLIPWCLPHTGNRHNHWAGLYGRLEWDGFFSTTVTNPEPMGKQG RVLHPEQHRWSVRECARSQGFPDTYRLFGNILDKHRQVGNAVPPPLAKAIGLEIKL CMLAKARE S AS AK I KEEEAAKD(the catalytic domain of human DNMT 1; SEQ ID NO: 12)48#14533290vlDNMT-like

[0205] In some aspects, the epigenetic effector domain may be a DNMT-like domain. A “DNMT-like” domain refers to a protein, or a functional fragment thereof, wherein the protein is a member of a DNMT family but does not possess DNA methylation activity. The DNMT-like protein typically activates or recruits other epigenetic effector domains.

[0206] An ETM of the present invention may, for example, comprise DNA (cytosine-5)-methyltransferase 3-like (DNMT3L), a catalytically inactive DNA methyltransferase that activates DNMT3A by binding to its catalytic domain. For example, an ETM of the present invention may comprise the sequence:MAAIPALDPEAEPSMDVILVGSSELSSSVSPGTGRDLIAYEVKANQRNIEDICICCG SLQVHTQHPLFEGGICAPCKDKFLDALFLYDDDGYQSYCSICCSGETLLICGNPDCT RCYCFECVDSLVGPGTSGKVHAMSNWVCYLCLPSSRSGLLQRRRKWRSQLKAFYDRE SENPLEMFETVPVWRRQPVRVLSLFEDIKKELTSLGFLESGSDPGQLKHWDVTDTV RKDVEEWGPFDLVYGATPPLGHTCDRPPSWYLFQFHRLLQYARPKPGSPRPFFWMFV DNLVLNKEDLDVASRFLEMEPVTIPDVHGGSLQNAVRVWSNIPAIRSRHWALVSEEE LSLLAQNKQSSKLAAKWPTKLVKNCFLPLREYFKYFSTELTSSL(human DNMT3L; SEQ ID NO: 13)HMT

[0207] In some aspects, the epigenetic effector domain may be a histone methyltransferase (HMT) domain, e.g., the catalytic domain. HMTs are histone modifying enzymes which catalyse the transfer of methyl groups to lysine and arginine residues of histone proteins.

[0208] Lysine-specific HMTs may contain a SET (Su(var)3-9, Enhancer of Zeste, Trithorax) domain or may be non-SET domain containing.

[0209] An example of an HMT is SET domain bifurcated 1 (SETDB1).

[0210] In early embryonic development, KAP1 is known to recruit SETDB1, a histone methyltransferase that deposits histone H3 lysine-9 di- and tri-methylation (H3K9me2 and H3K9me3, respectively), two histone marks associated with transcriptional repression. Concurrently, KAP1 binds to Heterochromatin Protein 1 alpha (HP1a), which reads H3K9me2 and H3K9me3 and stabilises the KAP1-containing complex. KAP1 can also interact with other well-known epigenetic silencers, such as lysine-specific histone demethylase 1 (LSD1) that inhibits49#14533290vltranscription by removing histone H3 lysine-4 methylation, and the nucleosome remodeling and deacetylase complex (NURD), which removes acetyl groups from histones. Finally, the KAP1 -containing complex contributes to the recruitment of the de novo DNA methyltransferase 3A (DNMT3A), which methylates cytosines at CpG sites (Jones, P. A. (2012) Nat. Rev. Genet. 13: 484-92). Together, these data suggest a model in which, in the pre-implantation embryo, the KAP1 complex ensures ERV silencing through the concerted action of histone modifying enzymes and DNA methylation. Then, after implantation, the DNA methylation previously targeted by KRAB-ZFPs to the ERVs becomes stable (Reik, W. (2007) Nature 447: 425-32), being inherited throughout mitosis and somatic cell differentiation without the need for continuous expression of ERVs-specific KRAB-ZFPs. Unlike in embryonic stem cells, the KAP1 complex is not able to efficiently induce DNA methylation in somatic cells, being only able to deposit H3K9 methylation. However, this histone mark is not maintained without continuous deposition at the targeted site by the KRAB-ZFPs (Hathaway, N. A. etal. (2012) Ce / / 149: 1447-60).

[0211] In some aspects, at least two epigenetic effector domains may be utilized, one based on, for example, the KRAB domain (e.g., the initiator of the epigenetic cascade occurring at ERVs in embryonic stem cells), and the other based on, for example, DNMT3A (e.g., the final lock of this process). This approach may allow recapitulating on a pre-selected target gene those repressive chromatin states established at ERVs in the pre-implantation embryo and then permanently inherited throughout mammalian development and adult life.

[0212] An ETM of the present invention may, for example, comprise a SETDB1 domain. For example, an ETM of the present invention may comprise any of the sequences:MSSLPGCIGLDAATATVESEEIAELQQAWEELGISMEELRHFIDEELEKMDCVQQR KKQLAELETWVIQKESEVAHVDQLFDDASRAVTNCESLVKDFYSKLGLQYRDSSSED ESSRPTEIIEIPDEDDDVLSIDSGDAGSRTPKDQKLREAMAALRKSAQDVQKFMDAV NKKSSSQDLHKGTLSQMSGELSKDGDLIVSMRILGKKRTKTWHKGTLIAIQTVGPGK KYKVKFDNKGKSLLSGNHIAYDYHPPADKLYVGSRWAKYKDGNQVWLYAGIVAETP NVKNKLRFLIFFDDGYASYVTQSELYP ICRPLKKTWEDIEDISCRDFIEEYVTAYPN RPMVLLKSGQLIKTEWEGTWWKSRVEEVDGSLVRILFLDDKRCEWI YRGSTRLEPMF SMKTSSASALEKKQGQLRTRPNMGAVRSKGPWQYTQDLTGTGTQFKPVEPPQPTAP PAPPFPPAPPLSPQAGDSDLESQLAQSRKQVAKKSTSFRPGSVGSGHSSPTSPALSE NVSGGKPGINQTYRSPLGSTASAPAPSALPAPPAPPVFHGMLERAPAEPSYRAPMEK LFYLPHVCSYTCLSRVRPMRNEQYRGKNPLLVPLLYDFRRMTARRRVNRKMGFHVIY50#14533290vlKTPCGLCLRTMQEIERYLFETGCDFLFLEMFCLDP YVLVDRKFQP YKPFYYILDITY GKEDVPLSCVNEIDTTPPPQVAYSKERIPGKGVFINTGPEFLVGCDCKDGCRDKSKC ACHQLTIQATACTPGGQINPNSGYQYKRLEECLPTGVYECNKRCKCDPNMCTNRLVQ HGLQVRLQLFKTQNKGWGIRCLDDIAKGSFVCI YAGKILTDDFADKEGLEMGDEYFA NLDHIESVENFKEGYESDAPCSSDSSGVDLKDQEDGNSGTEDPEESNDDSSDDNFCK DEDFSTSSVWRSYATRRQTRGQKENGLSETTSKDSHPPDLGPPHIPVPPSIPVGGCN PPSSEETPKNKVASWLSCNSVSEGGFADSDSHSSFKTNEGGEGRAGGSRMEAEKAST SGLGIKDEGDIKQAKKEDTDDRNKMSWTESSRNYGYNPSPVKPEGLRRPPSKTSMH QSRRLMASAQSNPDDVLTLSSSTESEGESGTSRKPTAGQTSATAVDSDDIQTISSGS EGDDFEDKKNMTGPMKRQVAVKSTRGFALKSTHGIAIKSTNMASVDKGESAPVRKNT RQFYDGEESCYI IDAKLEGNLGRYLNHSCSPNLFVQNVFVDTHDLRFPWVAFFASKR IRAGTELTWDYNYEVGSVEGKELLCCCGAIECRGRLL(human SETDB1; SEQ ID NO: 14) VGCDCKDGCRDKSKCACHQLTIQATACTPGGQINPNSGYQYKRLEECLPTGVYECNK RCKCDPNMCTNRLVQHGLQVRLQLFKTQNKGWGIRCLDDIAKGSFVCI YAGKILTDD FADKEGLEMGDEYFANLDHIESVENFKEGYESDAPCSSDSSGVDLKDQEDGNSGTED PEESNDDSSDDNFCKDEDFSTSSVWRSYATRRQTRGQKENGLSETTSKDSHPPDLGP PHIPVPPSIPVGGCNPPSSEETPKNKVASWLSCNSVSEGGFADSDSHSSFKTNEGGE GRAGGSRMEAEKASTSGLGIKDEGDIKQAKKEDTDDRNKMSWTESSRNYGYNPSPV KPEGLRRPPSKTSMHQSRRLMASAQSNPDDVLTLSSSTESEGESGTSRKPTAGQTSA TAVDSDDIQTISSGSEGDDFEDKKNMTGPMKRQVAVKSTRGFALKSTHGIAIKSTNM ASVDKGESAPVRKNTRQFYDGEESCYI IDAKLEGNLGRYLNHSCSPNLFVQNVFVDT HDLRFPWVAFFASKRIRAGTELTWDYNYEVGSVEGKELLCCCGAIECRGRLL(the catalytic domain of human SETDB1; SEQ ID NO: 15)

[0213] An ETM of the present invention may, for example, comprise an amino acid sequence that has at least 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identity to SEQ ID NO: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15, e.g., wherein the amino acid sequence substantially retains the natural function of the protein represented by SEQ ID NO: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15, respectively.

[0214] An ETM of the present invention may, for example, be encoded by a polynucleotide comprising a nucleic acid sequence which encodes the protein of SEQ ID NOs: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15, or a protein that has at least 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99% or 100% amino acid identity to SEQ ID NOs: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15, e.g., wherein the amino acid sequence substantially retains the natural function of the protein represented by SEQ ID NOs: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15, respectively. The coding sequence may be codon-optimized for optimal expression in human cells.51#14533290vl

[0215] An ETM of the present invention may, for example, comprise an amino acid sequence that has at least 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identity to SEQ ID NO: 4637, e.g., wherein the amino acid sequence substantially retains the natural function of the protein represented by SEQ ID NO: 4637.

[0216] An ETM of the present invention may, for example, be encoded by a polynucleotide comprising a nucleic acid sequence which encodes the protein of SEQ ID NO: 4637, or a protein that has at least 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99% or 100% amino acid identity to SEQ ID NO: 4637, e.g., wherein the amino acid sequence substantially retains the natural function of the protein represented by SEQ ID NO: 4637. The coding sequence may be codon-optimized for optimal expression in human cells.Endonuclease

[0217] An ETM (e.g., ETR) of the invention may comprise an endonuclease.

[0218] The endonuclease may be, for example, site-specific. As used herein, “sitespecific endonuclease” may refer to an enzyme which induces site-directed doublestrand breaks in DNA. The site-specific endonuclease enables the activity of the ETM (e.g., ETR) to be targeted to specific sites in a polynucleotide, for example the genome of a cell. For example, the endonuclease may be site-specific when used in combination with gRNAs, in other words, the endonuclease is capable of inducing site-directed DNA breaks when used in combination with gRNAs.

[0219] In an aspect, the endonuclease has exonuclease activity in addition to endonuclease activity.

[0220] The endonuclease may, e.g., bind to binding sites within a target gene or within regulatory sequences for the target gene, for example promoter or enhancer sequences.

[0221] The endonuclease may, e.g., bind to binding sites within splicing sites. Splicing variants of a given gene may be regulated by DNA methylation / demethylation at splicing sites. In turn, these modifications may cause exon exclusion / inclusion in the mature transcript. This exclusion / inclusion may have therapeutic relevance, such as in the case of Duchenne Muscular Dystrophy, in which exclusion (by genetic ablation or exon skipping) from the mature mRNA of an exon bearing the most frequent disease-causing mutation has been proposed for 52#14533290vltherapy (Ousterout, D. G. et al. (2015) Mol. Ther. 23: 523-32; Ousterout, D. G. et al. (2015) Nat. Commun. 6: 6244; Kole, R. et al. (2015) Adv. Drug Deliv. Rev. 87: 104-7; Touznik, A. et al. (2014) Expert Opin. Biol. Ther. 14: 809-19).

[0222] A number of suitable endonucleases are known in the art. For example, CRISPR / Cas systems (Sander, J. D. etal. (2014) Nat. Biotechnol. 32: 347-55) may be employed as suitable endonucleases in the ETMs (e.g., ETRs) of the present invention.

[0223] As used herein, “CRISPR / Cas system” refers to a clustered regularly interspaced short palindromic repeats / CRISPR associated nuclease system.

[0224] Clustered Regularly Interspaced Short Palindromic Repeats consist of short sequences that originate from viral genomes and have been incorporated into the bacterial genome. CRISPR associated proteins (Cas) process these sequences and cut matching viral DNA sequences. By introducing Cas and specifically constructed CRISPRs into eukaryotic cells, the eukaryotic genome can be cut at any desired position.

[0225] The CRISPR / Cas system is an RNA-guided DNA binding system (van der Oost et al. (2014) Nat. Rev. Microbiol. 12: 479-92), wherein the guide RNA (gRNA) may be selected to enable an ETM (e.g., ETR) comprising a Cas domain to be targeted to a specific sequence. Thus, to employ the CRISPR / Cas system as an endonuclease in the present invention, it is to be understood that an epigenetic effector domain may be operably linked to a Cas endonuclease such as a Cas9 endonuclease. The ETM (e.g., ETR) comprising the Cas endonuclease may be delivered to a target cell in combination with one or more gRNAs. The gRNAs are designed to target the ETM (e.g., ETR) to a target gene of interest or a regulatory element (e.g., a promoter, enhancer, or splicing site) of the target gene. Methods for the design of gRNAs are known in the art. Furthermore, fully orthogonal Cas9 proteins, as well as Cas9 / gRNA ribonucleoprotein complexes and modifications of the gRNA structure / composition to bind different proteins, have been developed to simultaneously and directionally target different effector domains to desired genomic sites of cells (Esvelt etal. (2013) Nat. Methods 10: 1116-21; Zetsche, B. etal. (2015) Cell pii: S0092-8674(15)01200-3; Zalatan, J. G. etal. (2015) Cell 160: 339-50; Paix, A. et al. (2015) Genetics 201: 47-54), and are suitable for use in the present invention.53#14533290vl

[0226] In an aspect, the ETM (e.g., ETR) comprises at least one endonuclease derived from type II CRISPR bacterial immune systems. In other words, the ETM (e.g., ETR) may comprise a Type II Cas.

[0227] Examples of Cas Type II enzymes include Cas9, Csn2 and Cas4.

[0228] Cas9 endonucleases typically comprise Reel, Recll, bridge helix, RuvC, HNH and PAM interacting domains.

[0229] The HNH and RuvC domains are nuclease domains. The Reel domain binds gRNA. The bridge helix initiates cleavage upon binding of target DNA. The PAM-interacting domain confers PAM specificity and is responsible for initiating binding to target DNA.

[0230] The endonuclease may comprise or consist of a Cas endonuclease. Thus, the endonuclease may have nuclease activity. For example, the endonuclease may be a catalytically active nuclease, bind gRNA, and bind to target DNA.

[0231] The endonuclease comprised in an ETM (e.g., ETR) according to the invention is a catalytically active endonuclease. In other words, the ETM (e.g., ETR) is capable of cleaving a target sequence, such as target DNA.

[0232] In an aspect, the endonuclease is catalytically active Cas nuclease.

[0233] In an aspect, the endonuclease is a modified or a variant endonuclease, such as a modified Cas or modified Cas9 enzyme. For example, it will be appreciated that the enzyme may be modified to recognise a specific PAM site suitable for a target gene. The modified PAM may be different to the PAM naturally recognised by the enzyme.

[0234] In an aspect, the ETM (e.g., ETR) according to the present invention does not comprise only catalytically inactive, or catalytically dead (dCas) nuclease. In an aspect, the ETM (e.g., ETR) according to the present invention does not comprise a catalytically inactive, or catalytically dead (dCas) nuclease, such as dCas9.

[0235] In an aspect, the endonuclease is a catalytically active Cas9 nuclease.

[0236] In an aspect, the endonuclease is a catalytically active Cas9 nuclease from Streptococcus pyogenes (SpCas9). In another aspect, the endonuclease is a catalytically active Cas9 nuclease from Staphylococcus aureus (SaCas9). In some aspects, the endonuclease comprises both SpCas9 and SaCas9. In some embodiments, the endonuclease comprises endonucleases derived from more than one species.54#14533290vl

[0237] Methods for determining whether a protein is a catalytically active nuclease are known in the art, for example using gel assays, Kunitz assays, radiolabel assays and fluorescence-based methods. Gel assays may be performed using purified recombinant target DNA as a substrate in an assay buffer. The protein to be tested may be incubated with the substrate, for example incubated at 37°C for 1 hour. The reaction products can be separated by electrophoresis, for example, on an agarose gel with ethidium bromide to visualize the products of the nuclease reaction. Other methods include, for example, fluorescence real-time quantification of DNA and RNA nuclease activity as reported in Sheppard, E. C., etal. Sci Rep 9, 8853 (2019) and cell free detection of Gas nucleases as reported in J. Cox etal., Chem Sci. 2019 Mar 7; 10(9): 2653-2662.

[0238] For example, an ETM (e.g., ETR) of the present invention may comprise the following catalytically active Cas9 sequence:DKKYSIGLDIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKNLIGALLFDSGET AEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEESFLVEEDKKHER HP IFGNIVDEVAYHEKYPTI YHLRKKLVDSTDKADLRLI YLALAHMIKFRGHFLIEG DLNPDNSDVDKLFIQLVQTYNQLFEENP INASGVDAKAILSARLSKSRRLENLIAQL PGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQIGDQY ADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVRQQL PEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKP ILEKMDGTEELLVKLNREDLLR KQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLAR GNSRFAWMTRKSEETITPWNFEEWDKGASAQSFIERMTNFDKNLPNEKVLPKHSLL YEYFTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKK IECFDSVEISGVEDRFNASLGTYHDLLKI IKDKDFLDNEENEDILEDIVLTLTLFED REMIEERLKTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKS DGFANRNFMQLIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVK WDELVKVMGRHKPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEH PVENTQLQNEKLYLYYLQNGRDMYVDQELDINRLSDYDVDHIVPQSFLKDDSIDNKV LTRSDKNRGKSDNVPSEEWKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDK AGFIKRQLVETRQITKHVAQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDF QFYKVREINNYHHAHDAYLNAWGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQ EIGKATAKYFFYSNIMNFFKTEITLANGEIRKRPLIETNGETGEIVWDKGRDFATVR KVLSMPQVNIVKKTEVQTGGFSKESILPKRNSDKLIARKKDWDPKKYGGFDSPTVAY SVLWAKVEKGKSKKLKSVKELLGITIMERSSFEKNP IDFLEAKGYKEVKKDLI IKL PKYSLFELENGRKRMLASAGELQKGNELALPSKYVNFLYLASHYEKLKGSPEDNEQK QLFVEQHKHYLDEI IEQISEFSKRVILADANLDKVLSAYNKHRDKP IREQAENI IHL FTLTNLGAPAAFKYFDTTIDRKRYTSTKEVLDATLIHQSITGLYETRIDLSQLGGDS(SEQ ID NO: 16)

[0239] An ETM (e.g., ETR) of the present invention may, for example, comprise an amino acid sequence that has at least 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99%55#14533290vlor 100% identity to SEQ ID NO: 16, e.g., wherein the amino acid sequence substantially retains the natural function e.g., endonuclease function) of the protein represented by SEQ ID NO: 16.

[0240] An ETM (e.g., ETR) of the present invention may, for example, be encoded by a polynucleotide comprising a nucleic acid sequence which encodes the protein of SEQ ID NO: 16, or a protein that has at least 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99% or 100% amino acid identity to SEQ ID NO: 16, e.g., wherein the amino acid sequence substantially retains the natural function of the protein represented by SEQ ID NO: 16. The coding sequence may be codon-optimized for optimal expression in human cells.

[0241] For comparison, the sequence of a catalytically dead Cas9 (dCas9) is:DKKYSIGLAIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKNLIGALLFDSGET AEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEESFLVEEDKKHER HP IFGNIVDEVAYHEKYPTI YHLRKKLVDSTDKADLRLI YLALAHMIKFRGHFLIEG DLNPDNSDVDKLFIQLVQTYNQLFEENP INASGVDAKAILSARLSKSRRLENLIAQL PGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQIGDQY ADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVRQQL PEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKP ILEKMDGTEELLVKLNREDLLR KQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLAR GNSRFAWMTRKSEETITPWNFEEWDKGASAQSFIERMTNFDKNLPNEKVLPKHSLL YEYFTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKK IECFDSVEISGVEDRFNASLGTYHDLLKI IKDKDFLDNEENEDILEDIVLTLTLFED REMIEERLKTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKS DGFANRNFMQLIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVK WDELVKVMGRHKPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEH PVENTQLQNEKLYLYYLQNGRDMYVDQELDINRLSDYDVDAIVPQSFLKDDSIDNKV LTRSDKNRGKSDNVPSEEWKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDK AGFIKRQLVETRQITKHVAQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDF QFYKVREINNYHHAHDAYLNAWGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQ EIGKATAKYFFYSNIMNFFKTEITLANGEIRKRPLIETNGETGEIVWDKGRDFATVR KVLSMPQVNIVKKTEVQTGGFSKESILPKRNSDKLIARKKDWDPKKYGGFDSPTVAY SVLWAKVEKGKSKKLKSVKELLGITIMERSSFEKNP IDFLEAKGYKEVKKDLI IKL PKYSLFELENGRKRMLASAGELQKGNELALPSKYVNFLYLASHYEKLKGSPEDNEQK QLFVEQHKHYLDEI IEQISEFSKRVILADANLDKVLSAYNKHRDKP IREQAENI IHL FTLTNLGAPAAFKYFDTTIDRKRYTSTKEVLDATLIHQSITGLYETRIDLSQLGGDS(catalytically dead Cas9; dCas9; SEQ ID NO: 17)

[0242] The above sequence contains D9A and H839A substitutions relative to its catalytically active (i.e., live) counterpart (SEQ ID NO: 16). A catalytically dead Cas9 (e.g., the above dCas9) may be used in the ETM for epi-editing of one or more target genes, without simultaneous genetic editing of another gene in a cell. For this use,56#14533290vlthe ETM (e.g., ETR) may, for example, comprise an amino acid sequence that has at least 70%, 80%, 90%, 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO: 17, e.g., wherein the amino acid sequence substantially retains the natural function of the protein represented by SEQ ID NO: 16, except for the endonuclease function. The ETM e.g., ETR) may, for example, be encoded by a polynucleotide comprising a nucleic acid sequence which encodes the protein of SEQ ID NO: 17, or a protein that has at least 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99% or 100% amino acid identity to SEQ ID NO: 17, e.g., wherein the amino acid sequence substantially retains the natural function of the protein represented by SEQ ID NO: 16 but for the endonuclease function. The coding sequence may be codon-optimized for optimal expression in human cells.gRNA

[0243] In an aspect, the present invention provides guide RNAs (gRNAs).

[0244] The gRNA targets the ETM (e.g., ETR) to a target gene. The gRNA may, for example, be an RNA sequence which recognises the target DNA region of interest and directs the endonuclease within the ETM (e.g., ETR) to that region.

[0245] A gRNA is typically made up of two parts:a) a spacer sequence (which may also be referred to as a targeting domain, guide sequence, or complementarity region, and which may constitute a CRISPR RNA (crRNA)); andb) a scaffold sequence (which may also be referred to as a tracrRNA in a CRISPR / Cas system).

[0246] The spacer and the scaffold sequences may, for example, be provided as separate molecules, or they may be linked, such as via a linker loop or other sequence or may be fused together.

[0247] For example, the gRNA may be constituted by two separate molecules, e.g., the spacer (crRNA) and the scaffold (tracrRNA). The 3’ end of the spacer (crRNA) may be complementary to the 5’ end of the scaffold (tracrRNA), which complementarity may lead to dimerization of the two molecules.57#14533290vl

[0248] In another example, the spacer (crRNA) and the scaffold (tracrRNA) may be fused, for example via a linker loop. This artificial configuration may also be known as a single guide RNA (sgRNA).

[0249] In some aspects, variants of the scaffold (tracrRNA) may be used. For example, the tetraloop and stem loop of the scaffold (tracrRNA) sequence may be modified to include RNA aptamers, which can be bound by specific protein domains. In some aspects, such modified gRNAs can be used to facilitate the recruitment of repressive or activating domains fused to the protein-interacting RNA aptamers.

[0250] Exemplary tracrRNA sequences include, without limitation:5 ' -GUUUAAGAGCUAUGCUGGAAACAGCAUAGCAAGUUUAAAUAAGGCUA GUCCGUUAUCAACUUGAAAAAGUGGCACCGAGUCGGUGCUUUUUUU- 3 '(SEQ ID NO: 4566), and5 ' -GUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAGGCUAGUCCGUUA UCAACUUGAAAAAGUGGCACCGAGUCGGUGCUUUU- 3 ' (SEQ ID NO: 4567)

[0251] As utilized herein, a “spacer” or “spacer sequence” refers to a sequence that may be fully complementary to a target domain (i.e., region) within a target sequence.

[0252] The 3’ end of the genomic target sequence generally comprises a protospacer adjacent motif (PAM) sequence. A “PAM” sequence is typically a 2 to 6 base pair DNA sequence immediately following the DNA sequence targeted by the nuclease. The PAM sequence is required for cleavage but is not part of the target of the gRNA sequence. The PAM sequence varies depending on the species of the nuclease. For example, the canonical PAM associated with the Cas9 nuclease of Streptococcus pyogenes is the sequence 5’-NGG-3’ where “N” is any nucleobase. Nuclease enzymes derived from different organisms or which have been engineered may recognise different PAM sequences.

[0253] For example, the Cas9 of Francisella novicida recognizes the canonical PAM sequence 5'-NGG-3', but has been engineered to recognize 5'-YG-3' (where "Y" is a pyrimidine), thus adding to the range of possible Cas9 targets. See, e.g., Hirano et al., (2016) Structure and Engineering of Francisella novicida Cas9, Cell. 164 (5): 950-961; the entire contents of which are incorporated herein by reference. The Cas12a (or Cpf 1 ) nuclease of Francisella novicida recognizes the PAM 5'-TTTN-3' or 5'-YTN-3'.58#14533290vl

[0254] The nucleotides upstream (towards the 5’ end of the target sequence) of the PAM sequence is the protospacer sequence.

[0255] A Cas9 nuclease will typically cleave approximately three bases upstream of the PAM.

[0256] It will be appreciated that one may choose a suitable nuclease of a particular context based on PAM specificity and the genomic target.

[0257] As utilized herein, a “scaffold” or “scaffold sequence” is a sequence necessary for endonuclease binding e.g., Cas binding.

[0258] In an aspect, the present invention provides single guide RNAs (sgRNAs). In an aspect, the gRNA according to the present invention is a sgRNA. sgRNAs are single RNA molecules which contain a crRNA sequence fused to the scaffold tracrRNA sequence. In nature, crRNAs and tracrRNAs exist as two separate RNA molecules, but sgRNAs have become a common format for CRISPR gRNAs in research.

[0259] In an aspect the gRNA comprises a spacer sequence which is 10 nucleotides in length. In an aspect the gRNA comprises a spacer sequence which is 11 nucleotides in length. In an aspect the gRNA comprises a spacer sequence which is 12 nucleotides in length. In an aspect the gRNA comprises a spacer sequence which is 13 nucleotides in length. In an aspect the gRNA comprises a spacer sequence which is 14 nucleotides in length. In an aspect the gRNA comprises a spacer sequence which is 15 nucleotides in length. In an aspect the gRNA comprises a spacer sequence which is 16 nucleotides in length. In an aspect the gRNA comprises a spacer sequence which is 17 nucleotides in length. In an aspect the gRNA / comprises a spacer sequence which is 18 nucleotides in length. In an aspect the gRNA comprises a spacer sequence which is 19 nucleotides in length. In an aspect the gRNA comprises a spacer sequence which is 20 nucleotides in length. In an aspect the gRNA comprises a spacer sequence which is 21 nucleotides in length.

[0260] Without wishing to be bound by theory, certain gRNAs e.g., gRNAs comprising a spacer sequence of around 20 nucleotides in length) may be used to induce gene editing by an ETM (e.g., ETR) whilst gRNAs comprising shorter spacer sequences (e.g., gRNAs comprising spacer sequences of around 16 nucleotides in length) may favor epigenetic editing such as epi-silencing by an ETM (e.g., ETR).59#14533290vlSee, for example, FIG. 2, which shows that gRNAs comprising spacer sequences of about 18 to 20 nucleotides in length induce NHEJ whilst gRNAs comprising spacer sequences of about 16 nucleotides in length or less do not induce NHEJ. FIG. 3 shows that gRNAs comprising spacer sequences of about 11 to 16 nucleotides in length are capable of inducing epigenetic modification, e.g., epi-silencing of B2M.

[0261] In some embodiments, the gRNA comprises a spacer sequence which is less than or equal to 15, 16, or 17 e.g., less than or equal to 17 or 16) nucleotides in length. In some embodiments, the gRNA comprises a spacer sequence which is 11 to 16 nucleotides in length, such as 12 to 16, 13 to 16, 14 to 16, 15 to 16, 12 to 17, 13 to 17, 14 to 17, 15 to 17, 16, or 17 nucleotides in length.

[0262] In some embodiments, the gRNA comprises a spacer sequence which is greater than or equal to 16, 17, or 18 (e.g., greater than or equal to 17 or 18) nucleotides in length, such as 18 or more, 19 or more, or 20 or more nucleotides in length. In some embodiments, the gRNA comprises a spacer sequence which is 17 to 30 nucleotides in length, such as 18 to 30, 19 to 30 or 20 to 30 nucleotides in length. In some embodiments, the gRNA comprises a spacer sequence which is 17 to 25 nucleotides in length, such as 18 to 25, 19 to 25 or 20 to 25 nucleotides in length. In some embodiments, the gRNA comprises a spacer sequence which is 17 to 20 nucleotides in length, such as 18 to 20 or 19 to 20 nucleotides in length.

[0263] An ETM according to the present invention may be capable of modifying the transcription, expression and / or activity (e.g., repressing transcription and / or expression) of multiple target genes within the same cell by epigenetic editing and by gene editing.

[0264] The present invention enables the selection of gRNAs which promote either gene editing or epigenetic editing of a target. In this manner, it is possible to choose to perform gene editing on gene targets which are not susceptible to epigenetic editing whilst simultaneously epigenetically targeting genes which are susceptible to epigenetic editing in a multiplexing approach.

[0265] In an aspect, a gRNA is capable of promoting epigenetic editing of a target. Epigenetic editing may be measured using methods known in the art. For example, as described in Example 2, the level of expression of a reporter gene may be measured as a model of epigenetic editing.60#14533290vl

[0266] In an aspect, a gRNA is capable of promoting gene editing of a target. Gene editing may be measured using methods known in the art. For example, as described in Example 1, the level of non-homologous end joining may be measured as a model of gene editing.

[0267] An exemplary sequence of a genomic target site (i.e., protospacer and PAM) recognised by gRNAs for use in targeting the [32-microglobulin (B2M) gene includes:5 ' -AGGGTAGGAGAGACTCACGCTGG-3 ' (SEQ ID NO: 21 )3' -TCCCATCCTCTCTGAGTGCGACC-5' (SEQ ID NO: 22)The underlined nucleotides are the PAM.

[0268] In an aspect, the present invention provides gRNAs which target the [32-microglobulin gene region set forth in SEQ ID NO: 21 or SEQ ID NO: 22 above.

[0269] Examples of spacer sequences which may be used in gRNAs targeting the [32-microglobulin gene, and in particular the target site above, include:TAGGGUAGGAGAGACUCACGC -21 -nt (SEQ ID NO: 23) AGGGUAGGAGAGACUCACGC -20-nt (SEQ ID NO: 24) - This spacer sequence may be incorporated in a gRNA and may be used for gene editing of B2M when used in combination with an ETM as shown in Example 2.GGGUAGGAGAGACUCACGC -19-nt (SEQ ID NO: 25) GGUAGGAGAGACUCACGC -18-nt (SEQ ID NO: 26)GUAGGAGAGACUCACGC -17-nt (SEQ ID NO: 27)UAGGAGAGACUCACGC -16-nt (SEQ ID NO: 28)AGGAGAGACUCACGC -15-nt (SEQ ID NO: 29)GGAGAGACUCACGC -14-nt (SEQ ID NO: 30)GAGAGACUCACGC -13-nt (SEQ ID NO: 31)AGAGACUCACGC -12-nt (SEQ ID NO: 32)GAGACUCACGC -11 -nt (SEQ ID NO: 33)AGACUCACGC -10-nt (SEQ ID NO: 34)61#14533290vl

[0270] In some aspects, the spacer sequence comprises a “T” at the 5’ end. In some aspects, the spacer sequence is modified to comprise a “G” nucleotide at the 5’ end. This “G” may, for example, not be part of the targeting sequence and may be necessary when the promoter that drives its expression is a LI6 promoter.

[0271] For example, the “T” at the 5’ end of SEQ ID NO: 23 can be changed to a “G” to drive expression from a LI6 promoter. Thus, it will be understood that if the spacer sequence in SEQ ID NO: 23 is driven by a LI6 promoter, a “G” at the 5’ end may be necessary.

[0272] In some aspects, the spacer sequences according to the present invention comprise a “G” nucleotide at the 5’ end.

[0273] Examples of a gRNA according to the present invention are:AGGGUAGGAGAGACUCACGCGUUUAAGAGCUAUGCUGGAAACAGCAUAGCAAGUUUA AAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCACCGAGUCGGUGCUUUUUUU(SEQ ID NO: 4479), and AGGGUAGGAGAGACUCACGCGUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAGGCUA GUCCGUUAUCAACUUGAAAAAGUGGCACCGAGUCGGUGCUUUU (SEQ ID NO: 4568 ),which comprise the spacer sequence SEQ ID NO: 24 (underlined above).

[0274] Alternative gRNAs for epi-silencing of B2M may be found, e.g., in Amabile etal., supra.

[0275] For example, an alternative spacer sequence which may be used in a gRNA according to the present invention is:GAGUAGCGCGAGCACAGCUA -20-nt (SEQ ID NO: 35 )

[0276] Examples of gRNA according to the present invention is:GAGUAGCGCGAGCACAGCUAGUUUAAGAGCUAUGCUGGAAACAGCAUAGCAAGUUUA AAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCACCGAGUCGGUGCUUUUUUU(SEQ ID NO: 4480 ), and GAGUAGCGCGAGCACAGCUAGUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAGGCUA GUCCGUUAUCAACUUGAAAAAGUGGCACCGAGUCGGUGCUUUU (SEQ ID NO: 4569).which comprise the spacer sequence SEQ ID NO: 35 (underlined above).

[0277] Truncated spacer sequences based on SEQ ID NO: 35 suitable for use in gRNAs according to the present invention include:62#14533290vlAGUAGCGCGAGCACAGCUA -19-nt (SEQ ID NO: 36) GUAGCGCGAGCACAGCUA -18-nt (SEQ ID NO: 37) UAGCGCGAGCACAGCUA -17-Flt (SEQ ID NO: 38) AGCGCGAGCACAGCUA -16-nt (SEQ ID NO: 39)GCGCGAGCACAGCUA -15-Flt (SEQ ID NO: 40)CGCGAGCACAGCUA -14-nt (SEQ ID NO: 41)GCGAGCACAGCUA -13-nt (SEQ ID NO: 42)CGAGCACAGCUA -12-nt (SEQ ID NO: 43)GAGCACAGCUA -11 -nt (SEQ ID NO: 44)AGCACAGCUA -10-nt (SEQ ID NO: 45)

[0278] Another spacer sequence (H8) which may be used in a gRNA according to the present invention is:CAUCGGCGCCCUCCGAUCUG -20-nt (SEQ ID NO: 2780)Examples of gRNAs having this spacer (underlined) are:CAUCGGCGCCCUCCGAUCUGGUUUAAGAGCUAUGCUGGAAACAGCAUAGCAAGUUUA AAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCACCGAGUCGGUGCUUUUUUU(SEQ ID NO: 4483 ), and CAUCGGCGCCCUCCGAUCUGGUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAGGCUA GUCCGUUAUCAACUUGAAAAAGUGGCACCGAGUCGGUGCUUUU (SEQ ID NO: 4570 ).Truncated spacer sequences based on SEQ ID NO: 2780 suitable for use in gRNAs according to the present invention include:AUCGGCGCCCUCCGAUCUG -19-nt (SEQ ID NO: 4484) UCGGCGCCCUCCGAUCUG -18-nt (SEQ ID NO: 4485) CGGCGCCCUCCGAUCUG -17-nt (SEQ ID NO: 4486) GGCGCCCUCCGAUCUG -16-nt (SEQ ID NO: 4487) GCGCCCUCCGAUCUG -15-nt (SEQ ID NO: 4488)CGCCCUCCGAUCUG -14-nt (SEQ ID NO: 4489)63#14533290vlGCCCUCCGAUCUG -13-nt (SEQ ID NO: 4490)CCCUCCGAUCUG -12-nt (SEQ ID NO: 4491)CCUCCGAUCUG -11 -nt (SEQ ID NO: 4492)CUCCGAUCUG -10-nt (SEQ ID NO: 4493)

[0279] Another spacer sequence (H10) which may be used in a gRNA according to the present invention is:GCGGGCCACCAAGGAGAACU -20-nt (SEQ ID NO: 2863)Examples of gRNAs having this spacer (underlined) are:GCGGGCCACCAAGGAGAACUGUUUAAGAGCUAUGCUGGAAACAGCAUAGCAAGUUUA AAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCACCGAGUCGGUGCUUUUUUU(SEQ ID NO: 4494 ), and GCGGGCCACCAAGGAGAACUGUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAGGCUA GUCCGUUAUCAACUUGAAAAAGUGGCACCGAGUCGGUGCUUUU (SEQ ID NO: 4571 ).Truncated spacer sequences based on SEQ ID NO: 2863 suitable for use in gRNAs according to the present invention include:CGGGCCACCAAGGAGAACU -19-nt (SEQ ID NO: 4495) GGGCCACCAAGGAGAACU -18-nt (SEQ ID NO: 4496) GGCCACCAAGGAGAACU -17-nt (SEQ ID NO: 4497) GCCACCAAGGAGAACU -16-nt (SEQ ID NO: 4498) CCACCAAGGAGAACU -15-nt (SEQ ID NO: 4499)CACCAAGGAGAACU -14-nt (SEQ ID NO: 4500)ACCAAGGAGAACU -13-nt (SEQ ID NO: 4501)CCAAGGAGAACU -12-nt (SEQ ID NO: 4502)CAAGGAGAACU -11 -nt (SEQ ID NO: 4503)AAGGAGAACU -10-nt (SEQ ID NO: 4504)

[0280] Another spacer sequence (H11 ) which may be used in a gRNA according to the present invention is:64#14533290vlCGAUAAGCGUCAGAGCGCCG -20-nt (SEQ ID NO: 2778)Examples of gRNAs having this spacer (underlined) are:CGAUAAGCGUCAGAGCGCCGGUUUAAGAGCUAUGCUGGAAACAGCAUAGCAAGUUUA AAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCACCGAGUCGGUGCUUUUUUU(SEQ ID NO: 4505 ), andC GAUAAGC GUC AGAGC GC C GGUUUUAGAGCUAGAAAUAGC AAGUUAAAAUAAGGCUA GUCCGUUAUCAACUUGAAAAAGUGGCACCGAGUCGGUGCUUUU (SEQ ID NO: 4572 ).Truncated spacer sequences based on SEQ ID NO: 2778 suitable for use in gRNAs according to the present invention include:GAUAAGCGUCAGAGCGCCG -19-nt (SEQ ID NO: 4506) AUAAGCGUCAGAGCGCCG -18-nt (SEQ ID NO: 4507) UAAGCGUCAGAGCGCCG -17-nt (SEQ ID NO: 4508) AAGCGUCAGAGCGCCG -16-nt (SEQ ID NO: 4509) AGCGUCAGAGCGCCG -15-nt (SEQ ID NO: 4510) GCGUCAGAGCGCCG -14-nt (SEQ ID NO: 4511)CGUCAGAGCGCCG -13-nt (SEQ ID NO: 4512)GUCAGAGCGCCG -12-nt (SEQ ID NO: 4513)UCAGAGCGCCG -11 -nt (SEQ ID NO: 4514)CAGAGCGCCG -10-nt (SEQ ID NO: 4515)

[0281] Another spacer sequence (H12) which may be used in a gRNA according to the present invention is:GAACGCGUGGAGGGGCGCUU -20-nt (SEQ ID NO: 2801)Examples of gRNAs having this spacer (underlined) are:GAACGCGUGGAGGGGCGCUUGUUUAAGAGCUAUGCUGGAAACAGCAUAGCAAGUUUA AAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCACCGAGUCGGUGCUUUUUUU(SEQ ID NO: 4516), and GAACGCGUGGAGGGGCGCUUGUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAGGCUA GUCCGUUAUCAACUUGAAAAAGUGGCACCGAGUCGGUGCUUUU (SEQ ID NO: 4573 ).65#14533290vlTruncated spacer sequences based on SEQ ID NO: 2801 suitable for use in gRNAs according to the present invention include:AACGCGUGGAGGGGCGCUU -19-nt (SEQ ID NO: 4517) ACGCGUGGAGGGGCGCUU -18-nt (SEQ ID NO: 4518) CGCGUGGAGGGGCGCUU -17-nt (SEQ ID NO: 4519) GCGUGGAGGGGCGCUU -16-nt (SEQ ID NO: 4520) CGUGGAGGGGCGCUU -15-nt (SEQ ID NO: 4521)GUGGAGGGGCGCUU -14-nt (SEQ ID NO: 4522)UGGAGGGGCGCUU -13-nt (SEQ ID NO: 4523)GGAGGGGCGCUU -12-nt (SEQ ID NO: 4524)GAGGGGCGCUU -11 -nt (SEQ ID NO: 4525)AGGGGCGCUU -10-nt (SEQ ID NO: 4526)

[0282] An example of a spacer sequence for use in a gRNA targeting the TRAC gene, includes:AGAGUCUCUCAGCUGGUACA (SEQ ID NO: 46).Examples of gRNAs having this spacer (underlined) are:AGAGUCUCUCAGCUGGUACAGUUUAAGAGCUAUGCUGGAAACAGCAUAGCAAGUUUA AAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCACCGAGUCGGUGCUUUUUUU(SEQ ID NO: 4574 ), and AGAGUCUCUCAGCUGGUACAGUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAGGCUA GUCCGUUAUCAACUUGAAAAAGUGGCACCGAGUCGGUGCUUUU (SEQ ID NO: 4575 ).

[0283] The present disclosure also provides variations of the above exemplified gRNAs in which the spacer sequences (those underlined) are truncated by, e.g., 1 to 9 e.g., 3 to 9) nucleotides at the 5’ end. The present disclosure also provides gRNAs in which the spacers (full-length or truncated versions) described herein are linked to the above-exemplified tracr RNA (the portions of the above gRNAs, e.g., SEQ ID NOs: 4574 and 4575, that are not underlined).66#14533290vl

[0284] In an aspect, the present invention provides a gRNA which comprises a spacer sequence which comprises or consists of a sequence set forth in any one of SEQ ID NOs: 23-46, 562-1076, 2778-4478, and 4553-4565, or a homologue thereof.

[0285] In an aspect, the present invention provides a gRNA which comprises a spacer sequence wherein the spacer sequence comprises or consists of a sequence set forth in any one of SEQ ID NOs: 23-46, 562-1076, 2778-4478, and 4553-4565 having one or more (such as two, or three, or four, or five) conservative substitutions. The spacer sequence comprising one or more conservative substitution(s) retains substantially the same activity as the spacer sequence having a sequence set forth in any one of SEQ ID NOs: 23-46, 562-1076, 2778-4478, and 4553-4565.

[0286] In an aspect, the present invention provides a gRNA which comprises a spacer sequence which comprises or consists of a sequence set forth in any one of SEQ ID NOs: 23-46, 562-1076, 2778-4478, and 4553-4565, or a fragment thereof.

[0287] Suitably, the spacer sequence may comprise or consist of a sequence set forth in any one of SEQ ID NO: 23-46, 562-1076, 2778-4478, and 4553-4565, and is 21 nucleotides in length or less (such as 20 nucleotides, such as 19 nucleotides, such as 18 nucleotides, such as 17 nucleotides, such as 16 nucleotides, such as 15 nucleotides, such as 14 nucleotides, such as 13 nucleotides, such as 12 nucleotides, such as 11 nucleotides, or such as 10 nucleotides).

[0288] In an aspect, the spacer sequence may comprise a sequence set forth in any one of SEQ ID NOs: 23-46, 562-1076, 2778-4478, and 4553-4565, or a fragment thereof that comprises or consists of 21 continuous nucleotides in length or less (such as 20 continuous nucleotides, such as 19 continuous nucleotides, such as 18 continuous nucleotides, such as 17 continuous nucleotides, such as 16 continuous nucleotides, such as 15 continuous nucleotides, such as 14 continuous nucleotides, such as continuous 13 nucleotides, such as 12 continuous nucleotides, such as 11 continuous nucleotides, or such as 10 continuous nucleotides) of SEQ ID NO: 23-46, 562-1076, 2778-4478, and 4553-4565. The fragment may be, e.g., a truncation of SEQ ID NO: 23-46, 562-1076, 2778-4478, and 4553-4565 from the 5’ end (i.e., nucleotides at the 5’ end are removed).

[0289] In some aspects, gRNA can be chemically modified. For example, chemical modification may increase the stability of the gRNA once administrated in a target cell as described for example in (Yin et al., Nat Biotechnol. 2017 Dec;35(12):1179- 67#14533290vl1187). Such chemical modifications are known in the literature and can comprise but are not limited to locked nucleic acids (LNA), phosphorothioate modified oligonucleotides, 2'-0-methoxyethyl modified oligonucleotides, and 2' O-methyl modified oligonucleotides.

[0290] In some aspects, the first three nucleosides and the last three nucleosides of a gRNA, regardless of the gRNA’s length, are 2’-O-methyl modified nucleosides. In some aspects, the first three internucleoside linkages and the last three internucleoside linkages of a gRNA, regardless of the gRNA’s length, are phosphorothioate linkages.

[0291] For gRNA sequences having the tracr RNA of Seq ID No: 4567 (which is 80 nucleotides in length), the tracr sequence portion of the full-length gRNA may be modified as follows (with nucleoside 1 being at the 5’ end of the tracr RNA sequence, and nucleoside 80 being at the 3’ end of the tracr RNA sequence):nucleosides 1-8: unmodified RNA nucleosides,nucleosides 9-20: 2’-O-Me modified nucleosides,nucleosides 21-48: unmodified RNA nucleosides, andnucleosides 49-80: 2’-O-Me modified nucleosides.The modified gRNA of SEQ ID NO: 4567 is shown in SEQ ID NO: 4647. In such a modified tracr sequence, the internucleoside linkages between nucleosides 77 and 78, 78 and 79, and 79 and 80 (i.e., the last three internucleoside linkages) may be phosphorothioate linkages. A spacer RNA may be attached at the 5’ end of this modified tracr sequence to form a full-length gRNA. In this full-length gRNA, the tracr portion of the gRNA sequence is modified as described above, and the spacer portion of the gRNA sequence is modified as follows:the first three nucleosides of the spacer sequence are 2’-O-Me nucleosides, andthe first three internucleoside linkages are phosphorothioate linkages.The general schematic for this full-length gRNA is shown below, wherein lowercase letters represent 2’-O-Me nucleosides, capital letters represent unmodified RNA nucleosides, s represents a phosphorothioate linkage, each X independently represents an A, C, G, or II nucleoside, and each x represents a 2’-O-Me A, C, G, or II nucleoside:5 ' -xsxsxs [X7-X17] GUUUUAGAgcuagaaauagcAAGUUAAAAUAAGGCUAGU 68#14533290vlCCGUUAUCAacuugaaaaaguggcaccgagucggugcusususu-3 ' (SEQ ID NO: 4638)

[0292] More specifically, for gRNA sequences having full-length spacer RNAs (i.e., 20 nucleotides) and the tracr RNA of Seq ID No: 4567 (which is 80 nucleotides in length, for a gRNA of 100 nucleotides in length), the gRNA may be modified as follows (with nucleoside 1 being at the 5’ end of the oligonucleotide, and nucleotide 100 being at the 3’ end of the oligonucleotide):nucleosides 1-3: 2’-O-Me modified nucleosides,nucleosides 4-28: Unmodified RNA nucleosides,nucleosides 29-40: 2’-O-Me modified nucleosides,nucleosides 41-68: Unmodified RNA nucleosides, andnucleosides 69-100: 2’-O-Me modified nucleosides.In such a modified gRNA, the internucleoside linkages between nucleosides 1 and 2, 2 and 3, 3 and 4, 97 and 98, 98 and 99, and 99 and 100 (i.e., the first three internucleoside linkages and the last three internucleoside linkages) may be phosphorothioate linkages. The remainder of the internucleoside linkages are phosphate linkages.

[0293] Similar modifications may be made to truncated gRNAs (e.g., a gRNA with a spacer that is 11 to 19 nucleotides). For example, the first three and the last three internucleoside linkages of the gRNA may be phosphorothioate linkages, and / or some or all of the nucleotides may be chemically modified, e.g., 2’-O-methyl nucleotides.

[0294] For example, the sequence of SEQ ID NO: 4568 can be modified as follows:5 ' -asgsgsGUAGGAGAGACUCACGCGUUUUAGAgcuagaaauagcAAGUUAAAAU AAGGCUAGUCCGUUAUCAacuugaaaaaguggcaccgagucggugcusususu-3' (gRNA ID F4_20; SEQ ID NO: 4648)where:N: RNA nucleosides; n: 2'-O-methyl nucleosides; s: phosphorothioate backbone modification between two nucleosides.

[0295] Another example is the modification of the sequence of SEQ ID NO: 456969#14533290vl5 ' -gsasgsUAGCGCGAGCACAGCUAGUUUUAGAgcuagaaauagcAAGUUAAAAU AAGGCUAGUCCGUUAUCAacuugaaaaaguggcaccgagucggugcusususu-3 ' (gRNA ID C8_20; SEQ ID NO: 4649)

[0296] Exemplary full-length modified gRNAs targeting B2M are shown below:5 ' -csasusCGGCGCCCUCCGAUCUGGUUUUAGAgcuagaaauagcAAGUUAAAA UAAGGCUAGUCCGUUAUCAacuugaaaaaguggcaccgagucggugcusususu-3 ' (modified version of SEQ ID NO: 4570; gRNA ID H8_20; SEQ ID NO: 4650)5 ' -gscsgsGGCCACCAAGGAGAACUGUUUUAGAgcuagaaauagcAAGUUAAAA UAAGGCUAGUCCGUUAUCAacuugaaaaaguggcaccgagucggugcusususu-3 ' (modified version of SEQ ID NO: 4571; gRNA ID H10_20; SEQ ID NO: 4651)5 ' -csgsasUAAGCGUCAGAGCGCCGGUUUUAGAgcuagaaauagcAAGUUAAAA UAAGGCUAGUCCGUUAUCAacuugaaaaaguggcaccgagucggugcusususu-3 ' (modified version of SEQ ID NO: 4572; gRNA ID Hll_20; SEQ ID NO: 4652)5 ' -gsasasCGCGUGGAGGGGCGCUUGUUUUAGAgcuagaaauagcAAGUUAAAA UAAGGCUAGUCCGUUAUCAacuugaaaaaguggcaccgagucggugcusususu-3 ' (modified version of SEQ ID NO: 4573; gRNA ID H12_20; SEQ ID NO: 4653)

[0297] Exemplary truncated modified gRNAs targeting B2M are shown below:5 ' - c s a s c s CAAGGAGAACUGUUUUAGAgcuagaaauagcAAGUUAAAA UAAGGCUAGUCCGUUAUCAacuugaaaaaguggcaccgagucggugcusususu-3 ' (SEQ ID NO: 4576; gRNA ID H10_14)5 ' -gscsgsCCCUCCGAUCUGGUUUUAGAgcuagaaauagcAAGUUAAAA UAAGGCUAGUCCGUUAUCAacuugaaaaaguggcaccgagucggugcusususu-3 ' (SEQ ID NO: 4577; gRNA ID H8_15)5 ' -as gs c s GCGAGCACAGCUAGUUUUAGAgcuagaaauagcAAGUUAAAA UAAGGCUAGUCCGUUAUCAacuugaaaaaguggcaccgagucggugcusususu-3 ' (SEQ ID NO: 4578; gRNA ID C8_16)5 ' -usasgsGAGAGACUCACGCGUUUUAGAgcuagaaauagcAAGUUAAAA UAAGGCUAGUCCGUUAUCAacuugaaaaaguggcaccgagucggugcusususu-3 ' (SEQ ID NO: 4579; gRNA ID F4_16)

[0298] An exemplary full-length modified gRNA targeting TRAC is shown below:5 ' -asgsasGUCUCUCAGCUGGUACAGUUUUAGAgcuagaaauagcAAGUUAAAA70#14533290vlUAAGGCUAGUCCGUUAUCAacuugaaaaaguggcaccgagucggugcusususu-3 ' (modified version of SEQ ID NO: 4575; SEQ ID NO: 4654)

[0299] Exemplary truncated modified gRNAs targeting TET2 are shown below:5 ' -cscsgsUGCAGUGGCGCGGUUUUAGAgcuagaaauagcAAGUUAAAA UAAGGCUAGUCCGUUAUCAacuugaaaaaguggcaccgagucggugcusususu-3 ' (SEQ ID NO: 4580; gRNA ID sgRNA TE13_15)5 ' -csgscsCGGCCUUUGUGCGUUUUAGAgcuagaaauagcAAGUUAAAA UAAGGCUAGUCCGUUAUCAacuugaaaaaguggcaccgagucggugcusususu-3 ' (SEQ ID NO: 4581; gRNA ID sgRNA TE14_15)5 ' -gscsgsGGGCCGGCGUCUGUUUUAGAgcuagaaauagcAAGUUAAAA UAAGGCUAGUCCGUUAUCAacuugaaaaaguggcaccgagucggugcusususu-3 ' (SEQ ID NO: 4582; gRNA ID sgRNA TE19_15)5 ' -usgsasAUAUUGAUGCGGGUUUUAGAgcuagaaauagcAAGUUAAAA UAAGGCUAGUCCGUUAUCAacuugaaaaaguggcaccgagucggugcusususu-3 ' SEQ ID NO: 4583; gRNA ID sgRNA TE20_15)

[0300] Exemplary truncated modified gRNAs targeting TGFBR2 are shown below:5 ' -uscscsUCGCCAACAGCUGUUUUAGAgcuagaaauagcAAGUUAAAA UAAGGCUAGUCCGUUAUCAacuugaaaaaguggcaccgagucggugcusususu-3 ' (SEQ ID NO: 4584; gRNA ID sgRNA TG7_15)5 ' -asgsusCACUCGCGCGCAGUUUUAGAgcuagaaauagcAAGUUAAAA UAAGGCUAGUCCGUUAUCAacuugaaaaaguggcaccgagucggugcusususu-3 ' (SEQ ID NO: 4585; gRNA ID sgRNA TG8_15)5 ' -ascsusCCCGUAGCUGCAGUUUUAGAgcuagaaauagcAAGUUAAAA UAAGGCUAGUCCGUUAUCAacuugaaaaaguggcaccgagucggugcusususu-3 ' (SEQ ID NO: 4586; gRNA ID sgRNA TG19_15)5 ' -usgsusUGGCCGCGUUCGGUUUUAGAgcuagaaauagcAAGUUAAAA UAAGGCUAGUCCGUUAUCAacuugaaaaaguggcaccgagucggugcusususu-3 ' (SEQ ID NO: 4587; gRNA ID sgRNA TG20_15)

[0301] Exemplary full-length modified gRNAs targeting TET2 are shown below:5 ' -gsgsasAUUAGCUCUGUAUCGGUGUUUUAGAgcuagaaauagcAAGUUAAAA UAAGGCUAGUCCGUUAUCAacuugaaaaaguggcaccgagucggugcusususu-3 ' (SEQ ID NO: 4588; gRNA ID TE1_2O)5 ' -asasasGUAAGGGCUCUUACGAGGUUUUAGAgcuagaaauagcAAGUUAAAA UAAGGCUAGUCCGUUAUCAacuugaaaaaguggcaccgagucggugcusususu-3 ' (SEQ ID NO: 4589; gRNA ID TE2_20)71#14533290vl5 ' -gsgscsGUCUCACAGAUUGAAAUGUUUUAGAgcuagaaauagcAAGUUAAAA UAAGGCUAGUCCGUUAUCAacuugaaaaaguggcaccgagucggugcusususu-3 ' (SEQ ID NO: 4590; gRNA ID TE3_20)5 ' -csgsgsUCAAUUUCCCAGUUUGUGUUUUAGAgcuagaaauagcAAGUUAAAA UAAGGCUAGUCCGUUAUCAacuugaaaaaguggcaccgagucggugcusususu-3 ' (SEQ ID NO: 4591; gRNA ID TE4_20)5 ' -asgscsGCUCCCCUGUUUCACCGGUUUUAGAgcuagaaauagcAAGUUAAAA UAAGGCUAGUCCGUUAUCAacuugaaaaaguggcaccgagucggugcusususu-3 ' (SEQ ID NO: 4592; gRNA ID TE5_20)5 ' - c s gs c s GGGCAACGGGAUCUAAAGUUUUAGAgcuagaaauagcAAGUUAAAA UAAGGCUAGUCCGUUAUCAacuugaaaaaguggcaccgagucggugcusususu-3 ' (SEQ ID NO: 4593; gRNA ID TE6_20)5 ' -csgscsAAGCGGAGGUGUGGUGCGUUUUAGAgcuagaaauagcAAGUUAAAA UAAGGCUAGUCCGUUAUCAacuugaaaaaguggcaccgagucggugcusususu-3 ' (SEQ ID NO: 4594; gRNA ID TE7_20)5 ' -gsusgsCGGGUACACUCCGGAGGGUUUUAGAgcuagaaauagcAAGUUAAAA UAAGGCUAGUCCGUUAUCAacuugaaaaaguggcaccgagucggugcusususu-3 ' (SEQ ID NO: 4595; gRNA ID TE8_20)5 ' -usgscsGCGGGACCUCGAAGUGGGUUUUAGAgcuagaaauagcAAGUUAAAA UAAGGCUAGUCCGUUAUCAacuugaaaaaguggcaccgagucggugcusususu-3 ' (SEQ ID NO: 4596; gRNA ID TE9_20)5 ' -asgscsAGAGCAAGCGCGAAGGUGUUUUAGAgcuagaaauagcAAGUUAAAA UAAGGCUAGUCCGUUAUCAacuugaaaaaguggcaccgagucggugcusususu-3 ' (SEQ ID NO: 4597; gRNA ID TE10_20)5 ' -usgscsAGCCCUCGGGAACCCCGGUUUUAGAgcuagaaauagcAAGUUAAAA UAAGGCUAGUCCGUUAUCAacuugaaaaaguggcaccgagucggugcusususu-3 ' (SEQ ID NO: 4598; gRNA ID TE11_2O)5 ' -gsusgsGUGCGCCCGGACCAGCGGUUUUAGAgcuagaaauagcAAGUUAAAA UAAGGCUAGUCCGUUAUCAacuugaaaaaguggcaccgagucggugcusususu-3 ' (SEQ ID NO: 4599; gRNA ID TE12_20)5 ' -uscsasCGCCGUGCAGUGGCGCGGUUUUAGAgcuagaaauagcAAGUUAAAA UAAGGCUAGUCCGUUAUCAacuugaaaaaguggcaccgagucggugcusususu-3 ' (SEQ ID NO: 4600; gRNA ID TE13_20)5 ' -gsgsusGCCGCCGGCCUUUGUGCGUUUUAGAgcuagaaauagcAAGUUAAAA UAAGGCUAGUCCGUUAUCAacuugaaaaaguggcaccgagucggugcusususu-3 ' (SEQ ID NO: 4601; gRNA ID TE14_20)5 ' -gscsasCCGGGCGUCCAGCACAAGUUUUAGAgcuagaaauagcAAGUUAAAA UAAGGCUAGUCCGUUAUCAacuugaaaaaguggcaccgagucggugcusususu-3 '72#14533290vl(SEQ ID NO: 4602; gRNA ID TE15_20)5 ' -asgsgsGAAUUAGCCCCCCGCACGUUUUAGAgcuagaaauagcAAGUUAAAA UAAGGCUAGUCCGUUAUCAacuugaaaaaguggcaccgagucggugcusususu-3 ' (SEQ ID NO: 4603; gRNA ID TE16_20)5 ' -asgsusGGCAGCGGCGAGAGCUUGUUUUAGAgcuagaaauagcAAGUUAAAA UAAGGCUAGUCCGUUAUCAacuugaaaaaguggcaccgagucggugcusususu-3 ' (SEQ ID NO: 4604; gRNA ID TE17_20)5 ' -ascsusUGCAUGCGAGCGGGACCGUUUUAGAgcuagaaauagcAAGUUAAAA UAAGGCUAGUCCGUUAUCAacuugaaaaaguggcaccgagucggugcusususu-3 ' (SEQ ID NO: 4605; gRNA ID TE18_20)5 ' -ascsusCAGCGGGGCCGGCGUCUGUUUUAGAgcuagaaauagcAAGUUAAAA UAAGGCUAGUCCGUUAUCAacuugaaaaaguggcaccgagucggugcusususu-3 ' (SEQ ID NO: 4606; gRNA ID TE19_20)5 ' -cscsusUAUGAAUAUUGAUGCGGGUUUUAGAgcuagaaauagcAAGUUAAAA UAAGGCUAGUCCGUUAUCAacuugaaaaaguggcaccgagucggugcusususu-3 ' (SEQ ID NO: 4607; gRNA ID TE20_20)

[0302] Exemplary full-length modified gRNAs targeting TGFBR2 are shown below:5 ' -ususcsUUUAGGUCGAAGUCUAGGUUUUAGAgcuagaaauagcAAGUUAAAA UAAGGCUAGUCCGUUAUCAacuugaaaaaguggcaccgagucggugcusususu-3 ' (SEQ ID NO: 4608; gRNA ID TG1_2O)5 ' -gsusgsCUCGCGACUCAAUAGAUGUUUUAGAgcuagaaauagcAAGUUAAAA UAAGGCUAGUCCGUUAUCAacuugaaaaaguggcaccgagucggugcusususu-3 ' (SEQ ID NO: 4609; gRNA ID TG2_20)5 ' -asascsGCAUCUCUAAAGCACCUGUUUUAGAgcuagaaauagcAAGUUAAAA UAAGGCUAGUCCGUUAUCAacuugaaaaaguggcaccgagucggugcusususu-3 ' (SEQ ID NO: 4610; gRNA ID TG3_20)5 ' -csusgsAUCUACUAGGGAAAACGGUUUUAGAgcuagaaauagcAAGUUAAAA UAAGGCUAGUCCGUUAUCAacuugaaaaaguggcaccgagucggugcusususu-3 ' (SEQ ID NO: 4611; gRNA ID TG4_20)5 ' -ususgsAGUAAAUACUUGGAGCGGUUUUAGAgcuagaaauagcAAGUUAAAA UAAGGCUAGUCCGUUAUCAacuugaaaaaguggcaccgagucggugcusususu-3 ' (SEQ ID NO: 4612; gRNA ID TG5_20)5 ' -asgsusCGGCCAAAGCUCUCGGAGUUUUAGAgcuagaaauagcAAGUUAAAA UAAGGCUAGUCCGUUAUCAacuugaaaaaguggcaccgagucggugcusususu-3 ' (SEQ ID NO: 4613; gRNA ID TG6_20)5 ' -gsasasACUCCUCGCCAACAGCUGUUUUAGAgcuagaaauagcAAGUUAAAA73#14533290vlUAAGGCUAGUCCGUUAUCAacuugaaaaaguggcaccgagucggugcusususu-3 ' (SEQ ID NO: 4614; gRNA ID TG7_20)5 ' -gsasgsUGAGUCACUCGCGCGCAGUUUUAGAgcuagaaauagcAAGUUAAAA UAAGGCUAGUCCGUUAUCAacuugaaaaaguggcaccgagucggugcusususu-3 ' (SEQ ID NO: 4615; gRNA ID TG8_20)5 ' -csgscsGUGCACCCGCUCGGGACGUUUUAGAgcuagaaauagcAAGUUAAAA UAAGGCUAGUCCGUUAUCAacuugaaaaaguggcaccgagucggugcusususu-3 ' (SEQ ID NO: 4616; gRNA ID TG9_20)5 ' -gsgsgsGCCUCCCCGCGCCUCGCGUUUUAGAgcuagaaauagcAAGUUAAAA UAAGGCUAGUCCGUUAUCAacuugaaaaaguggcaccgagucggugcusususu-3 ' (SEQ ID NO: 4617; gRNA ID TG10_20)5 ' -usgsgsCGAGCGGGCGCCACAUCGUUUUAGAgcuagaaauagcAAGUUAAAA UAAGGCUAGUCCGUUAUCAacuugaaaaaguggcaccgagucggugcusususu-3 ' (SEQ ID NO: 4618; gRNA ID TG11_2O)5 ' -uscsgsGUCUAUGACGAGCAGCGGUUUUAGAgcuagaaauagcAAGUUAAAA UAAGGCUAGUCCGUUAUCAacuugaaaaaguggcaccgagucggugcusususu-3 ' (SEQ ID NO: 4619; gRNA ID TG12_20)5 ' -cscsusGAGCAGCCCCCGACCCAGUUUUAGAgcuagaaauagcAAGUUAAAA UAAGGCUAGUCCGUUAUCAacuugaaaaaguggcaccgagucggugcusususu-3 ' (SEQ ID NO: 4620; gRNA ID TG13_20)5 ' -gsgsasCGAUGUGCAGCGGCCACGUUUUAGAgcuagaaauagcAAGUUAAAA UAAGGCUAGUCCGUUAUCAacuugaaaaaguggcaccgagucggugcusususu-3 ' (SEQ ID NO: 4621; gRNA ID TG14_20)5 ' -usgscsUGGCGAUACGCGUCCACGUUUUAGAgcuagaaauagcAAGUUAAAA UAAGGCUAGUCCGUUAUCAacuugaaaaaguggcaccgagucggugcusususu-3 ' (SEQ ID NO: 4622; gRNA ID TG15_20)5 ' -asascsGUGCGGUGGGAUCGUGCGUUUUAGAgcuagaaauagcAAGUUAAAA UAAGGCUAGUCCGUUAUCAacuugaaaaaguggcaccgagucggugcusususu-3 ' (SEQ ID NO: 4623; gRNA ID TG16_20)5 ' -gsascsUGUCAAGCGCAGCGGAGGUUUUAGAgcuagaaauagcAAGUUAAAA UAAGGCUAGUCCGUUAUCAacuugaaaaaguggcaccgagucggugcusususu-3 ' (SEQ ID NO: 4624; gRNA ID TG17_20)5 ' -csususUCCUCGUUUCCGCCCGGGUUUUAGAgcuagaaauagcAAGUUAAAA UAAGGCUAGUCCGUUAUCAacuugaaaaaguggcaccgagucggugcusususu-3 ' (SEQ ID NO: 4625; gRNA ID TG18_20)5 ' -gscscsCGACUCCCGUAGCUGCAGUUUUAGAgcuagaaauagcAAGUUAAAA UAAGGCUAGUCCGUUAUCAacuugaaaaaguggcaccgagucggugcusususu-3 ' (SEQ ID NO: 4626; gRNA ID TG19_20)74#14533290vl5 ' -csgsusUGUGUUGGCCGCGUUCGGUUUUAGAgcuagaaauagcAAGUUAAAA UAAGGCUAGUCCGUUAUCAacuugaaaaaguggcaccgagucggugcusususu-3 ' (SEQ ID NO: 4627; gRNA ID TG20_20)

[0303] An exemplary full-length modified gRNA targeting GFP is shown below:5 ' -csuscsCUCGCCCUUGCUCACCAGUUUUAGAgcuagaaauagcAAGUUAAAA UAAGGCUAGUCCGUUAUCAacuugaaaaaguggcaccgagucggugcusususu-3 ' (SEQ ID NO: 4628; gRNA ID GFP1)

[0304] In some aspects, the present invention utilizes two or more gRNAs.

[0305] Suitably, the two or more gRNAs may target the ETM (e.g., ETR) to different target genes. Suitably, the two or more gRNAs may comprise spacer sequences of different lengths. For example, the spacer sequences of different lengths may target the endonuclease of the ETM (e.g., ETR) to different target genes.

[0306] In some aspects, a two or more gRNAs may target the same target gene. For example, it may be beneficial to target the same gene with two gRNAs for optimal epigenetic modification e.g., epigenetic silencing.

[0307] In an aspect, at least one of the at least two gRNAs comprises a spacer sequence which is 18, 19 or 20 nucleotides in length.

[0308] In an aspect, at least one of the at least two gRNAs comprises a spacer sequence which is less than or equal to 17 nucleotides in length, such as 16 nucleotides in length, 15 nucleotides in length, such as 14 nucleotides in length, such as less than 13 nucleotides in length, such as 12 nucleotides in length, such as 11 nucleotides in length, or such as 10 nucleotides in length.Multiplexing - modifying multiple genes in the same cell

[0309] The present invention relates to the development of a combined gene editing and epigenetic editing strategy to modify the expression and / or activity of multiple target genes within the same cell. In particular, it may exploit an ETM e.g., ETR) which comprises an epigenetic effector domain and an endonuclease and gRNAs comprising spacer sequences of different lengths to promote epigenetic editing of one or more genes and genetic editing of another gene.75#14533290vl

[0310] As used herein “modify the expression and / or activity” refers to increasing or decreasing (e.g., decreasing) the expression and / or activity of a target gene.

[0311] In an aspect, transcription and / or expression of a target gene may be repressed.

[0312] In an aspect, a target gene may be silenced.

[0313] In an aspect, a target gene may be enhanced. In other words, the expression of the target gene may be increased. For example, the expression of an endogenous target gene may be increased.

[0314] In another example, an endogenous target (e.g., gene) may be modified (e.g., mutated) by gene editing and the expression of the modified target (e.g., gene) may be increased.

[0315] The effect of an ETM or combination of ETMs may be studied by comparing the transcription or expression of the target gene, for example a gene endogenous to a cell, in the presence and absence of the ETM or combination of ETMs. Methods of analysing transcription or expression of a gene are well known in the art.

[0316] The effect of an ETM or a combination of ETM and gRNAs may also be studied using a model system wherein the expression of a reporter gene, for example a gene encoding a fluorescent protein, is monitored. Suitable methods for monitoring expression of such reporter genes include flow cytometry, fluorescence-activated cell sorting (FACS) and fluorescence microscopy.

[0317] For example, a population of cells may be transfected with a vector which harbours a reporter gene. The vector may be constructed such that the reporter gene is expressed when the vector transfects a cell. Suitable reporter genes include genes encoding fluorescent proteins, for example green, yellow, cherry, cyan or orange fluorescent proteins. In addition, the population of cells may be transfected with vectors encoding the ETMs of interest and / or gRNAs. Subsequently, the number of cells expressing and not-expressing the reporter gene, as well as the level of expression of the reporter gene may be quantified using a suitable technique, such as FACS. The level of reporter gene expression may then be compared in the presence and absence of the ETM and / or gRNAs.

[0318] Methods for determining the transcription of a gene, for example the target of an ETM, are known in the art. Suitable methods include reverse transcription PCR 76#14533290vland Northern blot-based approaches. In addition to the methods for determining the transcription of a gene, methods for determining the expression of a gene are known in the art. Suitable additional methods include Western blot-based or flow cytometry approaches.Target gene transcription and expression

[0319] In some aspects, the product (e.g., ETM and / or gRNA) according to the present invention is used in a method which represses transcription and / or expression of at least one target gene. Suitably, the target gene may be an endogenous gene.

[0320] In an aspect, the target gene transcription and / or expression is repressed by epigenetic editing. In an aspect, the target gene transcription and / or expression is repressed by gene editing.

[0321] In some aspects, the product (e.g., ETM and / or gRNA) according to the present invention is used in a method which represses transcription and / or expression of at least two target genes. Suitably, at least one or both of the target genes may be an endogenous gene.

[0322] In an aspect, transcription and / or expression of only one gene is repressed by gene editing.

[0323] Following administration of an ETM (e.g., ETR) of the invention (e.g., with suitable gRNA(s)), the level of transcription or expression of the target gene may be reduced by, for example, at least 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 99% or 100% compared to the level of transcription or expression in the absence of the ETM (e.g., ETR).

[0324] In some aspects, the product (e.g., ETM and / or gRNA) according to the present invention is used in a method which silences at least one target gene.Suitably, the target gene may be an endogenous gene. Suitably, the target gene may be an exogenous gene, such as a viral gene.

[0325] In an aspect, the target gene is silenced by epigenetic editing. In an aspect, the target gene is silenced by gene editing.

[0326] In some aspects, the product (e.g., ETM and / or gRNA) according to the present invention is used in a method which silences at least two target genes.Suitably, at least one or both of the target genes may be an endogenous gene.77#14533290vl

[0327] In an aspect, only one gene is silenced by gene editing.

[0328] Without wishing to be bound by theory, restricting gene editing activity to one gene may reduce the potential for undesirable genomic translocations.

[0329] By “silencing a target gene”, it is to be understood that the expression of the target gene is reduced to an extent sufficient to achieve a desired effect. The reduced expression may be sufficient to achieve a therapeutically relevant effect, such as the prevention or treatment of a disease. For example, a dysfunctional target gene which gives rise to a disease may be repressed to an extent that there is either no expression of the target gene, or the residual level of expression of the target gene is sufficiently low to ameliorate or prevent the disease state. Furthermore, the reduced expression may allow for purification of the cells harbouring gene silencing.

[0330] The reduced expression may be sufficient to enable investigations to be performed into the gene’s function by studying cells reduced in or lacking that function.

[0331] The repression of the target gene may occur, e.g., following transient delivery or expression of the ETMs (e.g., ETRs) of the present invention to or in a cell (e.g., along with suitable gRNAs).Enhancing a target gene

[0332] By “enhancing a target gene”, it is to be understood that the expression of the target gene is increased to an extent sufficient to achieve a desired effect. The increased expression may be sufficient to achieve a therapeutically relevant effect, such as the prevention or treatment of a disease. For example, a dysfunctional target gene which gives rise to a disease may be enhanced to an extent that there is sufficient expression of the target gene to ameliorate or prevent the disease state. Alternatively, increased expression of the target gene may compensate for the dysfunctional activity of a disease-related gene. Furthermore, increased expression of the target gene may allow for selection of the cells expressing de novo that specific target gene.

[0333] Following administration of an ETM of the invention (e.g., with suitable gRNA(s)), the level of transcription or expression of the target gene may be increased by, for example, at least 50%, 60%, 70%, 80%, 90%, 95%, 99%, 100%,78#14533290vl200%, 300%, 400% or 500% compared to the level of transcription or expression in the absence of the ETM.

[0334] The enhancement of the target gene may occur, e.g., following transient delivery or expression of the ETMs of the present invention to or in a cell (along with suitable gRNAs).Transient expression

[0335] By “transient expression”, it is to be understood that the expression of the ETM e.g., ETR) is not stable over a prolonged period of time. For example, the polynucleotide encoding the ETM (e.g., ETR) may not integrate into the host genome. More specifically, transient expression may be expression which is substantially lost within 20 weeks following introduction of the polynucleotide encoding the ETM (e.g., ETR) into the cell. For example, expression may be substantially lost within 12, 6, 4, or 2 weeks following introduction of the polynucleotide encoding the ETM (e.g., ETR) into the cell.

[0336] Similarly, by “transient delivery”, it is to be understood that the ETM (e.g., ETR) substantially does not remain in the cell (i.e., is substantially lost by the cell) over a prolonged period of time. More specifically, transient delivery may result in the ETM (e.g., ETR) being substantially lost by the cell within 20 weeks following introduction of the ETM (e.g., ETR) into the cell. For example, the ETM (e.g., ETR) may be substantially lost within 12, 6, 4, or 2 weeks following introduction of the ETM (e.g., ETR) into the cell.

[0337] In an aspect, the ETM and / or gRNA may be delivered transiently. Transient delivery may result in permanent changes for example; transient delivery of the ETM and / or gRNA may lead to DNA methylation of a repressive regulatory element which in turn may lead to gene activation (e.g., given the stability of this epigenetic modification, permanent gene activation).

[0338] The target gene may, for example, be repressed, silenced, or enhanced permanently. By “permanent repression”, “permanent silencing” or “permanent enhancement” of a target gene, it is to be understood that transcription or expression of the target gene is reduced or increased (e.g., reduced or increased by at least 60%, at least 70%, at least 80%, at least 90% or 100%) compared to the level of transcription or expression in the absence of the ETM (e.g., ETR) for at least 279#14533290vlmonths, 6 months, 1 year, 2 year or the entire lifetime of the cell / organism. For example, a permanently repressed, silenced, or enhanced target gene may remain repressed, silenced, or enhanced for the remainder of the cell’s life.

[0339] In an aspect, the ETM and / or gRNA is stably expressed. For example, stable expression may be required to achieve permanent gene activation of some targets. The target gene may, for example, remain repressed, silenced, or enhanced in the progeny of the cell to which the product of the invention has been administered (i.e., the repression, silencing or enhancement of the target gene is inherited by the cell’s progeny). For example, the ETM (e.g., ETR) and gRNAs of the invention may be administered to a stem cell (e.g., a hematopoietic stem cell) to repress or silence a target gene in a stem cell and also in the stem cell’s progeny, which may include cells that have differentiated from the stem cell.Target gene

[0340] The target gene may, for example, give rise to a therapeutic effect when modified, e.g., repressed or silenced.

[0341] The products, of the present invention may be used to modify, e.g., repress or silence, genes without CpG islands (CGI). A CpG island generally refers to a nucleic acid sequence or chromosome region that comprises a high frequency of CpG dinucleotides, e.g., located at or near the transcription start site (TSS) of a gene (see, e.g., Deaton and Bird, CpG islands and the regulation of transcription. Genes Dev. 2011 May 15;25(10):1010-22, the entire contents of which are incorporated herein by reference. Genes without CGI include: TRAC TRBC; PDCD1; TIM-3; TIGIT; LAG3; CTLA4; AAVS1; RFX5; CD3D; CD3G and CCR5.

[0342] For example, targeting genes, such as genes without a CGI, may:produce allogenic products e.g., by targeting TRAC and / or TRABC); alter resistance to an immunosuppressive tumour microenvironment (e.g., by targeting of PDCD1, TIM-3, TIGIT, LAG3 and / or CTLA4); and / orallow CAR / transgenic TCR integration in a safe site (e.g., by targeting of AAVS1 and / or CCR5).80#14533290vlC1698.70059WO00

[0343] In an aspect, the present invention provides gRNAs which target a sequence set forth in any one of SEQ ID NOs: 47 to 561.

[0344] By way of example, target genes without CGI islands and exemplary gRNAs suitable for targeting said genes are presented in Table 1 below (SEQ indicates SEQ ID NO).Table 1. Target genes without CGI islands and exemplary gRNAs81#14533290vl82#14533290vl83#14533290vl84#14533290vl85#14533290vl86#14533290vl87#14533290vl88#14533290vl89#14533290vl90#14533290vl91#14533290vl

[0345] Products of the present invention may be used to modify, e.g., repress or silence, genes having CpG islands (CGI). Genes having CGI include: B2M; TET2; TGFBR2; A2AR; CISH; PTPN11; PTPN6; PTPA; PTPN2; JUNB; TOX; TOX2;NR4A1; NR4A2; NR4A3; MAP4K1; REL; IRF4; DGKA; PIK3CD; HLA-A; USP16; DCK; CD3Z; CUT A; ADORA2A; RFXAP and FAS.

[0346] For example, targeting genes, such as genes with a CGI, may:produce allogenic products e.g., by targeting B2M and / or HLA-A)alter resistance to an immunosuppressive tumour microenvironment (e.g., by targeting of TGFBR2, A2AR, PTPN11, PTPN6, PTPN2, and / or DGKA);allow CAR / transgenic TCR integration in a safe site (e.g., by targeting of AAVS1 and / or CCR5);provide resistance to exhaustion (e.g., by targeting of FAS, CISH, PTPA, PIK3CD, MAP4K1, NR4A1, NR4A2, NR4A3, JUNB, REL, TOX, TOX2, IRF4 and / or TET2); and / ordelay T cell senescence (e.g., by targeting USP16).

[0347] Silencing of the TCR genes, PDCD1 and CTLA4 may be used to improve efficacy of cancer immunotherapy approaches.

[0348] Silencing of B2M may be used to generate allogeneic HSPCs, T cells or mesenchymal cells to be used for transplantation.Table 2. Target genes having CGI islands and exemplary gRNAs92#14533290vl93#14533290vl94#14533290vl95#14533290vl96#14533290vl97#14533290vl98#14533290vl99#14533290vl100#14533290vl101#14533290vl102#14533290vl103#14533290vl104#14533290vl105#14533290vl106#14533290vl107#14533290vl108#14533290vl109#14533290vl110#14533290vl111#14533290vl112#14533290vl113#14533290vl114#14533290vl115#14533290vl116#14533290vl117#14533290vl118#14533290vl119#14533290vl120#14533290vl121#14533290vl122#14533290vl123#14533290vl124#14533290vl125#14533290vl126#14533290vl127#14533290vl128#14533290vl129#14533290vl130#14533290vl131#14533290vl132#14533290vl133#14533290vl134#14533290vl

[0349] In an aspect, the present invention provides gRNAs which target a sequence set forth in any one of SEQ ID NOs: 1077-2777, 4539-4545, and 4547-4552.135#14533290vl

[0350] By way of example, target genes having CGI and exemplary gRNAs suitable for targeting said genes are presented in Table 2 above (SEQ indicates: SEQ ID NO).

[0351] It will be appreciated that it may be beneficial to increase the expression of certain targets. For example, c-jun is a gene that when activated, may be beneficial; for example, increased expression in T cells may increase cell viability.Donor Template Nucleic Acids

[0352] In an aspect, the present invention provides one or more donor template nucleic acids for use as a HDR (homology-directed repair) template with an ETM described herein for targeted integration of the donor template nucleic acid into a locus in the genome (for example, a gene locus). In some embodiments, the locus is a genomic safe harbor locus (also referred to herein as a safe harbor genomic locus or a safe harbor locus) is following genes: TRAC, TRBC, CCR5, AAVS1, Rosa26, or any other suitable genomic safe harbor locus provided herein or known in the art. Some suitable genomic safe harbor loci are provided herein and additional suitable safe harbor loci will be apparent to the skilled artisan based on the present disclosure and the knowledge in the art, including, but not limited to those disclosed in Eyquem et al., Nature 2017 Mar 2;543(7643):113-117; Aznauryan et al., Cell Rep Methods. 2022; 2(1 ):100154; and Papapetrou and Schambach, Mol Ther. 2016 Apr;24(4):678-84.

[0353] As used herein, the term homology directed repair (HDR) refers to a cellular process in which cut or nicked ends of a nucleic acid e.g. DNA) strand, e.g., a genomic DNA strand in a target cell that is cut or nicked by an ETM provided herein, are repaired by polymerization from a homologous template nucleic acid, e.g., a donor template nucleic acid comprising a nucleic acid sequence homologous to the cut or nicked sequence at both the 5’ and 3’ ends, resulting in insertion of the homologous template nucleic acid into the cut site. Thus, the original targeted sequence is replaced with the sequence of the homologous donor template. If the homologous donor template nucleic acid comprises a heterologous sequence, for example, a heterologous expression cassette or a transgene flanked on both sides by sequences that are homologous to the cut or nicked site sequence (also sometimes referred to as ‘homology arms’), the insertion of the donor template 136#14533290vlnucleic acid results in the insertion of the heterologous sequence at the cut or nicked site. An exemplary structure of a suitable donor nucleic acid template comprising a transgene flanked by homology arms is provided below:5’- [5 ’homology arm] - [transgene] - [3’ homology arm]-3’.

[0354] Some exemplary suitable methods and modalities for generating donor templates and for the use of such donor templates in combination with DNA-cutting or -nicking nucleases, for example ETMs as provided herein, for HDR-mediated insertion of transgene sequences at a target site are provided herein. Additional suitable methods and modalities useful for donor templates and for targeting specific nucleic acid sequences will be apparent to those of skill in the art based on the present disclosure and the knowledge in the art, e.g., regarding HDR-mediated transgene insertion into specific nucleic acid target sites via CRISPR / Cas-mediated HDR (see, e.g., Liao et al., 2024, CRISPR-Cas9-mediated homology-directed repair for precise gene editing Molecular Therapy Nucleic Acids 35(4)-102344.In some embodiments of this disclosure, a donor template nucleic acid (also referred to herein as a donor template, a homologous donor template, or a homologous donor nucleic acid) is provided in combination with an ETM that cuts or nicks a target nucleic acid, e.g., a target nucleic acid sequence comprised in the genomic DNA of a target cell, at a specific site.

[0355] In some embodiments, a transgene is delivered via HDR using a methods and / or modality provided herein, e.g., via HDR-mediated strand break repair of a target nucleic acid cut or nicked by an ETM provided herein, In some such embodiments, the transgene comprises a nucleic acid sequence that encodes a therapeutic protein, for example, a protein the expression of which ameliorates a symptom of a disease or disorder in a subject in need thereof. Any suitable transgene encoding a therapeutic protein can be delivered to target cells in this manner, including, for example, a transgene encoding a therapeutic chimeric antigen receptor (CAR), a therapeutic T Cell receptor (TCR), a therapeutic binder e.g., an antibody, or antibody fragment), or a protein the loss of function of which is associated with a disease or disorder in a subject (an approach also sometimes referred to as gene replacement or gene addition). Some suitable exemplary transgenes which can be used as donor template nucleic acids for use in137#14533290vlcombination with ETMs provided herein, include transgenes encoding a chimeric antigen receptor (CAR), which, when expressed in a lymphocyte, e.g., a T-cell, NK-cell, or macrophage, can direct the cytotoxic activity of such a CAR-expressing cell towards malignant cells expressing the binding target of the CAR. A number of suitable CARs are known in the art. For example, as described in Mitra et al., Front Immunol 2023 14:1188049. CAR Suitable CARs that can be expressed by delivering a CAR-encoding donor template nucleic acid to such lymphocytes include CARs binding CD19 e.g., for targeting lymphocytes such as T-cells or NK-cells to B-cell leukemia and lymphoma cells), CARs binding CD20 (e.g., for targeting lymphocytes such as T cells or NK cells to B-cell leukemia and lymphoma cells), CARs binding BCMA (e.g., for targeting lymphocytes such as T cells or NK cells to myeloma cells), CARs binding PSMA (e.g., for targeting lymphocytes such as T cells or NK cells to prostate tumor cells), CARs binding EGFR (e.g., EGFRvlll for targeting lymphocytes such as T cells or NK cells to certain cancer cells expressing EGFRvlll), CARs binding HER2 (e.g., for targeting lymphocytes such as T cells or NK cells to certain cancer cells expressing HER2), and CARs binding MSLN (e.g., for targeting lymphocytes such as T cells or NK cells to certain ovarian, lung, and pancreatic cancer cells expressing MSLN).

[0356] Suitable transgenes for gene replacement or gene addition include, for example, a cystic fibrosis transmembrane conductance regulator (CFTR) gene, a beta-sarcoglycan gene, an aspartoacylase (ASPA) gene, or a Hemoglobin subunit beta (HBB) gene.

[0357] In an aspect, the donor template nucleic acid provides a CAR sequence for targeted knock-in. Suitable CAR sequences for use with the present invention are well known in the art. Non-limiting exemplary CARs can be found in Bhaskar et al., Clin Hematol Int. 202423;6(4):93-99; Vera et al., Hum Vaccin Immunother. 2024 20(1):2378543; Zugasti et al., Signal Transduction and Targeted Therapy 10: 210 (2025); Curr Probl Cancer 2022;46(1):100826; Brudno et al., JAMA. 2024 332(22):1924-1935; each of which are incorporated herein by reference in their entirety.

[0358] In other aspects the donor template nucleic acid provides a functional gene replacement sequence for replacement of a mutated or otherwise defective or dysfunctional gene sequence for use in gene therapy. Suitable gene replacement 138#14533290vlsequences for use with the present invention are well known in the art. Non-limiting gene replacement examples can be found in Azhagiri et al., Stem Cell Research & Therapy 12, 500 (2021 ); Butt et al., Mol Ther. 2025;33(5):2154-2171; Park et al., Transfus Apher Sci. 2021;60(1 ):103060; each of which are incorporated herein by reference in their entirety.Cell

[0359] In an aspect, the present invention provides a cell comprising an ETM (e.g., ETR) according to the present invention, at least one gRNA according the present invention, a combination according to the present invention, a polynucleotide according to the present invention, a nucleic acid construct according to the present invention, a vector according to the present invention or a kit of polynucleotides according to the present invention.

[0360] The cell may be any cell which can be used to express the product of the invention.

[0361] The cell may be an immune effector cell. An “immune effector cell” is a cell which has differentiated into a form capable of modulating or effecting a specific immune response. Immune effector cells may include alpha / beta T cells, gamma / delta T cells, B cells, natural killer (NK) cells, neutrophils, basophils, eosinophils, and macrophages. Suitably, the cell may be an alpha / beta T cell.Suitably, the cell may be a B cell. Suitably, the cell may be a gamma / delta T cell. Suitably, the cell may be a T cell, such as a cytolytic T cell, e.g., a CD8+ T cell.Suitably, the cell may be an NK cell, such as a cytolytic NK cell. Suitably, the cell may be a macrophage.

[0362] In an aspect, the cell may be a stem cell. A “stem cell” refers to an undifferentiated cell which is capable of indefinitely giving rise to more stem cells of the same type, and from which other, specialized cells may arise by differentiation. Adult stem cells are usually multipotent, while induced or embryonic-derived stem cells are pluripotent.

[0363] In another aspect, the cell may be a progenitor cell. A “progenitor cell” refers to a cell which is able to differentiate to form one or more types of cells but has limited self-renewal in vitro and in vivo.139#14533290vl

[0364] Suitably, the cell may be capable of being differentiated into a T cell.Suitably, the cell may be capable of being differentiated into an NK cell. Suitably, the cell may be capable of being differentiated into a macrophage. Suitably, the cell may be an embryonic stem cell (ESC). Suitably, the cell may be a hematopoietic stem cell or hematopoietic progenitor cell. Suitably, the cell may be an induced pluripotent stem cell (iPSC). Suitably, the cell may be obtained from umbilical cord blood.Suitably, the cell may be obtained from adult peripheral blood or mobilized form the bone marrow.

[0365] A “hematopoietic stem and progenitor cell” or “HSPC” refers to a cell which expresses the antigenic marker CD34 (CD34+) and populations of such cells. In particular embodiments, the term “HSPC” refers to a cell identified by the presence of the antigenic marker CD34 (CD34+) and the absence of lineage (lin) markers. The population of cells comprising CD34+ and / or Lin(-) cells includes hematopoietic stem cells and hematopoietic progenitor cells.

[0366] HSPCs can be obtained or isolated from bone marrow of adults, which includes femurs, hip, ribs, sternum, and other bones. Bone marrow aspirates containing HSPCs can be obtained or isolated directly from the hip using a needle and syringe. Other sources of HSPCs include umbilical cord blood, placental blood, mobilized peripheral blood, Wharton's jelly, placenta, fetal blood, fetal liver, or fetal spleen. In particular embodiments, harvesting a sufficient quantity of HSPCs for use in therapeutic applications may require mobilizing the stem and progenitor cells in the subject.

[0367] As used herein, the term “induced pluripotent stem cell” or “iPSC” refers to a non-pluripotent cell that has been reprogrammed to a pluripotent state. Once the cells of a subject have been reprogrammed to a pluripotent state, the cells can then be programmed to a desired cell type, such as a hematopoietic stem or progenitor cell (HSC and HPC respectively).

[0368] As used herein, the term “reprogramming” refers to a method of increasing the potency of a cell to a less differentiated state and “programming” refers to a method of decreasing the potency of a cell or differentiating the cell to a more differentiated state.140#14533290vl

[0369] Suitably, the cell may be matched or is autologous to the subject. The cell may be generated ex vivo either from a patient’s own peripheral blood, or from donor peripheral blood.

[0370] Suitably, the cell may be autologous to the subject. In some aspects, the cell may be derived from ex vivo differentiation of inducible progenitor cells or embryonic progenitor cells to the immune cell.

[0371] In these instances, cells are generated by introducing DNA or RNA coding for the ETM (e.g., ETR) of the present invention by one of any means including transduction with a viral vector or transfection with DNA or RNA.

[0372] In some aspects, the cell further comprises a polynucleotide, such as an integrating vector, which encodes an agent:i) which promotes the survival, proliferation and / or activity of a cell, such as a cell which comprises the polynucleotide or a cell which does not comprise the polynucleotide; and / orii) which is detrimental to the survival, proliferation, activity, chemoresistance and / or chemotaxis of a cell, such as a cell which comprises the polynucleotide or a cell which does not comprise the polynucleotide and / oriii) which enables selection of a cell, such as a cell which comprises the polynucleotide or a cell which does not comprise the polynucleotide.Combinations

[0373] In an aspect, the present invention provides a combination (e.g., a system) comprising an ETM (e.g., ETR) according to the present invention, and at least one gRNA which targets the endonuclease of the ETM (e.g., ETR) to a target gene.

[0374] The combination may comprise at least two gRNAs (such as at least three, at least four, at least five, at least six, at least seven, or at least eight gRNAs).

[0375] The combination may comprise gRNAs which target the endonuclease to at least two different target genes.141#14533290vl

[0376] In some embodiments, one target gene may be targeted with two or more gRNAs. For example, it may be beneficial to target the same gene with several gRNAs for optimal epigenetic modification, e.g., epigenetic silencing.

[0377] The combination may comprise at least two gRNAs which comprise spacer sequences of different lengths. Suitably, at least one gRNA comprises a spacer sequence which is 15, 16, 17, 18, 19 or 20 nucleotides in length. Suitably, at least one of the at least two gRNAs comprises a spacer sequence which is less than or equal to 17 e.g., less than or equal to 16) nucleotides in length. Suitably, at least one of the at least two gRNAs comprises a spacer sequence which is less than or equal to 17 (e.g., less than or equal to 16) nucleotides in length and at least one of the at least two gRNAs comprises a spacer sequence which is more than 17 nucleotides in length.

[0378] Without wishing to be bound by theory, the gRNAs comprising spacer sequences of different lengths may target the ETM (e.g., ETR) to different target genes, wherein a first target gene is modified by gene editing and at least a second target gene is modified by epigenetic editing.

[0379] In an aspect, the combination comprises at least one gRNA according to the present invention. Suitably, the combination may comprise at least two gRNAs according to the present invention.

[0380] Suitably, the combination may comprise a first gRNA and a second gRNA having the sequences of C8 and F4, respectively, optionally wherein the combination further comprises a third gRNA having the sequence of H8, H10, H11, or H12.

[0381] Suitably, the combination may comprise a first gRNA and a second gRNA having the sequences of C8 and H8, respectively, optionally wherein the combination further comprises a third gRNA having the sequence of F4, H10, H11, or H12.

[0382] Suitably, the combination may comprise a first gRNA and a second gRNA having the sequences of C8 and H10, respectively, optionally wherein the combination further comprises a third gRNA having the sequence of F4, H8, H11, or H12.

[0383] Suitably, the combination may comprise a first gRNA and a second gRNA having the sequences of C8 and H11, respectively, optionally wherein the142#14533290vlcombination further comprises a third gRNA having the sequence of F4, H8, H10, or H12.

[0384] Suitably, the combination may comprise a first gRNA and a second gRNA having the sequences of C8 and H12, respectively, optionally wherein the combination further comprises a third gRNA having the sequence of F4, H8, H10, or H11.

[0385] Suitably, the combination may comprise a first gRNA and a second gRNA having the sequences of F4 and H8, respectively, optionally wherein the combination further comprises a third gRNA having the sequence of C8, H10, H11, or H12.

[0386] Suitably, the combination may comprise a first gRNA and a second gRNA having the sequences of F4 and H10, respectively, optionally wherein the combination further comprises a third gRNA having the sequence of C8, H8, H11, or H12.

[0387] Suitably, the combination may comprise a first gRNA and a second gRNA having the sequences of F4 and H11, respectively, optionally wherein the combination further comprises a third gRNA having the sequence of C8, H8, H10, or H12.

[0388] Suitably, the combination may comprise a first gRNA and a second gRNA having the sequences of F4 and H12, respectively, optionally wherein the combination further comprises a third gRNA having the sequence of C8, H8, H10, or H11.

[0389] Suitably, the combination may comprise a first gRNA and a second gRNA having the sequences of H8 and H10, respectively, optionally wherein the combination further comprises a third gRNA having the sequence of C8, F4, H11, or H12.

[0390] Suitably, the combination may comprise a first gRNA and a second gRNA having the sequences of H10 and H11, respectively, optionally wherein the combination further comprises a third gRNA having the sequence of C8, F4, H8, or H12.

[0391] Suitably, the combination may comprise a first gRNA and a second gRNA having the sequences of H10 and H12, respectively, optionally wherein the combination further comprises a third gRNA having the sequence of C8, F4, H8, or H11.143#14533290vl

[0392] Suitably, the combination may comprise a first gRNA and a second gRNA having the sequences of H11 and H12, respectively, optionally wherein the combination further comprises a third gRNA having the sequence of C8, F4, H8, or H10.

[0393] The combination may, for example, have gRNAs comprising or consisting of H8+F4, H8+H10, C8+H10, F4+H10, F4+H8+H10, or C8+F4+H10. In a particular case, the gRNAs may comprise or consist of F4+H8+H10.

[0394] In an aspect, the combination further comprises an agent:i) which promotes the survival, proliferation and / or activity of a cell, such as a cell which comprises the combination or a cell which does not comprise the combination; and / orii) which is detrimental to the survival, proliferation, activity, chemoresistance and / or chemotaxis of a cell, such as a cell which comprises the combination or a cell which does not comprise the combination; and / oriii) which enables selection of a cell, such as a cell which comprises the combination or a cell which does not comprise the combination.

[0395] The combination may further comprise an agent which modifies the tissue microenvironment.

[0396] The agent may be a protein, such as a cytokine or chemokine, which promotes the survival, proliferation and / or activity of a cell according to the present invention.

[0397] As used herein, “agent which promotes the survival, proliferation and / or activity of a cell” means that in the presence of the agent, the survival, proliferation, or activity of a cell which comprises a product according to the present invention is increased.

[0398] The agent may be, for example, beneficial for certain cells and detrimental to other cells.

[0399] The agent may play a role in homeostasis, for example, blood coagulation; an example of a suitable agent may be coagulation factor IX or FVIII.144#14533290vl

[0400] The agent may, for example, allow selection of cells. An example of a suitable agent is Delta low-affinity nerve growth factor (LNGFR).

[0401] The agent may, for example, be detrimental for the cell. The agent may be a thymidine kinase (TK) or a caspase, such as CASP9. Activation of these agents can be used for in vivo removal of cells which comprise the agent, e.g., if it is desirable to remove engineered T cells from a subject.

[0402] Suitably, in the presence of the agent, the survival, proliferation and / or activity of the cell which comprises a product according to the present invention e.g., a cell according to the present invention) may be increased by at least 5%, at least 10%, 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 95% or at least 99%.

[0403] The combination may comprise an agent which is detrimental to the survival, proliferation, activity, chemoresistance and / or chemotaxis of a cell such as a tumour cell.

[0404] As used herein “agent which is detrimental to” means that in the presence of the agent, the survival, proliferation, or activity of a cell which does not comprise a product according to the present invention (e.g., a tumour cell) is compromised, reduced, or completely abolished.

[0405] Suitably, in the presence of the detrimental agent, the survival, proliferation and / or activity of the cell which does not comprise a product according to the present invention (e.g., a tumour cell) may be reduced by at least 5%, at least 10%, 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 95% or at least 99%.

[0406] Cell survival and proliferation may be measured by methods known in the art. Suitable methods include measuring the size of the cell population (e.g., by counting cells using a marker specific for the cell population, i.e., a tumour specific marker or an engineered cell specific marker, such as a CAR or transgenic TCR); by performing cell cycle analysis using 5-bromo-2'-deoxyuridine (Brdll) which becomes incorporated into newly made DNA and / or propidium iodide (PI) and analysing by flow cytometry in combination with a cell population specific marker; and / or by measuring the number of viable cells, e.g., by measuring apoptosis by 7AAD and / or Annexin V staining using flow cytometry.145#14533290vl

[0407] In an aspect, the combination further comprises a CAR. In an aspect, the combination further comprises a transgenic TCR.

[0408] The agent, e.g., which promotes the survival, proliferation and / or activity of a cell (or population of cells) or allows selection of the cell, such as the cell (or population of cells) which expresses an ETM (e.g., ETR); and / or which is detrimental to the survival, proliferation, activity, chemoresistance and / or chemotaxis of a cell which does not express an ETM (e.g., ETR), may be introduced into the genome of the cell by any method. The method may include, for example, using an integrating vector (a procedure independent from the multiplexing strategy performed by the ETM (e.g., ETR) according to the invention); or by targeting the agent (e.g., CAR or transgenic TCR) within the site recognized by the nuclease (a procedure depending on the nuclease activity of the ETM (e.g., ETR) according to the present invention).

[0409] Thus in some aspects, the combination further comprises a polynucleotide, such as an integrating vector which encodes an agent which allows selection or promotes the survival, proliferation and / or activity of a cell (or population of cells), such as the cell (or population of cells) which comprises the polynucleotide; and / or which is detrimental to the survival, proliferation, activity, chemoresistance and / or chemotaxis of a cell which does not comprise the polynucleotide; and / or which is beneficial for the survival, proliferation and / or activity of a cell, tissue or organ, such as a cell, tissue or organ which does not comprise the combination.Polynucleotides

[0410] In an aspect, the present invention provides a polynucleotide encoding at least one ETM (e.g., ETR) according to the present invention.

[0411] Polynucleotides of the invention may comprise DNA or RNA. They may be single-stranded or double-stranded. It will be understood by a skilled person that numerous different polynucleotides can encode the same polypeptide as a result of the degeneracy of the genetic code. In addition, it is to be understood that the skilled person may, using routine techniques, make nucleotide substitutions that do not affect the polypeptide sequence encoded by the polynucleotides of the invention to reflect the codon usage of any particular host organism in which the polypeptides of the invention are to be expressed.146#14533290vl

[0412] The polynucleotides may be modified by any method available in the art. Such modifications may be carried out in order to enhance the in vivo activity or lifespan of the polynucleotides of the invention.

[0413] Polynucleotides such as DNA polynucleotides may be produced recombinantly, synthetically or by any means available to those of skill in the art. They may also be cloned by standard techniques.

[0414] Longer polynucleotides will generally be produced using recombinant means, for example using PGR cloning techniques. This will involve making a pair of primers (e.g., of about 15 to 30 nucleotides) flanking the target sequence which it is desired to clone, bringing the primers into contact with mRNA or cDNA obtained from an animal or human cell, performing a polymerase chain reaction under conditions which bring about amplification of the desired region, isolating the amplified fragment (e.g., by purifying the reaction mixture with an agarose gel) and recovering the amplified DNA. The primers may be designed to contain suitable restriction enzyme recognition sites so that the amplified DNA can be cloned into a suitable vector. Constructs

[0415] In an aspect, the present invention provides a nucleic acid construct comprising a nucleic acid sequence encoding at least one ETM (e.g., ETR) according to the present invention.

[0416] The nucleic acid construct may further comprise a nucleic acid sequence which encodes an agent:i) which promotes the survival, proliferation and / or activity of a cell, such as a cell which expresses said nucleic acid construct or a cell which does not express said nucleic acid construct; and / orii) which is detrimental to the survival, proliferation, activity, chemoresistance and / or chemotaxis of a cell, such as a cell which expresses said nucleic acid construct or a cell which does not express said nucleic acid construct; and / oriii) which enables selection of a cell, such as a cell which comprises the nucleic acid construct or a cell which does not comprise the construct.147#14533290vlProteins

[0417] As used herein, the term “protein” includes single-chain polypeptide molecules as well as multiple-polypeptide complexes where individual constituent polypeptides are linked by covalent or non-covalent means. As used herein, the terms “polypeptide” and “peptide” refer to a polymer in which the monomers are amino acids and are joined together through peptide or disulfide bonds.Variants, derivatives, analogues, homologues and fragments

[0418] In addition to the specific proteins and nucleotides mentioned herein, the present invention also encompasses the use of variants, derivatives, analogues, homologues, and fragments thereof.

[0419] In the context of the present invention, a variant of any given sequence is a sequence in which the specific sequence of residues (whether amino acid or nucleic acid residues) has been modified in such a manner that the polypeptide or polynucleotide in question substantially retains at least one of its endogenous functions. A variant sequence can be obtained by addition, deletion, substitution, modification, replacement and / or variation of at least one residue present in the naturally-occurring protein.

[0420] The term “derivative” as used herein, in relation to proteins or polypeptides of the present invention, includes any substitution of, variation of, modification of, replacement of, deletion of and / or addition of one (or more) amino acid residues from or to the sequence providing that the resultant protein or polypeptide substantially retains at least one of its endogenous functions.

[0421] The term “analogue” as used herein, in relation to polypeptides or polynucleotides, includes any mimetic, that is, a chemical compound that possesses at least one of the endogenous functions of the polypeptides or polynucleotides which it mimics.

[0422] Typically, amino acid substitutions may be made, for example from 1, 2 or 3 to 10 or 20 substitutions provided that the modified sequence substantially retains the required activity or ability. Amino acid substitutions may include the use of non-naturally occurring analogues.148#14533290vl

[0423] Proteins used in the present invention may also have deletions, insertions or substitutions of amino acid residues which produce a silent change and result in a functionally equivalent protein. Deliberate amino acid substitutions may be made on the basis of similarity in polarity, charge, solubility, hydrophobicity, hydrophilicity, and / or the amphipathic nature of the residues as long as the endogenous function is retained. For example, negatively charged amino acids include aspartic acid and glutamic acid; positively charged amino acids include lysine and arginine; and amino acids with uncharged polar head groups having similar hydrophilicity values include asparagine, glutamine, serine, threonine, and tyrosine.

[0424] Conservative substitutions may be made, for example according to the table below. Amino acids in the same block in the second column and, in particular examples, in the same line in the third column may be substituted for each other:

[0425] The term “homologue” as used herein means an entity having a certain homology with the wild type amino acid sequence or the wild type nucleotide sequence. The term “homology” can be equated with “identity”.

[0426] A homologous sequence may include an amino acid sequence which may be at least 50%, 55%, 65%, 75%, 85% or 90% identical, for example at least 95% or 97% or 99% identical, to the subject sequence. Typically, the homologues will comprise the same active sites, etc., as the subject amino acid sequence. Although homology can also be considered in terms of similarity (i.e., amino acid residues having similar chemical properties / functions), in the context of the present invention it is preferred to express homology in terms of sequence identity.

[0427] A homologous sequence may include a nucleotide sequence which may be at least 50%, 55%, 65%, 75%, 85% or 90% identical, for example at least 95% or 97% or 99% identical, to the subject sequence. Although homology can also be considered in terms of similarity, in the context of the present invention it is preferred to express homology in terms of sequence identity.149#14533290vl

[0428] Reference to a sequence which has a percent identity to any one of the SEQ ID NOs detailed herein may refer, for example to a sequence which has the stated percent identity over the entire length of the SEQ ID NO referred to.

[0429] Homology comparisons can be conducted by eye or, more usually, with the aid of readily available sequence comparison programs. These commercially available computer programs can calculate percentage homology or identity between two or more sequences.

[0430] Percentage homology may be calculated over contiguous sequences, i.e., one sequence is aligned with the other sequence and each amino acid in one sequence is directly compared with the corresponding amino acid in the other sequence, one residue at a time. This is called an “ungapped” alignment. Typically, such ungapped alignments are performed only over a relatively short number of residues.

[0431] Although this is a very simple and consistent method, it fails to take into consideration that, for example, in an otherwise identical pair of sequences, one insertion or deletion in the nucleotide sequence may cause the following codons to be put out of alignment, thus potentially resulting in a large reduction in percent homology when a global alignment is performed. Consequently, most sequence comparison methods are designed to produce optimal alignments that take into consideration possible insertions and deletions without penalizing unduly the overall homology score. This is achieved by inserting “gaps” in the sequence alignment to try to maximize local homology.

[0432] However, these more complex methods assign “gap penalties” to each gap that occurs in the alignment so that, for the same number of identical amino acids, a sequence alignment with as few gaps as possible, reflecting higher relatedness between the two compared sequences, will achieve a higher score than one with many gaps. “Affine gap costs” are typically used that charge a relatively high cost for the existence of a gap and a smaller penalty for each subsequent residue in the gap. This is the most commonly used gap scoring system. High gap penalties will of course produce optimized alignments with fewer gaps. Most alignment programs allow the gap penalties to be modified. However, it is preferred to use the default values when using such software for sequence comparisons. For example, when150#14533290vlusing the GCG Wisconsin Bestfit package, the default gap penalty for amino acid sequences is -12 for a gap and -4 for each extension.

[0433] Calculation of maximum percentage homology therefore firstly requires the production of an optimal alignment, taking into consideration gap penalties. A suitable computer program for carrying out such an alignment is the GCG Wisconsin Bestfit package (University of Wisconsin, U. S. A.; Devereux etal. (1984) Nucleic Acids Res. 12: 387). Examples of other software that can perform sequence comparisons include, but are not limited to, the BLAST package (see Ausubel etal. (1999) ibid- Ch. 18), FASTA (Atschul etal. (1990) J. Mol. Biol. 403-410) and the GENEWORKS suite of comparison tools. Both BLAST and FASTA are available for offline and online searching (see Ausubel etal. (1999) ibid, pages 7-58 to 7-60). However, for some applications, it is preferred to use the GCG Bestfit program. Another tool, called BLAST 2 Sequences is also available for comparing protein and nucleotide sequences (see FEMS Microbiol. Lett. (1999) 174: 247-50; FEMS Microbiol. Lett. (1999) 177: 187-8).

[0434] Although the final percentage homology can be measured in terms of identity, the alignment process itself is typically not based on an all-or-nothing pair comparison. Instead, a scaled similarity score matrix is generally used that assigns scores to each pairwise comparison based on chemical similarity or evolutionary distance. An example of such a matrix commonly used is the BLOSUM62 matrix -the default matrix for the BLAST suite of programs. GCG Wisconsin programs generally use either the public default values or a custom symbol comparison table if supplied (see the user manual for further details). For some applications, it is preferred to use the public default values for the GCG package, or in the case of other software, the default matrix, such as BLOSUM62.

[0435] Once the software has produced an optimal alignment, it is possible to calculate percentage homology, e.g., percentage sequence identity. The software typically does this as part of the sequence comparison and generates a numerical result.

[0436] As used herein, “fragments” also includes variants and the term typically refers to a selected region of the polypeptide or polynucleotide that is of interest either functionally or, for example, in an assay. “Fragment,” as used herein, thus151#14533290vlrefers to an amino acid or nucleic acid sequence that is a portion of a full-length polypeptide or polynucleotide.

[0437] Such variants may be prepared using standard recombinant DNA techniques such as site-directed mutagenesis. Where insertions are to be made, synthetic DNA encoding the insertion together with 5' and 3' flanking regions corresponding to the naturally-occurring sequence either side of the insertion site may be made. The flanking regions will contain convenient restriction sites corresponding to sites in the naturally-occurring sequence so that the sequence may be cut with the appropriate enzyme(s) and the synthetic DNA ligated into the cut. The DNA is then expressed in accordance with the invention to make the encoded protein. These methods are only illustrative of the numerous standard techniques known in the art for manipulation of DNA sequences and other known techniques may also be used.Codon optimisation

[0438] The polynucleotides used in the present invention may be codon-optimized. Codon optimization has previously been described in WO 1999 / 41397 and WO 2001 / 79518. Different cells differ in their usage of particular codons. This codon bias corresponds to a bias in the relative abundance of particular tRNAs in the cell type. By altering the codons in the sequence so that they are tailored to match with the relative abundance of corresponding tRNAs, it is possible to increase expression. By the same token, it is possible to decrease expression by deliberately choosing codons for which the corresponding tRNAs are known to be rare in the particular cell type. Thus, an additional degree of translational control is available. Vectors

[0439] In an aspect, the present invention provides a vector comprising a polynucleotide according the present invention, or a nucleic acid construct according to the present invention.

[0440] As used herein, a vector is a tool that allows or facilitates the transfer of an entity from one environment to another. In accordance with the present invention, and by way of example, some vectors used in recombinant nucleic acid techniques allow entities, such as a segment of nucleic acid (e.g., a heterologous DNA segment,152#14533290vlsuch as a heterologous cDNA segment), to be transferred into a target cell. The vector may serve the purpose of maintaining the heterologous nucleic acid (DNA or RNA) within the cell, facilitating the replication of the vector comprising a segment of nucleic acid, or facilitating the expression of the protein encoded by a segment of nucleic acid. Vectors may be non-viral or viral. Examples of vectors used in recombinant nucleic acid techniques include, but are not limited to, plasmids, mRNA molecules (e.g., in vitro transcribed mRNAs), chromosomes, artificial chromosomes, and viruses. The vector may also be, for example, a naked nucleic acid (e.g., DNA). In its simplest form, the vector may itself be a nucleotide of interest.

[0441] The vectors used in the invention may be, for example, plasmid, mRNA, or virus vectors and may include a promoter for the expression of a polynucleotide and optionally a regulator of the promoter.

[0442] Vectors comprising polynucleotides used in the invention may be introduced into cells using a variety of techniques known in the art, such as transfection, transformation, and transduction. Several such techniques are known in the art, for example infection with recombinant viral vectors, such as retroviral, lentiviral (e.g., integration-defective lentiviral), adenoviral, adeno-associated viral, baculoviral and herpes simplex viral vectors; direct injection of nucleic acids and biolistic transformation.

[0443] Non-viral delivery systems include but are not limited to DNA or RNA transfection methods. Here, transfection includes a process using a non-viral vector to deliver a gene to a target cell. Typical transfection methods include electroporation, DNA biolistics, lipid-mediated transfection, compacted DNA-mediated transfection, liposomes, immunoliposomes, lipofectin, cationic agent-mediated transfection, cationic facial amphiphiles (CFAs) Nat. Biotechnol. (1996) 14: 556) and combinations thereof.

[0444] As used herein, the term “transfection” is to be understood as encompassing the delivery of polynucleotides to cells by both viral and non-viral delivery.153#14533290vlProtein transduction

[0445] As an alternative to the delivery of polynucleotides to cells, products and ETMs (e.g., ETRs) of the present invention may be delivered to cells by protein transduction.

[0446] Protein transduction may be via vector delivery (Cai, Y. et al. (2014) Elife 3: e01911; Maetzig, T. etal. (2012) Curr. Gene Then 12: 389-409). Vector delivery involves the engineering of viral particles (e.g., lentiviral particles) to comprise the proteins to be delivered to a cell. Accordingly, when the engineered viral particles enter a cell as part of their natural life cycle, the proteins comprised in the particles are carried into the cell.

[0447] Protein transduction may be via protein delivery (Gaj, T. etal. (2012) Nat. Methods 9: 805-7). Protein delivery may be achieved, for example, by utilizing a vehicle (e.g., liposomes) or even by administering the protein itself directly to a cell.Delivery to target cells

[0448] The products of the invention, such as, for example, the ETMs (e.g., ETRs), gRNAs, donor template nucleic acids provided herein, alone or in any combination thereof, can be delivered into a target cell as proteins, nucleic acids, or as a combination of proteins and nucleic acids, e.g., as a combination of ETR protein and one or more gRNAs; as a combination of ETR protein, one or more gRNAs, and one or more template DNAs for insertion into a target gene. The products of the invention may be delivered to target cells in vitro, ex vivo, or in vivo. Some suitable methods and modalities for the delivery of the products of the invention are provided herein, including electroporation, nucleic acid transfection, and protein transduction for in vitro and ex vivo delivery, and delivery via viral vectors, viral-like particles (VLPs), or LNPs in vivo.

[0449] A number of non-limiting examples of suitable LNPs for delivery of the ETMs e.g., ETRs), gRNAs, donor template nucleic acids provided herein can be found in International Publication Nos. WO2025166314A1; W02025090525A1;WO2024102770A1; and WO2024102769A2; including, for example, targeted lipid nanoparticles (tLNPs) may be employed for delivering Targeted LNP mRNA for chimeric antigen receptors (CAR) to specific T cells, including to specific T cell154#14533290vlsubsets (see, e.g., Hunter et al. Science. 2025;388(6753):1311-1317, the entire contents of which are incorporated herein by reference).

[0450] Additional suitable delivery methods and modalities include, for example, those described in the section entitled “Carriers for delivering CRISPR technology” of Li et al., CRISPR / Cas9 therapeutics: progress and prospects. Sig Transduct Target Ther 8, 36 (2023), the contents of which are incorporated herein by reference.

[0451] Additional suitable delivery methods and modalities will be apparent to the skilled artisan based on the present disclosure and the knowledge in the art. The disclosure is not limited in this respect.Composition

[0452] The products of the invention such as ETMs {e.g., ETRs), gRNAs, combinations, polynucleotides, nucleic acid constructs, vectors, cells, and kits of polynucleotides of the present invention may be provided in a composition.

[0453] The products of the invention such as combinations, ETMs e.g., ETRs), gRNAs, polynucleotides, nucleic acid constructs, vectors, compositions, and cells of the present invention may be formulated for administration to subjects with a pharmaceutically acceptable carrier, diluent, or excipient. Suitable carriers and diluents include isotonic saline solutions, for example, phosphate-buffered saline, and potentially contain human serum albumin.

[0454] Handling of the cell therapy products may be performed in compliance with the Foundation for the Accreditation of Cellular Therapy and the Joint Accreditation Committee - International Society Cell & Gene Therapy (ISCT) and European Society for Blood and Marrow Transplantation (EBMT) (FACT-JACIE) International Standards for cellular therapy.

[0455] In an aspect, there is provided a combination of chemically modified mRNA encoding for an ETM or ETR plus a chemically modified gRNA.

[0456] In another aspect, there is provided a ribonucleic complex of protein-RNA that includes the ETR protein attached to a chemically modified gRNA.Kit

[0457] In an aspect, the present invention provides a kit of polynucleotides comprising:155#14533290vla) at least one polynucleotide encoding at least one ETM (e.g., ETR) according to the present invention; andb) a polynucleotide providing at least one gRNA as described herein; and optionally,c) further comprising a nucleic acid sequence which encodes an agent:i) which promotes the survival, proliferation and / or activity of a cell, such as a cell which comprises the polynucleotides or a cell which does not comprise the polynucleotides; and / orii) which is detrimental to the survival, proliferation, activity, chemoresistance and / or chemotaxis of a cell, such as a cell which comprises said polynucleotides or a cell which does not comprise said polynucleotides; and / oriii) which enables selection of a cell, such as a cell which comprises the polynucleotides or a cell which does not comprise the polynucleotides.

[0458] The kit may also include instructions for use, for example instructions for the simultaneous, sequential, or separate administration of at least one ETM (e.g., ETR) and at least two gRNAs, to a subject in need thereof.Use

[0459] In an aspect, the present invention provides the use of an ETM (e.g., ETR) according to the present invention, at least one gRNA according to the present invention, a combination according to the present invention, a polynucleotide according to the present invention, a nucleic acid construct according to the present invention, a vector according to the present invention or a kit of polynucleotides according to the present invention for modifying the activity and / or expression of at least one target gene, e.g., wherein the use is in vitro or ex vivo use.

[0460] Suitably, the use may repress transcription and / or expression of e.g., silence) at least one target gene. Suitably, the use may repress transcription and / or expression of (e.g., silence) at least two target genes. For example, transcription156#14533290vland / or expression of a first gene may be repressed (e.g., silenced) by gene editing and transcription and / or expression of a second target gene may be repressed (e.g., silenced) by epigenetic editing.

[0461] Suitably, the use may enhance at least one target gene.

[0462] In another aspect, the present invention provides a method of repressing transcription and / or expression of (e.g., silencing) at least one target gene in a cell comprising the step of administering an ETM (e.g., ETR) according to the present invention, at least one gRNA according to the present invention, a combination according to the present invention, a polynucleotide according to the present invention, a nucleic acid construct according to the present invention, a vector according to the present invention or a kit of polynucleotides according to the present invention to a cell.

[0463] Suitably, transcription and / or expression of at least two target genes may be repressed (e.g., silenced), wherein at least one of the at least two target genes is epigenetically repressed (e.g., silenced) and at least one of the at least two target genes is repressed (e.g., silenced) by gene editing, wherein at least one ETM (e.g., ETR) and at least two gRNAs are administered to said cell simultaneously, sequentially, or separately.

[0464] In another aspect, the present invention provides the products, ETMs (e.g., ETRs), gRNAs, combinations, polynucleotides, nucleic acid constructs, vectors, kits of polynucleotides, cells, and pharmaceutical compositions of the present invention for use in therapy.

[0465] The use in therapy may, for example, be a use for the preparation of “universally” allogeneic transplantable cells (e.g., by the silencing of [32-microglobulin, B2M). This use may, for example, be applied to the preparation of hematopoietic stem and / or progenitor cells (HSPCs), whole organ transplantation and cancer immunotherapy.

[0466] The ETM (e.g., ETR) (or polynucleotide, nucleic acid construct, or vector encoding therefor) and gRNAs may be administered simultaneously, in combination, sequentially or separately (as part of a dosing regimen).

[0467] By “simultaneously”, it is to be understood that the two or more agents are administered concurrently, whereas the term “in combination” is used to mean they are administered, if not simultaneously, then “sequentially” within a time frame that 157#14533290vlthey both are available to act therapeutically within the same time frame. Thus, administration “sequentially” may permit one agent to be administered within 5 minutes, 10 minutes, or a matter of hours after the other provided the circulatory halflife of the first administered agent is such that they are both concurrently present in therapeutically effective amounts. The time delay between administration of the components will vary depending on the exact nature of the components, the interaction there-between, and their respective half-lives.

[0468] In contrast to “in combination” or “sequentially”, “separately” is to be understood as meaning that the gap between administering one agent and the other agent is significant, i.e., the first administered agent may no longer be present in the bloodstream in a therapeutically effective amount when the second agent is administered.

[0469] In another aspect, the present invention provides a method for treating and / or preventing a disease or condition, which comprises the step of administering any of the products of the invention (e.g., ETMs (e.g., ETRs), gRNAs, combinations, polynucleotides, nucleic acid constructs, vectors, kits of polynucleotides, cells, or pharmaceutical compositions according to the present invention) to a subject in need thereof.

[0470] Suitably, the ETM (e.g., ETR) and gRNAs may be administered to a subject simultaneously, sequentially, or separately.

[0471] In an aspect, the present invention provides a method of gene therapy which comprises the steps of:(i) isolation of a cell containing sample;(ii) introduction of a polynucleotide according to the present invention, a nucleic acid construct according to the present invention, at least one gRNA according to the present invention, an ETM (e.g., ETR) according to the present invention, a vector according to the present invention or a kit of polynucleotides according to the present invention to the cell(s); and(iii) administering the cell(s) from step (ii) to a subject.

[0472] The nucleic acid construct or vector may be introduced by transduction or transfection.158#14533290vl

[0473] The cell may, for example, be autologous. The cell may, for example, be allogeneic.

[0474] In an aspect, the present invention provides a method of gene therapy which comprises the steps of:(i) introduction of a polynucleotide according to the present invention, a nucleic acid construct according to the present invention, at least one gRNA according to the present invention, an ETM (e.g., ETR) according to the present invention, a vector according to the present invention or a kit of polynucleotides according to the present invention to a cell in a subject.

[0475] The nucleic acid construct or vector may be introduced by transduction or transfection.

[0476] The cell may, for example, be autologous. The cell may, for example, be allogeneic.

[0477] Some aspects of this disclosure provide methods in which the products of the invention, such as, for example, the ETMs e.g., ETRs), gRNAs, donor template nucleic acids provided herein, alone or in any combination thereof, are administered to a subject in need thereof via a parenteral administration route (e.g., intravenous, intramuscular, subcutaneous, or intrathecal), as a pharmaceutical formulation (or pharmaceutical composition) comprising one or more products of the invention, e.g., a combination of ETR protein and one or more gRNAs; as a combination of ETR protein, one or more gRNAs, and one or more template DNAs for insertion into a target gene, in addition to one or more pharmaceutically acceptable excipient(s), e.g., as described below.

[0478] The term “excipient” is used herein to describe any ingredient other than the compound(s) of the present disclosure. The choice of excipient(s) will to a large extent depend on factors such as the particular mode of administration, the effect of the excipient on solubility and stability, and the nature of the dosage form. As used herein, “pharmaceutically acceptable excipient” includes any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like that are physiologically compatible. Some examples of pharmaceutically acceptable excipients are water, saline, phosphate 159#14533290vlbuffered saline, dextrose, glycerol, ethanol and the like, as well as combinations thereof. In many cases, it will be preferable to include isotonic agents, for example, sugars, polyalcohols such as mannitol, sorbitol, or sodium chloride in the composition. Additional examples of pharmaceutically acceptable substances are wetting agents or minor amounts of auxiliary substances such as wetting or emulsifying agents, preservatives, or buffers, which enhance the shelf life or effectiveness of the antibody.

[0479] Formulations of a pharmaceutical composition suitable for parenteral administration typically comprise the active ingredient combined with a pharmaceutically acceptable carrier, such as sterile water or sterile isotonic saline. Such formulations may be prepared, packaged, or sold in a form suitable for bolus administration or for continuous administration. In some embodiments, the epigenetic editor or its component(s) are introduced to target cells in the form of nucleic acid molecule(s) encoding the epigenetic editor or its component(s); accordingly, the pharmaceutical compositions herein comprise the nucleic acid molecule(s). Such nucleic acid molecule(s) may be, for example, DNA, RNA or mRNA, and / or modified nucleic acid sequence(s) (e.g., with chemical modifications, a 5’ cap, or one or more 3’ modifications). In some embodiments, the nucleic acid molecule(s) may be delivered as naked DNA or RNA, for instance by means of transfection or electroporation, or can be conjugated to molecules (e.g., N-acetylgalactosamine) promoting uptake by target cells. In some embodiments, the nucleic acid molecule(s) may be in nucleic acid expression vector(s), which may include expression control sequences such as promoters, enhancers, transcription signal sequences, transcription termination sequences, introns, polyadenylation signals, Kozak consensus sequences, internal ribosome entry sites (IRES), etc. Such expression control sequences are well known in the art. A vector may also comprise a sequence encoding a signal peptide (e.g., for nuclear localization, nucleolar localization, or mitochondrial localization), associated with (e.g., inserted into or fused to) a sequence coding for a protein.

[0480] Examples of viral vectors include, but are not limited to, plasmid vectors; viral vectors based on vaccinia virus, poliovirus, adenovirus, adeno-associated virus, SV40, herpes simplex virus, human immunodeficiency virus, retrovirus (e.g., Murine Leukemia Virus, or spleen necrosis virus, vectors derived from retroviruses such as 160#14533290vlRous Sarcoma Virus, Harvey Sarcoma Virus, avian leukosis virus, a lentivirus, human immunodeficiency virus, myeloproliferative sarcoma virus, and mammary tumor virus); and other recombinant vectors. In certain embodiments, the vector is a plasmid or a viral vector. Viral particles may be used to deliver products of the invention, such as, for example, the ETMs (e.g., ETRs), gRNAs, and / or donor template nucleic acids provided herein, either as proteins (e.g., ETM or ETR proteins) or as nucleic acid molecule(s) (e.g., gRNAs or DNA comprising sequence(s) encoding gRNAs, and / or DNA comprising donor template nucleic acid sequence(s)) as described herein. In addition, “empty” viral particles, also sometimes referred to as viral-like particles (VLPs), can be assembled to contain any suitable cargo.

[0481] Suitable VLPs can comprise one or more virus-derived proteins, such as a structural protein of VLPs and an envelope protein. In some cases, the virus-derived protein is present as part of a chimeric protein that forms the VLP. Suitable VLPs can comprise structural proteins (e.g., gag protein).

[0482] In some embodiments, the structural protein of VLP (e.g., plasma membrane localization protein) is a polyprotein derived from a virus, a homologue thereof, a fragment thereof, a variant thereof, or any combination thereof. For instance, the structural protein of VLP (e.g., plasma membrane localization protein) comprises a retroviral gag protein, e.g., a retroviral polyprotein that comprises one or more of a matrix (MA) polypeptide, an RNA-binding phosphoprotein polypeptide, a capsid (CA) polypeptide, or a nucleocapsid (NC) polypeptide. In some cases, the gag protein is derived from Friend murine leukemia virus (FMLV). In some cases, the retroviral gag polyprotein is a gag polyprotein of an alpha retrovirus, a beta retrovirus, a gamma retrovirus, a delta retrovirus, an epsilon retrovirus, or a spumavirus. In some cases, the retroviral gag polyprotein is a gag polyprotein of a human immunodeficiency virus.

[0483] Non-limiting examples of the structural protein of VLP e.g., plasma membrane recruitment element) comprises Human Papillomavirus (HPV) LI protein, HPV L2 protein, Hepatitis B virus (HBV) core protein, Chikungunya virus (CHIKV) C-E3-E2-6k-EI, human immunodeficiency virus (HIV) gag-pol, HIV gag, Respiratory syncytial virus (RSV) M, RSV NP, Human metapneumovirus (HMPV) M, Influenza Ml, Zika virus (ZIKV) C, ZIKV prM / M, Dengaue virus (DENV) C-prM, West Nile Virus 161#14533290vl(WNV) prME protein, WNV CprME protein, Filovirus VP40 orZ protein, Baculovirus P 1 protein, Rotavirus VP7, Rotavirus VP2 protein, Rotavirus VP6 protein, SARS M protein, SARS E protein, SARS N protein, Porcine Circovirus Type 2 (PCV2) capsid, baculovirus VP2 protein, baculovirus VP5 protein, baculovirus VP3 protein, or baculovirus VP7 protein, Hepatitis C virus (HCV) core protein, Ebola nucleocapsid, Parovirus VP1 protein, Parovirus VP2 protein, Newcastle disease virus (NDV) M protein, hepatitis E virus (HeV) M protein, Nipah virus (NIV) M protein, Human polyomavirus 2 (JCPyV) VP1 protein, Human parainfluenza virus type 3 (HPIV3) M protein, HPIV3N protein, or Mumps virus (MuV) M proteins, a homologue thereof, a fragment thereof, a variant thereof, or any combination thereof.

[0484] Suitable VLPs can have reduced or no immunogenicity to human subjects, e.g., non-viral human endogenous viral-like particles (heVLPs), that comprise human endogenous viral components.

[0485] Various VLP formulations and methods of VLP delivery that can be used to deliver the products of the invention will be apparent to those skilled in the art from the present disclosure and the state of the art. Non-limiting exemplary suitable compositions and methods can be found in International Publication Nos.WO2023102537; WO2023102538; WO2023102550; WO2023225572;WO2024138033; and WO2024254544; and Bernadin et al., Blood Adv. 2019 Feb 12;3(3):461-475.

[0486] Viral vectors and viral particles may also be engineered to incorporate targeting ligands to alter target tissue specificity.

[0487] In certain embodiments, a product of the invention, such as, for example, the ETMs (e.g., ETRs), gRNAs, donor template nucleic acids provided herein, alone or in any combination thereof, is delivered into a target cell (e.g., as protein, nucleic acid, or as a combination of protein(s) and nucleic acid(s), e.g., as a combination of ETR protein and one or more gRNAs; as a combination of ETR protein, one or more gRNAs, and one or more template DNAs for insertion into a target gene) via one or more viral vectors. Non-limiting examples of viral vectors include adeno-associated viral 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., HIV and FIV-based vectors), and herpesvirus vectors (e.g., HSV-2).162#14533290vl

[0488] In some embodiments, delivery of a product of the invention is effected in vivo via an adeno-associated virus (AAV) vector. AAV vector delivery may be particularly useful where the DNA-binding domain of an epigenetic editor fusion protein is a zinc finger array. Without wishing to be bound by any theory, the smaller size of zinc finger arrays compared to larger DNA-binding domains such as Gas protein domains may allow such a fusion protein to be conveniently packed in viral vectors such as an AAV vector.

[0489] Any AAV serotype, e.g., human AAV serotype, can be used for an AAV vector as 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, an AAV variant has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% amino acid sequence identity to a wildtype AAV. In certain embodiments, the AAV variant may be engineered such that its capsid proteins have reduced immunogenicity or enhanced transduction ability in humans. In some instances, 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 may comprise inverted terminal repeats (ITRs) that are of a heterologous serotype compared to the serotype of the capsid. The resulting chimeric AAV can have a different antigenic reactivity or recognition compared to its parental serotypes. In some embodiments, a chimeric variant of an AAV includes amino acid sequences from 2, 3, 4, 5, or more different AAV serotypes.

[0490] Non-viral delivery systems are also contemplated for in vitro, ex vivo, and in vivo delivery as described herein. Non-viral systems suitable for delivery one or more products of the invention, such as, for example, an ETM e.g., an ETR), a gRNA, and / or a donor template nucleic acid provided herein, alone or in any combination thereof, is delivered into a target cell (e.g., as protein, nucleic acid, or as a combination of protein(s) and nucleic acid(s), e.g., as a combination of ETR protein and one or more gRNAs; as a combination of ETR protein, one or more gRNAs, and one or more template DNAs for insertion into a target gene) include, but are not limited to, nucleic acid transfection methods including electroporation, sonoporation,163#14533290vlcalcium phosphate transfection, microinjection, DNA biolistics, lipid-mediated transfection, transfection through heat shock, compacted DNA-mediated transfection, lipofection, cationic agent-mediated transfection, and transfection with liposomes, immunoliposomes, or cationic facial amphiphiles (CFAs).

[0491] In some embodiments, one or more products of the invention, such as, for example, an ETM (e.g., an ETR), a gRNA, and / or a donor template nucleic acid provided herein, alone or in any combination thereof, is delivered into a target cell (e.g., as protein, nucleic acid, or as a combination of protein(s) and nucleic acid(s), e.g., as a combination of ETR protein and one or more gRNAs; as a combination of ETR protein, one or more gRNAs, and one or more template DNAs for insertion into a target gene) is delivered to a target cell in vivo via lipid nanoparticles (LNPs).

[0492] Suitable LNP compositions are typically sized on the order of micrometers or smaller and may include a lipid bilayer. In some embodiments, a LNP refers to any particle that has a diameter of less than 1000 nm, 500 nm, 250 nm, 200 nm, 150 nm, 100 nm, 75 nm, 50 nm, or 25 nm. Nanoparticle compositions encompass lipid nanoparticles (LNPs), liposomes e.g., lipid vesicles), and lipoplexes.

[0493] An LNP as described herein may be made from cationic, anionic, or neutral lipids. In some embodiments, an LNP may comprise neutral lipids, such as the fusogenic phospholipid 1,2-Dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE) or the membrane component cholesterol, as helper lipids to enhance transfection activity and nanoparticle stability. In some embodiments, an LNP may comprise hydrophobic lipids, hydrophilic lipids, or both hydrophobic and hydrophilic lipids. Any lipid or combination of lipids that are known in the art can be used to produce an LNP. The lipids may be combined in any molar ratios to produce the LNP. In some embodiments, the LNP is a liver-targeting (e.g., preferentially or specifically targeting the liver) LNP.

[0494] LNP formulations and methods of LNP delivery that can be used to deliver the products of the invention will be apparent to those skilled in the art from the present disclosure and the state of the art. Non-limiting exemplary suitable compositions and methods can be found in Shah, R., Eldridge, D., Palombo, E., and Harding, I., Lipid Nanoparticles: Production, Characterization and Stability, Springer, 2015, ISBN-13978-3319107103; Mitchell, M. J., Billingsley, M. M., Haley, R. M. et al. Engineering precision nanoparticles for drug delivery, Nat Rev Drug Discov 20, 101- 164#14533290vl124 (2021); Hou, X., Zaks, T., Langer, R. et al. Lipid nanoparticles for mRNA delivery. Nat Rev Mater 6, 1078-1094 (2021); Lipid-Nanoparticle-Based Delivery of CRISPR / Cas9 Genome-Editing Components, Pardis Kazemian, Si-Yue Yu, Sarah B. Thomson, Alexandra Birkenshaw, Blair R. Leavitt, and Colin J. D. Ross. Molecular Pharmaceutics 2022 19 (6), 1669-1686; Cullis PR, Hope MJ. Lipid Nanoparticle Systems for Enabling Gene Therapies, Mol Ther. 2017 Jul 5;25(7):1467-1475; Hatit, M. Z. C., Lokugamage, M. P., Dobrowolski, C. N. et al. Species-dependent in vivo mRNA delivery and cellular responses to nanoparticles, Nat. Nanotechnol. 17, 310— 318 (2022); Lam, K., Schreiner, P., Leung, A., Stainton, P., Reid, S., Yaworski, E., Lutwyche, P. and Heyes, J. (2023), Optimizing Lipid Nanoparticles for Delivery in Primates, Adv. Mater; Dilliard, S. A., Siegwart, D. J. Passive, active and endogenous organ-targeted lipid and polymer nanoparticles for delivery of genetic drugs, Nat Rev Mater (2023); Kasiewicz, L. N., et.al., Lipid nanoparticles incorporating a GalNAc ligand enable in vivo liver ANGPTL3 editing in wild-type and somatic LDLR knockout non-human primates, bioRxiv 2021.11.08.467731; Tombacz, L, et.al., Highly efficient CD4+ T cell targeting and genetic recombination using engineered CD4+ cellhoming mRNA-LNPs, Molecular Therapy, Volume 29, Issue 11, 2021, 3293-3304; Cheng, Q., Wei, T., Farbiak, L. et al. Selective organ targeting (SORT) nanoparticles for tissue-specific mRNA delivery and CRISPR-Cas gene editing, Nat. Nanotechnol.15, 313-320 (2020); Zhang, Y., et.al., Lipids and Lipid Derivatives for RNA Delivery, Chemical Reviews 2021 121 (20); Lam, K., et.al, Unsaturated, Trialkyl Ionizable Lipids are Versatile Lipid-Nanoparticle Components for Therapeutic and Vaccine Applications, Adv. Mater. 2023, 35; Han, X., Zhang, H., Butowska, K. et al. An ionizable lipid toolbox for RNA delivery, Nat Commun 12, 7233 (2021); US Patent No. 9,364,435; US Patent No. 8,058,069; US Patent No. 8,822,66; US Patent No. 8,492,359; US Patent No. 11,141,378; US Patent No. 9,518,272; US Patent No. 9,404,127; US Patent No. 9,006,417; US Patent No. 7,901,708; US Patent No.9,005,654; US Patent No. 9,878,042; US Patent No. 9,682,139; US Patent No.8,642,076; US Patent No. 9,593,077; US Patent No. 9,415,109; US Patent No.9,701,623; US Patent No. 10,369,226; US Patent No. 9,999,673; US Patent No. 9,301,923; US Patent No. 10,342,761; US Patent No. 10,137,201; International Publication No. WO2015199952A1; International Publication No.WO2017075531 A1; International Publication No. W02018081480A1; International 165#14533290vlPublication No. WO2016081029A1; European Application No. EP3852911 A2; each of which are incorporated herein by reference in their entirety.

[0495] Other methods of delivery to target cells will be known to those skilled in the art and can be used with the compositions of the present disclosure. For example, targeted lipid nanoparticle (tLNP) may be employed for delivering mRNA for chimeric antigen receptors (CAR) to T cells, including to specific T cell subsets (Hunter etal. (2025) Science. 388: 1311-1317).

[0496] Any type of cell may be targeted for delivery of a product of the invention, or component(s) or combinations thereof as described herein. For example, the cells may be eukaryotic or prokaryotic. In some embodiments, the cells are mammalian (e.g., human) cells. Human cells may include, for example, neurons, skin cells, muscle cells, blood cells (e.g., hematopoietic stem cells, lymphocytes, T-cells, B-cells, NK cells, macrophages), hepatocytes, biliary epithelial cells (cholangiocytes), stellate cells, Kupffer cells, and liver sinusoidal endothelial cells.

[0497] In some embodiments, product of the invention described herein, or component(s) or combination(s) thereof, are delivered to a host cell for transient expression, e.g., via a transient expression vector. In some embodiments, transient expression of an ETM (e.g., ETR) provided herein results in permanent editing of a first target gene (e.g., via integration of a donor nucleic acid template) as well as prolonged or permanent epigenetic modification of one or more additional target gene(s), e.g., target gene(s) comprising a CGI. For example, the epigenetic modification may be stable for at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10. 11, or 12 weeks or more; or 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 months or more, after introduction of the epigenetic editor into the host cell. The epigenetic modification may be maintained after one or more mitotic and / or meiotic events of the host cell. In particular embodiments, the epigenetic modification is maintained across generations in offspring generated or derived from the host cell.

[0498] It is to be appreciated that all references herein to treatment include curative, palliative and prophylactic treatment; although in the context of the present invention references to preventing are more commonly associated with prophylactic treatment. The treatment of mammals, particularly humans, is preferred. Both human and veterinary treatments are within the scope of the present invention.166#14533290vlDiseases and conditions

[0499] By way of example, the products, ETMs (e.g., ETRs), polynucleotides and cells of the present invention may be used in the treatment of, for example, Huntington’s disease, spinocerebellar ataxias, collagenopathies, hemaglobinopathies, and diseases caused by trinucleotide expansions. Furthermore, the product of the present invention may be used in the treatment or prevention of certain infectious diseases (e.g., CCR5-tropic HIV infections) by inactivating either pathogen-associated gene products or host genes that are necessary for the pathogen life cycle.

[0500] In addition, or in the alternative, the products, ETMs (e.g., ETRs), polynucleotides and cells of the present invention may be useful in the treatment of the disorders listed in WO 1998 / 005635. For ease of reference, part of that list is now provided: cancer, inflammation or inflammatory disease, dermatological disorders, fever, cardiovascular effects, haemorrhage, coagulation and acute phase response, cachexia, anorexia, acute infection, HIV infection, shock states, graft-versus-host reactions, autoimmune disease, reperfusion injury, meningitis, migraine and aspirin-dependent anti-thrombosis; tumour growth, invasion and spread, angiogenesis, metastases, malignant, ascites and malignant pleural effusion; cerebral ischaemia, ischaemic heart disease, osteoarthritis, rheumatoid arthritis, osteoporosis, asthma, multiple sclerosis, neurodegeneration, Alzheimer's disease, atherosclerosis, stroke, vasculitis, Crohn's disease and ulcerative colitis; periodontitis, gingivitis; psoriasis, atopic dermatitis, chronic ulcers, epidermolysis bullosa; corneal ulceration, retinopathy and surgical wound healing; rhinitis, allergic conjunctivitis, eczema, anaphylaxis; restenosis, congestive heart failure, endometriosis, atherosclerosis or endosclerosis.

[0501] In addition, or in the alternative, the products, ETMs (e.g., ETRs), polynucleotides and cells of the present invention may be useful in the treatment of the disorders listed in WO 1998 / 007859. For ease of reference, part of that list is now provided: cytokine and cell proliferation / differentiation activity; immunosuppressant or immunostimulant activity (e.g., for treating immune deficiency, including infection with human immune deficiency virus; regulation of lymphocyte growth; treating cancer and many autoimmune diseases, and to prevent167#14533290vltransplant rejection or induce tumour immunity); regulation of haematopoiesis, e.g., treatment of myeloid or lymphoid diseases; promoting growth of bone, cartilage, tendon, ligament and nerve tissue, e.g., for healing wounds, treatment of burns, ulcers and periodontal disease and neurodegeneration; inhibition or activation of follicle-stimulating hormone (modulation of fertility); chemotactic / chemokinetic activity e.g., for mobilising specific cell types to sites of injury or infection); haemostatic and thrombolytic activity (e.g., for treating haemophilia and stroke); anti-inflammatory activity (for treating e.g., septic shock or Crohn's disease); as antimicrobials; modulators of e.g., metabolism or behaviour; as analgesics; treating specific deficiency disorders; in treatment of e.g., psoriasis, in human or veterinary medicine.

[0502] In addition, or in the alternative, the products, ETMs (e.g., ETRs), polynucleotides and cells of the present invention may be useful in the treatment of the disorders listed in WO 1998 / 009985. For ease of reference, part of that list is now provided: macrophage inhibitory and / or T cell inhibitory activity and thus, antiinflammatory activity; anti-immune activity, i.e., inhibitory effects against a cellular and / or humoral immune response, including a response not associated with inflammation; inhibit the ability of macrophages and T cells to adhere to extracellular matrix components and fibronectin, as well as up-regulated fas receptor expression in T cells; inhibit unwanted immune reaction and inflammation including arthritis, including rheumatoid arthritis, inflammation associated with hypersensitivity, allergic reactions, asthma, systemic lupus erythematosus, collagen diseases and other autoimmune diseases, inflammation associated with atherosclerosis, arteriosclerosis, atherosclerotic heart disease, reperfusion injury, cardiac arrest, myocardial infarction, vascular inflammatory disorders, respiratory distress syndrome or other cardiopulmonary diseases, inflammation associated with peptic ulcer, ulcerative colitis and other diseases of the gastrointestinal tract, hepatic fibrosis, liver cirrhosis or other hepatic diseases, thyroiditis or other glandular diseases, glomerulonephritis or other renal and urologic diseases, otitis or other oto-rhino-laryngological diseases, dermatitis or other dermal diseases, periodontal diseases or other dental diseases, orchitis or epididimo-orchitis, infertility, orchidal trauma or other immune-related testicular diseases, placental dysfunction, placental insufficiency, habitual abortion, eclampsia, pre-eclampsia and other immune and / or inflammatory-related gynaecological diseases, posterior uveitis, intermediate uveitis,168#14533290vlanterior uveitis, conjunctivitis, chorioretinitis, uveoretinitis, optic neuritis, intraocular inflammation, e.g., retinitis or cystoid macular oedema, sympathetic ophthalmia, scleritis, retinitis pigmentosa, immune and inflammatory components of degenerative fondus disease, inflammatory components of ocular trauma, ocular inflammation caused by infection, proliferative vitreo-retinopathies, acute ischaemic optic neuropathy, excessive scarring, e.g., following glaucoma filtration operation, immune and / or inflammation reaction against ocular implants and other immune and inflammatory-related ophthalmic diseases, inflammation associated with autoimmune diseases or conditions or disorders where, both in the central nervous system (CNS) or in any other organ, immune and / or inflammation suppression would be beneficial, Parkinson's disease, complication and / or side effects from treatment of Parkinson's disease, AIDS-related dementia complex HIV-related encephalopathy, Devic's disease, Sydenham chorea, Alzheimer's disease and other degenerative diseases, conditions or disorders of the CNS, inflammatory components of stokes, post-polio syndrome, immune and inflammatory components of psychiatric disorders, myelitis, encephalitis, subacute sclerosing pan-encephalitis, encephalomyelitis, acute neuropathy, subacute neuropathy, chronic neuropathy, Guillaim-Barre syndrome, Sydenham chora, myasthenia gravis, pseudo-tumour cerebri, Down's Syndrome, Huntington's disease, amyotrophic lateral sclerosis, inflammatory components of CNS compression or CNS trauma or infections of the CNS, inflammatory components of muscular atrophies and dystrophies, and immune and inflammatory related diseases, conditions or disorders of the central and peripheral nervous systems, post-traumatic inflammation, septic shock, infectious diseases, inflammatory complications or side effects of surgery, bone marrow transplantation or other transplantation complications and / or side effects, inflammatory and / or immune complications and side effects of gene therapy, e.g., due to infection with a viral carrier, or inflammation associated with AIDS, to suppress or inhibit a humoral and / or cellular immune response, to treat or ameliorate monocyte or leukocyte proliferative diseases, e.g., leukemia, by reducing the amount of monocytes or lymphocytes, for the prevention and / or treatment of graft rejection in cases of transplantation of natural or artificial cells, tissue and organs such as cornea, bone marrow, organs, lenses, pacemakers, natural or artificial skin tissue.169#14533290vl

[0503] For example, the present invention may be used to treat inherited disease such as [3-haemoglobinopathies by targeting hemoglobin F (HBF) or haemoglobin subunit beta (HBB); or to treat severe combined immunodeficiency disease (SCID), Wiskott-Aldrich syndrome protein (WASP), sickle cell disease (SCD) or adenosine deaminase deficiency (ADA).

[0504] The skilled person will understand that they can combine any or all features of the invention disclosed herein without departing from the scope of the invention as disclosed.FURTHER ASPECTS

[0505] The present invention also provides further aspects as defined in the following numbered aspects.1. An engineered transcriptional modulator (ETM) comprising: (a) at least one epigenetic effector domain; operably linked to (b) an endonuclease.2. An ETM according to aspect 1, wherein the at least one epigenetic effector domain comprises a Kruppel-associated box (KRAB) domain, a DNA methyltransferase (DNMT) domain, a DNMT-like domain, and / or a histone methyltransferase (HMT) domain.3. An ETM according to aspect 1 or aspect 2, wherein the at least one epigenetic effector domain is selected from the group consisting of: DNMT1, DNMT3A, DNMT3B, DNMT3L and SETDB1.4. An ETM according to any preceding aspect, wherein the endonuclease comprises an RNA binding domain.5. An ETM according to any preceding aspect, wherein the endonuclease is a Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) / Cas system.6. An ETM according to any preceding aspect, wherein the endonuclease is a Cas9 endonuclease.7. An ETM according to any preceding aspect, wherein the ETM comprises or consists of: a Cas9-KRAB, Cas9-DNMT3A or Cas9-DNMT3L fusion protein.8. An ETM according to any preceding aspect, wherein the ETM is bi- or tri-partite fusion protein.170#14533290vl9. A gRNA comprising a spacer sequence which comprises or consists of the sequence of any one of SEQ ID NOs: 23-46, 562-1076, 2778-4478, and 4553-4565, or a fragment thereof.10. A combination comprising an ETM according to any one of aspects 1-8, and at least one guide RNA (gRNA).11. A combination according to aspect 10, which comprises one or more ETMs, wherein each ETM is a fusion protein comprising a catalytically active CRISPR / Cas endonuclease domain.12. A combination according to aspect 10 or aspect 11, which comprises one to three ETMs.13. A combination according to any one of aspects 10-12, wherein at least one epigenetic effector domain is a transcriptional repressor domain, and / or wherein at least one epigenetic effector domain is a DNMT3L domain.14. A combination according to any one of aspects 10-13, wherein the one or more ETMs collectively comprise a transcriptional repressor domain and a DNMT3L domain.15. A combination according to any one of aspects 10-14, which comprises at least two gRNAs.16. A combination according to aspect 15, wherein the gRNAs target the ETM to at least two different target genes.17. A combination according to aspect 15 or aspec 16, wherein the at least two gRNAs comprise spacer sequences which are of different lengths.18. A combination according to any one of aspects 10-13, wherein at least one gRNA comprises a spacer sequence which is 15, 16, 17, 18, 19 or 20 nucleotides in length.19. A combination according to any one of aspects 15-18, wherein one of the at least two gRNAs comprises a spacer sequence which is less than or equal to 17 (e.g., less than or equal to 16) nucleotides in length.20. A combination according to any one of aspects 10-19, wherein the at least one target gene is selected from: genes without CpG Islands (CGI), such as: TRAC; TRBC; PDCD1; TIM-3; TIGIT; LAG3; CTLA4; AAVS1; RFX5; CD3D; CD3Gand CCR5; and / or genes having CGI, such as: B2M; TET2; TGFBR2; A2AR; CISH;PTPN11; PTPN6; PTPA; PTPN2; JUNB; TOX; TOX2; NR4A1; NR4A2; NR4A3;171#14533290vlMAP4K1; REL IRF4; DGKA; PIK3CD; HLA-A; USP16; DCK; CD3Z; CIITA;ADORA2A; RFXAP and FAS.21. A combination according to any one of aspects 10-20, which comprises: one or more guide RNAs (gRNAs) having a spacer sequence with a length that allows epigenetic editing and not gene editing of a first gene in the cell, optionally wherein the first gene comprises a CpG island (CGI); and one or more gRNAs having a spacer sequence with a length that allows gene editing of a second gene in the cell.22. A combination according to aspect 21, wherein the one or more guide RNAs (gRNAs) having a spacer sequence with a length that allows epigenetic editing and not gene editing of a first gene in the cell has a spacer sequence of:(a) less than or equal to 17 nucleotides (e.g., less than or equal to 16 nucleotides); or(b) 11 to 17 nucleotides (e.g., 11 to 16 nucleotides).23. A combination according to aspect 21 or aspect 22, wherein the one or more gRNAs having a spacer sequence with a length that allows gene editing of a second gene in the cell has a spacer sequence of:(a) 17 or more nucleotides (e.g., 18 or more nucleotides); or(b) 17 to 30 nucleotides, optionally 18 to 25 nucleotides (e.g., 18 to 21 nucleotides).24. A combination comprising one or more polynucleotides coding for the ETM(s) (e.g., fusion proteins) and / or gRNAs as defined in any one of aspects s 10-23.25. A combination according to any one of aspects 21-24, further comprising a donor DNA (e.g., a donor template nucleic acid) comprising 5’ and 3’ arms that are homologous to sequences in the second gene.26. A combination according to any one of aspects 10-25, wherein the endonuclease domain is derived from a Cas9 protein, optionally SpCas9.27. A combination according to any one of aspects 21-26, whereinthe first gene is selected from B2M, TET2, TGFBR2, A2AR, CISH, PTPN11, PTPN6, PTPA, PTPN2, JUNB, TOX, TOX2, NR4A1, NR4A2, NR4A3, MAP4K1, REL, IRF4, DGKA, PIK3CD, HLA-A, LISP 16, DCK, CD3Z, CIITA, ADORA2A, RFXAP, and FAS; and / orthe second gene is selected from TRAC, TRBC, PDCD1, TIM-3, TIGIT, LAG3, CTLA4, AAVS1, and CCR5.172#14533290vl28. A combination according to any one of aspects 21-27, wherein the second gene is a TRAC gene, optionally wherein the one or more gRNAs targeting the TRAC gene comprise a spacer having the sequence of one of SEQ ID NOs: 562-611, optionally SEQ ID NO: 604.29. A combination according to any one of aspects 21-28, wherein the first gene is a B2 / W gene, optionally wherein the one or more gRNAs targeting the B2M gene each comprise a spacer having the sequence of one of SEQ ID NOs: 28-33 and 39-44; or the sequence of one of SEQ ID NOs: 2778-2878 with a 3 to 9 nucleotide truncation at the 5’ end, optionally one of SEQ ID NOs: 2778, 2780, 2801, and 2863 with a 3 to 9 nucleotide truncation at the 5’ end, selected from SEQ ID NOs: 4486-4492, 4497-4503, 4508-4514, and 4519-4525.30. A combination according to any one of aspects 21-28, wherein the first gene is a TGFBR2 gene, optionally wherein the one or more gRNAs targeting the TGFBR2 gene each comprise a spacer having the sequence of one of SEQ ID NOs: 2929-2978 and 4553-4559 with a 3 to 9 nucleotide truncation at the 5’ end.31. A combination according to any one of aspects 21 -28, wherein the first gene is a TET2 gene, optionally wherein the one or more gRNAs targeting the TET2 gene each comprise a spacer having the sequence of one of SEQ ID NOs: 4429-4478 and 4560-4565 with a 3 to 9 nucleotide truncation at the 5’ end.32. A combination according to any one of aspects 10-31 for modifying transcription, expression and / or activity of one or more e.g. two or more) gene in a cell, wherein the cell is a mammalian cell, optionally a human cell, optionally wherein the cell is a human immune cell, or a human T cell.33. A combination according to any one of aspects 10 to 32, further comprising an agent:i) which promotes the survival, proliferation and / or activity of a cell, such as a cell which comprises the combination or a cell which does not comprise the combination; and / orii) which is detrimental to the survival, proliferation, activity, chemoresistance and / or chemotaxis of a cell, such as a cell which comprises the combination or a cell which does not comprise the combination and / oriii) which enables selection of a cell, such as a cell which comprises the combination or a cell which does not comprise the combination.173#14533290vl34. A combination according to any one of aspects 10 to 33, comprising at least one gRNA according to aspect 9.35. The combination of any one of aspects 20-34, wherein the gene comprising a CGI is a B2M gene and the gRNAs targeting it are two or three gRNAs each independently comprising a spacer having the sequence ofC8 (SEQ ID NO: 35),F4 (SEQ ID NO: 24),H8 (SEQ ID NO: 2780),H10 (SEQ ID NO: 2863),H11 (SEQ ID NO: 2778), orH12 (SEQ ID NO: 2801),optionally with a 1 to 9, optionally 3 to 9, nucleotide truncation at the 5’ end.36. The combination of aspect 35, wherein the B2M-targeting gRNAs comprise (i) a gRNA comprising a spacer having the sequence of F4 optionally with a 1 to 9, optionally 3 to 9, nucleotide truncation at the 5’ end,a gRNA comprising a spacer having the sequence of H8 optionally with a 1 to 9, optionally 3 to 9, nucleotide truncation at the 5’ end, anda gRNA comprising a spacer having the sequence of H10 optionally with a 1 to 9, optionally 3 to 9, nucleotide truncation at the 5’ end;(ii) a gRNA comprising a spacer having the sequence of C8 optionally with a 1 to 9, optionally 3 to 9, nucleotide truncation at the 5’ end,a gRNA comprising a spacer having the sequence of H8 optionally with a 1 to 9, optionally 3 to 9, nucleotide truncation at the 5’ end, anda gRNA comprising a spacer having the sequence of H10 optionally with a 1 to 9, optionally 3 to 9, nucleotide truncation at the 5’ end;(iii) a gRNA comprising a spacer having the sequence of F4 optionally with a 1 to 9, optionally 3 to 9, nucleotide truncation at the 5’ end, anda gRNA comprising a spacer having the sequence of H8 optionally with a 1 to 9, optionally 3 to 9, nucleotide truncation at the 5’ end;(iv) a gRNA comprising a spacer having the sequence of F4 optionally with a 1 to 9, optionally 3 to 9, nucleotide truncation at the 5’ end, anda gRNA comprising a spacer having the sequence of H10 optionally with a 1 to 9, optionally 3 to 9, nucleotide truncation at the 5’ end; or174#14533290vl(v) a gRNA comprising a spacer having the sequence of H8 optionally with a 1 to 9, optionally 3 to 9, nucleotide truncation at the 5’ end, anda gRNA comprising a spacer having the sequence of H10 optionally with a 1 to 9, optionally 3 to 9, nucleotide truncation at the 5’ end.37. The combination of any one of aspects 21-36, wherein the ETM(s) (e.g., one or more fusion proteins) collectively further comprise a DNMT1, DNMT3A, DNMT3B, or SETDB1 domain, optionally DNMT3A.38. The combination of any one of aspects 10-37, wherein the combination comprises(i) a first fusion protein comprising a transcriptional repressor domain and a Cas endonuclease domain, and a second fusion protein comprising a DNMT3L domain and a Cas endonuclease domain, or(ii) a fusion protein comprising, optionally from N-terminus to C-terminus, a transcriptional repressor domain, a Cas endonuclease domain, and a DNMT3L domain.39. The combination of any one of aspects 10-37, wherein the combination comprises(i) a first fusion protein comprising a transcriptional repressor domain and a Cas endonuclease domain, a second fusion protein comprising a DNMT3L domain and a Cas endonuclease domain, and a third fusion protein comprising a DNMT3A domain and a Cas endonuclease domain, or(ii) a fusion protein comprising a transcriptional repressor domain, a Cas endonuclease domain, a DNMT3L domain, and a DNMT3A domain.40. The combination of any one of aspects 10-39, wherein the epigenetic effector domain (e.g. transcriptional repressor domain) is a Kruppel-associated box (KRAB) domain, optionally derived from human Kox1 or ZIM3.41. The combination of any one of aspects 10-40, wherein the combination comprises a fusion protein comprising, optionally from N terminus to C terminus, a KRAB domain derived from ZIM3, a catalytically active Cas9 domain, and a DNMT3L domain, optionally comprising an amino acid sequence of SEQ ID NO: 4482.42. The combination of any one of aspects 10-41, further comprising gRNAs for targeting one or more additional genes in the cell.175#14533290vl43. The combination of any one of aspects 10-42, wherein the gRNA(s) are chemically modified, optionally wherein the chemically modified gRNA(s) comprise phosphorothioate internucleoside linkages at the 5’ and / or 3’ ends, and / or 2’-O-methyl nucleotides.44. A polynucleotide encoding at least one ETM according to any one of aspects 1 to 8 or as defined in any one of aspects 10-43.45. A nucleic acid construct comprising a nucleic acid sequence encoding at least one ETM according to any one of aspects 1 to 8 or as defined in any one of aspects 10-43.46. A nucleic acid construct according to aspect 45, further comprising a nucleic acid sequence:i) which promotes the survival, proliferation and / or activity of a cell, such as a cell which expresses said nucleic acid construct or a cell which does not express said nucleic acid construct; and / orii) which is detrimental to the survival, proliferation, activity, chemoresistance and / or chemotaxis of a cell, such as a cell which expresses said nucleic acid construct or a cell which does not express said nucleic acid construct; and / or iii) which enables selection of a cell, such as a cell which comprises the nucleic acid construct or a cell which does not comprise the construct.47. A vector comprising a polynucleotide according to aspect 44 or a nucleic acid construct according to aspect 45 or 46.48. A kit of polynucleotides comprising:a) at least one polynucleotide encoding at least one ETM according to any one of aspects 1 to 8 or as defined in any one of aspects 10-43; and b) a polynucleotide providing at least one gRNA as described in any one of aspects 9 or 10 to 32 or 35 to 43; and optionally,c) a further polynucleotide comprising a nucleic acid sequence which encodes an agent:i) which promotes the survival, proliferation and / or activity of a cell, such as a cell which comprises the polynucleotides or a cell which does not comprise the polynucleotides; and / orii) which is detrimental to the survival, proliferation, activity, chemoresistance and / or chemotaxis of a cell, such as a cell which 176#14533290vlcomprises said polynucleotides or a cell which does not comprise said polynucleotides; and / oriii) which enables selection of a cell, such as a cell which comprises the polynucleotides or a cell which does not comprise the polynucleotides.49. A cell comprising an ETM according to any one of aspects 1 to 8, at least one gRNA according to aspect 9, a combination according to any one of aspects 10 to 43, a polynucleotide according to aspect 44, a nucleic acid construct according to aspect 45 or aspect 46, a vector according to aspect 47 or a kit of polynucleotides according to aspect 48.50. A cell wherein the cell is a progeny of the cell of aspect 49.51. A composition comprising an ETM according to any one of aspects 1 to 8, at least one gRNA according to aspect 9, a combination according to any one of aspect 10 to 43, a polynucleotide according to aspect 44, a nucleic acid construct according to aspect 45 or aspect 46, a vector according to aspect 47, a kit of polynucleotides according to aspect 48 or a cell according to aspect 49 or aspect 50.52. A pharmaceutical composition comprising an ETM according to any one of aspect 1 to 8, at least one gRNA according to aspect 9, a combination according to any one of aspects 10 to 43, a polynucleotide according to aspect 44, a nucleic acid construct according to aspect 45 or aspect 46, a vector according to aspect 47, a kit of polynucleotides according to aspect 48 or a cell according to aspect 49 or aspect 50.53. Use of an ETM according to any one of aspects 1 to 8, at least one gRNA according to aspect 9, a combination according to any one of aspects 10 to 43, a polynucleotide according to aspect 44, a nucleic acid construct according to aspect 45 or aspect 46, a vector according to aspect 47, a kit of polynucleotides according to aspect 48 or a cell according to aspect 49 or aspect 50 for modifying the transcription, expression and / or activity of (e.g. repressing or silencing) at least one target gene in a cell.54. A method of modifying the transcription, expression and / or activity of e.g. repressing or silencing) at least one target gene in a cell comprising the step of administering an ETM according to any one of aspects 1 to 8, at least one gRNA according to aspect 9, a combination according to any one of aspects 10 to 43, a polynucleotide according to aspect 44, a nucleic acid construct according to aspect 177#14533290vl45 or aspect 46, a vector according to aspect 47 or a kit of polynucleotides according to aspect 48 to a cell.55. The use or method of aspects 53 or 54, wherein the cell is a T cell.56. The use or method of any one or aspects 53-55, wherein the ETM, at least one gRNA, combination, polynucleotide, nucleic acid construct, vector or a kit of polynucleotides is introduced into the cell in vitro or ex vivo.57. A method according to any one of aspects 54-56, wherein at least two target genes are silenced, wherein at least one of the at least two target genes is epigenetically silenced and at least one of the at least two target genes is silenced by gene editing, wherein at least one ETM and at least two gRNAs are administered to said cell simultaneously, sequentially or separately.58. A cell obtained by the use or method of any one of aspects 53-57, or a progeny of the cell.59. The cell of any one of aspects 49, 50 or 58, wherein the cell is a human T cell, optionally engineered to express a recombinant antigen receptor, optionally selected from a recombinant T cell receptor (TCR) or a chimeric antigen receptor (CAR). 60. An ETM according to any one of aspects 1 to 8, at least one gRNA according to aspect 9, a combination according to any one of aspect 10 to 43, a polynucleotide according to aspect 44, a nucleic acid construct according to aspect 45 or aspect 46, a vector according to aspect 47, a kit of polynucleotides according to aspect 48, a cell according to aspect 49, 50, 58 or 59 or a pharmaceutical composition according to aspect 52 for use in therapy (e.g. for use in treating a human in need thereof). 61. Use of an ETM according to any one of aspects 1 to 8, at least one gRNA according to aspect 9, a combination according to any one of aspects 10 to 43, a polynucleotide according to aspect 44, a nucleic acid construct according to aspect 45 or aspect 46, a vector according to aspect 47, a kit of polynucleotides according to aspect 48, a cell according to aspect 49, 50, 58 or 59 or a pharmaceutical composition according to aspect 52 in the manufacture of medicament for treating a human in need thereof.62. An ETM, combination, polynucleotide, nucleic acid construct, vector, kit of polynucleotides, cell or pharmaceutical composition for use according to aspect 60, or the use of aspect 61, wherein at least one ETM (e.g. fusion protein) and at least178#14533290vltwo gRNAs are administered to a cell or subject simultaneously, sequentially or separately.63. A method for treating and / or preventing a disease (e.g. in a human in need thereof), which comprises the step of administering an ETM according to any one of aspects 1 to 8, at least one gRNA according to aspect 9, a combination according to any one of aspects 10 to 43, a polynucleotide according to aspect 44, a nucleic acid construct according to aspect 45 or aspect 46, a vector according to aspect 47, a kit of polynucleotides according to aspect 48, a cell according to aspect 49, 50, 58 or 59 or a pharmaceutical composition according to aspect 52 to a subject in need thereof.64. A method for treating and / or preventing a disease according to aspect 63, wherein at least one ETM e.g. fusion protein) and at least two gRNAs are administered to a cell or subject simultaneously, sequentially or separately.65. A method of gene therapy which comprises the steps:(i) isolation of a cell containing sample;(ii) introduction of an ETM according to any one of aspects 1 to 8, at least one gRNA according to aspect 9, the combination according to any one of aspects 10 to 43, the polynucleotide as defined in aspect 44, the nucleic acid construct according to aspect 45 or aspect 46, a vector according to aspect 47 and / or a kit of polynucleotides according to aspect 48 to the cell(s); and(iii) administering the cell(s) from step (ii) to a subject.66. The method according to aspect 65, wherein the polynucleotide, nucleic acid construct and / or vector is introduced by transduction or transfection.67. An ETM, combination, polynucleotide, nucleic acid construct, vector, kit of polynucleotides, cell or pharmaceutical composition for use according to aspect 60 or 62, the use of aspect 61 or 62, or the method according to any one of aspects 63-66, wherein the cell is autologous.68. An ETM, combination, polynucleotide, nucleic acid construct, vector, kit of polynucleotides, cell or pharmaceutical composition for use according to aspect 60 or 62, the use of aspect 61 or 62, or the method according to any one of aspects 63-66, wherein the cell is allogeneic.

[0506] Unless otherwise defined herein, scientific, and technical terms used in connection with the present disclosure shall have the meanings that are commonly 179#14533290vlunderstood by those of ordinary skill in the art. Exemplary methods and materials are described below, although 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.Generally, nomenclature used in connection with, and techniques of, medicine, medicinal and pharmaceutical chemistry, and cell biology described herein are those well-known and commonly used in the art. Enzymatic reactions and purification techniques are performed according to manufacturer’s specifications, as commonly accomplished in the art or as described herein. Further, unless otherwise required by context, singular terms shall include pluralities and plural terms shall include the singular. Throughout this specification and embodiments, the words “have” and “comprise,” or variations such as “has,” “having,” “comprises,” or “comprising,” will be understood to imply the inclusion of a stated integer or group of integers but not the exclusion of any other integer or group of integers. It should also be noted that the term “or” is generally employed in its sense including “and / or” unless the content clearly dictates otherwise. As used herein the term “about” refers to a numerical range that is 10%, 5%, or 1% plus or minus from a stated numerical value within the context of the particular usage. Further, headings provided herein are for convenience only and do not interpret the scope or meaning of the claimed embodiments.

[0507] The practice of the present invention will employ, unless otherwise indicated, conventional techniques of chemistry, biochemistry, molecular biology, microbiology, and immunology, which are within the capabilities of a person of ordinary skill in the art. Such techniques are explained in the literature. See, for example, Sambrook, J., Fritsch, E. F. and Maniatis, T. (1989) Molecular Cloning: A Laboratory Manual, 2nd Edition, Cold Spring Harbor Laboratory Press; Ausubel, F. M. et al. (1995 and periodic supplements) Current Protocols in Molecular Biology, Ch. 9, 13 and 16, John Wiley & Sons; Roe, B., Crabtree, J. and Kahn, A. (1996) DNA Isolation and Sequencing: Essential Techniques, John Wiley & Sons; Polak, J. M. and McGee, J. O’D. (1990) In Situ Hybridization: Principles and Practice, Oxford University Press; Gait, M. J. (1984) Oligonucleotide Synthesis: A Practical Approach, IRL Press; and Lilley, D. M. and Dahlberg, J. E. (1992) Methods in Enzymology: DNA Structures Part A: Synthesis and Physical Analysis of DNA, Academic Press.180#14533290vl

[0508] All publications and other references mentioned herein are incorporated by reference in their entirety. Although a number of 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.

[0509] In order that this invention may be better understood, the following examples are set forth. These examples are for purposes of illustration only and are not to be construed as limiting the scope of the invention in any manner.EXAMPLESEXAMPLE 1 - gRNAs comprising truncated spacer sequences promote epigenetic silencing without causing mutagenesis

[0510] To assess the feasibility of using gRNAs comprising truncated spacer sequences to promote ETR-mediated epi-silencing of B2M while sparing the gene from mutagenesis, we first designed a 20nt-long gRNA against B2M (named F4; SEQ ID NO: 24) and a corresponding panel of 5’-truncated B2M gRNAs with spacer sequences of different lengths, and then we tested them in the B2MtdTomatoK-562 cell line (Amabile et al., supra).

[0511] In particular, the gRNAs comprising spacer sequences spanning from 21 to 10 nt in length and comprising the same seed and PAM sequence (FIG. 1) were individually delivered into the cells together with either Cas9 or the dCas9-based ETR combination, the latter containing KRAB, DNMT3A or DNMT3L. The cells were then analysed for genetic traces of Cas9 activity at the B2M gene or expression of tdTomato, the latter used as a proxy for B2M epigenetic silencing.

[0512] gRNAs comprising the standard 20 nt-long B2M spacer sequence plus Cas9 or dCas9-ETRs were included as positive controls for gene disruption or epigenetic silencing, respectively. Molecular analyses of the B2M target site in Cas9-treated cells showed a threshold effect: gRNAs comprising a spacer sequence of >17nt in length mediated high and comparable levels of B2M editing (-30%) while gRNAs comprising a spacer sequence <1 Ont resulted in undetectable gene editing (FIG. 2). Flow cytometry analyses of ETR-treated cells showed a different trend: all gRNAs except the gRNA comprising the 10nt-long spacer sequence were able to 181#14533290vlinduce efficient epigenetic silencing of B2M, although at different levels (from 30 to 48% of tdTomato-negative cells; FIG. 3). Importantly, the gRNAs comprising truncated spacer sequences that were ineffective in promoting gene editing with Cas9 (i.e., <16nt) were highly effective in mediating epigenetic silencing with the dCas9-ETRs. To assess if these findings were portable to other gRNAs, we performed a similar truncation experiment using three other gRNAs (named H8_20 (spacer SEQ ID NO: 2780; gRNA SEQ ID NO: 4570), C8_20 (spacer SEQ ID NO: 2813; gRNA SEQ ID NO: 4569) and H10_20 (spacer SEQ ID NO: 2863; gRNA SEQ ID NO: 4571) and found that the spacer length at which Cas9 lost its activity depended on the specific gRNA used, ranging between <15 and <17nt in length (FIG. 4; left panel). In accordance to what we showed above, ETRs were able to induce epi-silencing of B2Meven with truncated gRNAs (FIG. 4; right panel).

[0513] Overall, these data indicate that gRNAs comprising a truncated spacer sequence of <17nt promote epigenetic silencing of B2M while sparing this gene from mutagenesis induced by Cas9-based ETRs. Furthermore, they provide the first demonstration that epi-silencing can be imposed also when using gRNAs comprising truncated spacer sequences. In parallel to these experiments, we also produced a gRNA comprising a 20nt spacer sequence capable of inducing gene editing at the TRAC locus (FIG. 5).EXAMPLE 2 - A combination of ETR and gRNAs enables simultaneous inactivation of two genes without inducing chromosomal translocations

[0514] Based on these data, we then constructed ETRs equipped with a catalytically active Cas9 (hereafter referred as to Cas9-ETRs, containing KRAB, DNMT3A or DNMT3L) and assessed their multiplexing efficiency with gRNAs comprising truncated or full-length spacer sequences in the B2MtdTomatoK-562 cells. In particular, we co-transfected the cells with the triple Cas9-ETR combination plus the F4-derived B2MgRNA comprising 16nt-long spacer sequences (see FIG.s 2 and 3) and the TRAC gRNA with the 20nt-long spacer. The following controls were also included in the experiment: (i) cells co-transfected with the just mentioned gRNA combination plus either Cas9 or the standard triple dCas9-ETRs, used as positive control for either genetic disruption of TRAC or epi-silencing of B2 / W and disruption of TRAC, respectively; (ii) cells co-transfected with two gRNAs comprising 20nt-long 182#14533290vlspacer sequences, one against B2 / W and the other against TRAC, plus either Cas9 or Cas9-ETRs, were used here as positive controls for co-disruption of B2M and TRAC. The latter conditions were also included to assess if simultaneous gene editing of the two loci may lead to reciprocal chromosomal translocations. Upon transfection, the cells were longitudinally monitored by flow cytometry for tdTomato expression for up to 25 days.

[0515] As shown in FIG. 6 and FIG. 7, when delivered with the gRNA comprising a 16nt spacer sequence, Cas9- and dCas9-based ETRs performed equally in terms of B2Mepi-silencing (14 vs. 21%, respectively). Cas9 promoted B2M inactivation only when coupled with the gRNA comprising a 20nt-long spacer sequence and not with its truncated counterpart (24% vs. 1.7% of tdTomato negative cells), further confirming the results of FIG. 2 and FIG. 3. The use of the gRNA comprising a 20nt-long B2M spacer sequence with Cas9-ETRs resulted in a percentage of tdTomato negative cells that was higher than that found in all other conditions (up to 44%), a finding expected considering the additive effect of gene and epigenetic editing on this locus. Of note, silencing was stable long-term in all analyzed conditions (FIG.8), indicating that also the Cas9-ETRs with the gRNA comprising a 16nt spacer sequence are able to instruct mitotically inherited epigenetic modifications.

[0516] We then analyzed the cells for gene editing (FIG. 9) and found that both Cas9 and Cas9-ETRs induced efficient editing of TRAC (up to 37%). On the other hand, gene editing of B2M was limited to the conditions in which Cas9 or Cas9-ETRs were co-delivered with the gRNA comprising the 20nt-long B2M spacer sequence. Finally, we performed a PGR analysis with primers specific for reciprocal chromosomal translocations between B2M and TRAC and found occurrence of these events exclusively in the conditions co-treated with the two gRNAs comprising 20nt-long spacer sequences, but not when the gRNA comprising the 16nt spacer sequence was used (FIG. 10).

[0517] Overall, these data show that Cas9-ETRs perform as their dCas9-based counterparts in terms of silencing efficiency and stability. Yet, adoption of Cas9-ETRs in combination with gRNAs comprising a truncated and a full-length spacer sequence can be safely used to inactivate simultaneously two genes without inducing chromosomal translocations.183#14533290vlEXAMPLE 3 - Optimization of the B2 / W epi-silencing procedure in human primary T lymphocytes

[0518] Inactivation of B2M'\s emerging as a promising approach to generate allogenic T cell products. To assess feasibility of B2M epi-silencing in human primary T cells, we first expanded our repertoire of gRNAs against this gene to include 2 other guides: H11_20 (spacer SEQ ID NO: 2778; gRNA SEQ ID NO: 4572) and H12_20 (spacer SEQ ID NO: 2801; gRNA SEQ ID NO: 4573) (FIG. 11). We then delivered each of these 6 gRNAs with mRNAs encoding for the triple ETR combination in T cells. Time course flow cytometry analyses of treated cells were then used to assess efficiency and stability of B2M epi-silencing. Unexpectedly, at day 12 post-treatment, all but one of the tested gRNAs failed to induce epi-silencing of B2M (FIG. 12). The only working gRNAs (namely gRNA C8) resulted in up to 2% of B2M-negative cells, which, however, were lost upon T cell restimulation (analysis at day 25).

[0519] We then tested whether combined delivery of gRNAs would improve epi-silencing efficiency. To this end, we combined either gRNA C8 or H8 with all other gRNAs and delivered these dual gRNA combinations together with the triple ETR combination in T cells. Flow cytometry analyses at day 12 post-treatment revealed that all gRNA combinations were able to induce epi-silencing of B2M, although at different levels (FIG. 12). For instance, gRNA combination H12+H8 induced limited silencing, while the F4+H8 combination resulted in up to 28% of B2M-negative cells. Importantly, for some of these gRNA combinations, epi-silencing resisted the T cell restimulation process, ranging from 11 to 20% of long-term stable B2M-negative cells (FIG.s 12 and 13). Extended time course flow cytometry analyses over a timeframe of 37 days and spanning two rounds of T cell restimulations showed that most of the tested gRNA combinations induced an initial wave of B2M epi-silencing, which then declined after the first T cell restimulation (day 12) to reach near stability until the second round of T cell restimulation (day 25) (FIG. 14). Then, the percentage of B2M increased until termination of the experiment (day 37). Of note, the efficiency of epi-silencing was dependent on the combination of gRNAs used, with H8+F4 being the most effective at long-term (up to 30% of B2M-negative cells) while H8+H11 and H8+H12 resulting in barely detectable, if any, epi-silencing.184#14533290vl

[0520] Epi-silencing stability was also dependent on gRNA combination (FIG. 15).Indeed, by comparing the percentage of B2M-negative cells between day 25 (just before to the second round of T cell restimulation) and day 12 (just before the first round of T cell restimulation), we found that some gRNA combinations were poorly resistant (fold reduction in B2M-negative cells <0.5) while others were more resistant (fold reduction in B2M-negative cells > 0.5), although none of them were able to result in fully stable gene silencing. Among the most stable, combinations H8+F4 and H8+H10 were the best performing ones.

[0521] We then performed a similar experiment, in which we excluded the ineffective gRNA combinations H8+H11 and H8+H12 and included the new dual-gRNA combination F4+H10. Furthermore, we also included triple gRNA combinations (namely, C8+F4+H8, C8+F4+H10, C8+H8+H10 and F4+H8+H10) to assess if these were able to further improve epi-silencing efficiency and stability. Among the dual-gRNA combinations tested, the most effective at long-term (day 32) was F4+H10, reaching up to 36.5% of B2M-negative cells. Among the triple gRNA combinations tested, the F4+H8+H10 outperformed the others by 1.6-fold, reaching up to 66% of B2M-negative cells at termination of the experiment (FIG. 16). As observed in the previous experiment, the first round of T cell restimulation caused a marked reduction in the percentage of B2M negative cells for most of the gRNA combinations (FIG. 16). Noticeable exceptions to this were the gRNA combinations containing F4+H10 (including the triple C8+F4+H10 and F4+H8+H10), for which the percentage of B2M-negative cells at day 28 and 14 were nearly superimposable (fold reduction in B2M-negative cells ~1; FIG. 17). Similar findings were obtained for the dual C8+H10 gRNA combination (FIG. 17). Overall, these data show that epi-silencing efficiency and durability of B2M depends on which gRNA combination is used, with the triple based on the F4+H8+H10 being the best-preforming one.

[0522] With the aim of reducing the molecular complexity of the technology, we then asked whether all the components of the triple ETR combination were required for epi-silencing of B2M. To this end, we transiently delivered to T cells the dual-gRNA combination containing C8 and F4 together with mRNAs encoding either: (i) the triple ETR combination, taken here as reference for epi-silencing efficiency of B2M-, (ii) the double ETR combination containing the KRAB and DNMT3L effector domains; (iii) the double ETR combination containing the DNMT3A and DNMT3L 185#14533290vleffector domains; or (iv) the double ETR combination containing the KRAB and DNMT3L effector domains. The T cells were then analysed for B2M expression by flow cytometry until day 37 post-treatment (FIGs. 18A and 18B). This experiment showed that, among all the double ETR combinations tested, only the one based on the KRAB and DNMT3L effector domains induced long-term silencing, at efficiencies superimposable to those observed with the triple ETR combination (up to 14% of B2M-negative cells). The double ETR combination based on KRAB and DNMT3A induced only transient B2M repression, which, after the first round of T cell restimulation, returned to the levels observed in untreated T cells. Unexpectedly, the double ETR combination based on DNMT3A and DNMT3L failed to induce any B2M silencing, even at early time points post-treatment.

[0523] Based on these results, we then performed a similar experiment to that shown in FIG. 16 but using the double ETR combination containing KRAB and DNMT3L, confirming that this combination performed as efficiently as the triple one for all gRNA combinations tested (FIG. 19). As for the triple ETR combination, the conditions in which the gRNAs F4 and H10 were co-present were the most resistant to T cell restimulation (FIG. 19). Overall, these data show that the double ETR combination containing KRAB and DNMT3L performs as efficiently as the canonical triple ETR combination in silencing B2M\r T cells.

[0524] Based on these results, we then compared the efficiencies of B2M episilencing between the double ETR combination containing KRAB and DNMT3L and an all-in-one bi-partite ETR equipped with the KRAB domain homolog of the Zinc finger imprinted 3 (ZIM3) protein (Alerasool et al., Nat Methods (2020) 17(11 ):1093-6) and DNMT3L (FIG. 20A, left schematic), hereafter referred as to the ZIM:dCas9: DNMT3L fusion. The amino acid sequence of this fusion protein is shown below, wherein the SV40 nuclear localization signals (NLSs) are in box, the ZIM3 KRAB repressor domain is in boldface, the flexible linkers are in underlined boldface, dCas9 is underlined and the DNMT3L domain is in italics (only):M[pKKKRK |GGGASMGRVTFEDVTVNFTQGEWQRLNPEQRNLYRDVMLENYSNLVSVGQGETTKPDVIL RLEQGKEPWLEEEEVLGSGRAEKNGDIGGQIWKPKDVKESLGGGGSGGGGSGGGGSGGGGSLEDKKYS IGLAIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKNLIGALLFDSGETAEATRLKRTARRRYTR RKNRICYLQEIFSNEMAKVDDSFFHRLEESFLVEEDKKHERHP IFGNIVDEVAYHEKYPTIYHLRKKL VDSTDKADLRLIYLALAHMIKFRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKA ILSARLSKSRRLENLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNL186#14533290vlLAQIGDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVRQQLPEK YKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLRKQRTFDNGSIPHQI HLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARGNSRFAWMTRKSEETITPWNFEEVV DKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEYFTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVD LLFKTNRKVTVKQLKEDYFKKIECFDSVEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILED IVLTLTLFEDREMIEERLKTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSD GFANRNFMQLIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGRH KPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLYLYYLQNGRDM YVDQELDINRLSDYDVDAIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVPSEEWKKMKNYWRQLLNAK LITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKHVAQILDSRMNTKYDENDKLIREVKVITL KSKLVSDFRKDFQFYKVREINNYHHAHDAYLNAWGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSE QEIGKATAKYFFYSNIMNFFKTEITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNI VKKTEVQTGGFSKESILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLWAKVEKGKSKKLKSVKE LLGITIMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNELALPS KYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILADANLDKVLSAYNKHR DKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVLDATLIHQSITGLYETRIDLSQLG GDSPKKKRJ \GVDGSGGGMAAIPALDPEAEPSMDVILVGSSELSSSVSPGTGRDLIAYEVKANQRNIE DICICCGSLQVHTQHPLFEGGICAPCKDKFLDALFLYDDDGYQSYCSICCSGETLLICGNPDCTRCYC FECVDSLVGPGTSGKVHAMSNWVCYLCLPSSRSGLLQRRRKWRSQLKAFYDRESENPLEMFETVPVWR R QP VR VL SLFED IKKEL TSL GFLES GSDP GQLKHWD VTD TVRKD VEEWGPFDL VYGA TPPLGHTCDR PPSWYLFQFHRLL Q YARPKP GSPRPFFWMFVDNL VLNKEDLD VASRFLEMEP VTIPD VHGGSL QNAVR VW SNIP A IR SRHWAL VS EE EL SLLA QNKQSSKLAAKWP TKL VKNCFLPLRE YFKYFS TEL TSSL(SEQ ID NO: 4481)

[0525] In these experiments, the T cells were co-transfected with the mRNAs encoding the ETRs and (i) the gRNAs F4 or C8, to assess if the bi-partite ETR was able to rescue epi-silencing efficiency of individual gRNAs; (ii) the dual-gRNA combination C8+F4; or (iii) the best-performing triple gRNA combination F4+H8+H10. Cells were then analysed by flow cytometry until day 55. To avoid any confounding effects due to the delivery of different amounts of mRNAs encoding the ETRs, these experiments were performed by using 1.5 pg of each ETR for the double combination and 1.5 pg of the ZIM:dCas9: DNMT3L fusion. As such, matched amounts of epigenetic effectors were used. In accordance with our previous data, individual gRNAs were ineffective with the double ETR combination, and adoption of the ZIM:dCas9: DNMT3L fusion only slightly increased B2M epi-silencing efficiency and exclusively for gRNA C8 (FIG. 20A, right graph). On the other hand, a marked increase in B2M epi-silencing was found when comparing the double ETR combination and the ZIM:dCas9: DNMT3L fusion with the dual-gRNA combination C8+F4 (from 11% to 70%, respectively; FIG.s 20A-B). Similar results were obtained for the triple gRNA combination F4+H8+H10, although the differences between the double ETR combination and the ZIM:dCas9: DNMT3L fusion were less pronounced (86 and 95% of B2M-negative cells for the double ETR combination and the187#14533290vlZIM:dCas9: DNMT3L fusion, respectively; FIG.s 20A-B). This effect was likely due to the already high epi-silencing efficiency of the double ETR combination. For all conditions with the triple gRNA combination, B2M epi-silencing proved to be durable, resisting 2 rounds of T cell restimulations. Notable was the stability observed with the ZIM:dCas9: DNMT3L fusion and the triple gRNA combination, which reached 95% of B2M-negative cells at day 8 post-treatment to then remain stable until day 55.Finally, to assess if the mRNA dose of the ZIM:dCas9: DNMT3L fusion was at saturation, we performed a dose titration experiment in T cells and found that one third of the standard doses (1 vs. 1.5 pg) was already sufficient to obtain efficient epi-silencing of B2M (FIG. 21).

[0526] Overall, these data show that adoption of the fusion protein ZIM:dCas9: DNMT3L improves epi-silencing in T cells, achieving up to 95% of B2M-negative cells. Interesting features of this fusion protein include the reduced costs of production as compared to the triple or double ETR combinations and the fact that it can depose efficient silencing at one third of the dose of the double ETR combination.EXAMPLE 4 - Orthogonal editing of B2M and TRAC in human primary T cells without inducing reciprocal chromosomal translocations

[0527] Based on the above data, we then tested if co-delivery of Cas9-based ETRs together with truncated gRNAs against B2 / W and the full-length gRNA against TRAC (SEQ ID NO: 4575) can induce orthogonal edits (namely epi-silencing of B2M and targeted integration into the TRAC gene) in human primary T cells without causing reciprocal chromosomal translocations. To mediate epi-silencing of B2M, we used some of the truncated gRNAs described above (see FIGs. 2-4), namely truncated C8 (C8_16; 16nt-long spacer; gRNA SEQ ID NO: 4578), truncated F4 (F4_16; 16nt-long spacer; gRNA SEQ ID NO: 4579) and truncated H8 (H8_15; 15nt-long spacer; gRNA SEQ ID NO: 4577), which we co-delivered in T cells as a triple combination. All truncations herein start from the 5’ end of the full-length sequence. In these experiments, we also used the reduced ETR combination / architecture identified above, namely the double ETRs containing KRAB and DNMT3L or the cognate all-in-one fusion protein with a ZIM3 KRAB domain and DNMT3L, both of which were modified to contain the catalytically active Cas9. The all-in-one fusion 188#14533290vlwith ZIM3 KRAB, active Cas9 and DNMT3L domains has the following amino acid sequence, wherein the SV40 NLSs are in box, the ZIM3 KRAB repressor domain is in boldface, the flexible linkers are in underlined boldface, Cas9 is underlined and the DNMT3L domain is in italics (only):M[pKKKRK |GGGASMGRVTFEDVTVNFTQGEWQRLNPEQRNLYRDVMLENYSNLVSVGQGETTKPDVIL RLEQGKEPWLEEEEVLGSGRAEKNGDIGGQIWKPKDVKESLGGGGSGGGGSGGGGSGGGGSLEDKKYS IGLDIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKNLIGALLFDSGETAEATRLKRTARRRYTR RKNRICYLQEIFSNEMAKVDDSFFHRLEESFLVEEDKKHERHP IFGNIVDEVAYHEKYPTIYHLRKKL VDSTDKADLRLIYLALAHMIKFRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKA ILSARLSKSRRLENLIAQLPGEKKNGLFG...

Claims

CLAIMS1. A combination for modifying transcription, expression and / or activity of one or more gene in a cell, the combination comprising:(A) one or more fusion proteins each comprising a catalytically active CRISPR / Cas endonuclease domain, wherein the one or more fusion proteins collectively comprise a transcriptional repressor domain and a DNMT3L domain, or polynucleotide(s) encoding the one or more fusion proteins;(B) one or more guide RNAs (gRNAs) having a spacer sequence with a length that allows epigenetic editing and not gene editing of a first gene in the cell, wherein the first gene comprises a CpG island (CGI), or polynucleotide(s) coding for the one or more gRNAs; and(C) one or more gRNAs having a spacer sequence with a length that allows gene editing of a second gene in the cell, or polynucleotide(s) coding for the one or more gRNAs.

2. The combination of claim 1, wherein:(a) the one or more gRNAs in (B) has a spacer sequence of 11 to 17 nucleotides; and / or(b) the one or more gRNAs in (C) has a spacer sequence of 18 to 21 nucleotides.

3. The combination of claim 1 or 2, wherein the combination comprises one to three fusion proteins.

4. The combination of any one of claims 1 -3, further comprising a donor DNA comprising 5’ and 3’ arms that are homologous to sequences in the second gene.

5. The combination of any one of claims 1 -4, wherein the endonuclease domain is derived from a Cas9 protein, optionally SpCas9.

6. The combination of any one of claims 1 -5, wherein214#14533290vlthe first gene is selected from B2M, TET2, TGFBR2, A2AR, CISH, PTPN11, PTPN6, PTPA, PTPN2, JUNB, TOX, TOX2, NR4A1, NR4A2, NR4A3, MAP4K1, REL, IRF4, DGKA, PIK3CD, HLA-A, USP16, DCK, and FAS and / orthe second gene is selected from TRAC, TRBC, PDCD1, TIM-3, TIGIT, LAG3, CTLA4, AAVS1, and CCR5.

7. The combination of claim 6, wherein the second gene is a TRAC gene, optionally wherein the one or more gRNAs targeting the TRAC gene comprise a spacer having the sequence of one of SEQ ID NOs: 562-611.

8. The combination of any one of claims 1 -7, wherein the first gene is a B2M gene, optionally wherein the one or more gRNAs targeting the B2M gene each comprise a spacer having the sequence of one of SEQ ID NOs: 28-33 and 39-44, or the sequence of one of SEQ ID NOs: 2778-2878 with a 3 to 9 nucleotide truncation at the 5’ end.

9. The combination of any one of claims 1 -7, wherein the first gene is a TGFBR2 gene, optionally wherein the one or more gRNAs targeting the TGFBR2 gene each comprise a spacer having the sequence of one of SEQ ID NOs: 2929-2978 and 4553-4559 with a 3 to 9 nucleotide truncation at the 5’ end.

10. The combination of any one of claims 1 -7, wherein the first gene is a TET2 gene, optionally wherein the one or more gRNAs targeting the TET2 gene each comprise a spacer having the sequence of one of SEQ ID NOs: 4429-4478 and 4560-4565 with a 3 to 9 nucleotide truncation at the 5’ end.

11. The combination of any one of claims 1 -10, wherein the cell is a mammalian cell, optionally a human cell, further optionally wherein the cell is a human immune cell, or a human T cell.

12. A combination for regulating one or more gene in a human cell, optionally an immune cell or a T cell, the combination comprising:215#14533290vlone to three fusion proteins each comprising a catalytically inactive Cas9, optionally SpCas9, endonuclease domain, wherein the one to three fusion proteins collectively comprise a transcriptional repressor and a DNMT3L domain, or polynucleotide(s) encoding the one to three fusion proteins, wherein the gene comprises a CpG island (CGI) and is(i) a B2M gene and the combination further comprises two or more gRNAs each comprising a spacer having the sequence of one of SEQ ID NOs: 2778-2878 optionally with a 1 to 9 nucleotide truncation at the 5’ end, or comprises polynucleotide(s) coding for the gRNAs;(ii) a TGFBR2 gene and the combination further comprises a gRNA that comprises a spacer having the sequence of any one of SEQ ID NOs: 2929-2978 and 4553-4559 optionally with a 1 to 9 nucleotide truncation at the 5’ end, or comprises polynucleotide(s) coding for the gRNA; or(iii) a TET2 gene and the combination further comprises a gRNA that comprises a spacer having the sequence of any one of SEQ ID NOs: 4429-4478 and 4560-4565 optionally with a 1 to 9 nucleotide truncation at the 5’ end, or comprises polynucleotide(s) coding for the gRNA.

13. The combination of any one of claims 1 -12, wherein the gene comprising a CGI is a B2M gene and the gRNAs targeting it are two or three gRNAs each independently comprising a spacer having the sequence ofC8 (SEQ ID NO: 35),F4 (SEQ ID NO: 24),H8 (SEQ ID NO: 2780),H10 (SEQ ID NO: 2863),H11 (SEQ ID NO: 2778), orH12 (SEQ ID NO: 2801),optionally with a 1 to 9, optionally 3 to 9, nucleotide truncation at the 5’ end.216#14533290vl14. The combination of claim 13, wherein the B2M-targeting gRNAs comprise (i) a gRNA comprising a spacer having the sequence of F4 optionally with a 1 to 9, optionally 3 to 9, nucleotide truncation at the 5’ end,a gRNA comprising a spacer having the sequence of H8 optionally with a 1 to 9, optionally 3 to 9, nucleotide truncation at the 5’ end, anda gRNA comprising a spacer having the sequence of H10 optionally with a 1 to 9, optionally 3 to 9, nucleotide truncation at the 5’ end;(ii) a gRNA comprising a spacer having the sequence of C8 optionally with a 1 to 9, optionally 3 to 9, nucleotide truncation at the 5’ end,a gRNA comprising a spacer having the sequence of H8 optionally with a 1 to 9, optionally 3 to 9, nucleotide truncation at the 5’ end, anda gRNA comprising a spacer having the sequence of H10 optionally with a 1 to 9, optionally 3 to 9, nucleotide truncation at the 5’ end;(iii) a gRNA comprising a spacer having the sequence of F4 optionally with a 1 to 9, optionally 3 to 9, nucleotide truncation at the 5’ end, anda gRNA comprising a spacer having the sequence of H8 optionally with a 1 to 9, optionally 3 to 9, nucleotide truncation at the 5’ end;(iv) a gRNA comprising a spacer having the sequence of F4 optionally with a 1 to 9, optionally 3 to 9, nucleotide truncation at the 5’ end, anda gRNA comprising a spacer having the sequence of H10 optionally with a 1 to 9, optionally 3 to 9, nucleotide truncation at the 5’ end; or(v) a gRNA comprising a spacer having the sequence of H8 optionally with a 1 to 9, optionally 3 to 9, nucleotide truncation at the 5’ end, anda gRNA comprising a spacer having the sequence of H10 optionally with a 1 to 9, optionally 3 to 9, nucleotide truncation at the 5’ end.

15. The combination of any one of claims 1 -12, wherein the gene comprising a CGI is a TGFBR2 gene and the combination comprises one or more gRNAs targeting it, or coding sequences of the one or more gRNAs, the one or more gRNAs each independently comprising a spacer having the sequence ofTG1 (SEQ ID NO: 4553),TG2 (SEQ ID NO: 4554),217#14533290vlTG3 (SEQ ID NO: 4555),TG4 (SEQ ID NO: 4556),TG5 (SEQ ID NO: 4557),TG6 (SEQ ID NO: 2940),TG7 (SEQ ID NO: 2937),TG8 (SEQ ID NO: 2930),TG9 (SEQ ID NO: 2955),TG10 (SEQ ID NO: 4558),TG11 (SEQ ID NO: 2957),TG12 (SEQ ID NO: 2929),TG13 (SEQ ID NO: 4559),TG14 (SEQ ID NO: 2945),TG15 (SEQ ID NO: 2931),TG16 (SEQ ID NO: 2942),TG17 (SEQ ID NO: 2939),TG18 (SEQ ID NO: 2935),TG19 (SEQ ID NO: 2938), orTG20 (SEQ ID NO: 2932),optionally with a 1 to 9, optionally 3 to 9, nucleotide truncation at the 5’ end.

16. The combination of claim 15, wherein the TGFBR2-targeting gRNAs comprise (i) a gRNA comprising a spacer having the sequence of TG7 optionally with a 1 to 9, optionally 3 to 9, nucleotide truncation at the 5’ end, anda gRNA comprising a spacer having the sequence of TG8 optionally with a 1 to 9, optionally 3 to 9, nucleotide truncation at the 5’ end; or(ii) a gRNA comprising a spacer having the sequence of TG19 optionally with a 1 to 9, optionally 3 to 9, nucleotide truncation at the 5’ end, anda gRNA comprising a spacer having the sequence of TG20 optionally with a 1 to 9, optionally 3 to 9, nucleotide truncation at the 5’ end.

17. The combination of any one of claims 1 -12, wherein the gene comprising a CGI is a TET2 gene and the combination comprises one or more gRNAs targeting it,218#14533290vlor coding sequences of the one or more gRNAs, the one or more gRNAs each independently comprising a spacer having the sequence ofTE1 (SEQ ID NO: 4560),TE2 (SEQ ID NO: 4561),TE3 (SEQ ID NO: 4562),TE4 (SEQ ID NO: 4563),TE5 (SEQ ID NO: 4443),TE6 (SEQ ID NO: 4434),TE7 (SEQ ID NO: 4466),TE8 (SEQ ID NO: 4438),TE9 (SEQ ID NO: 4429),TE10 (SEQ ID NO: 4469),TE11 (SEQ ID NO: 4564),TE12 (SEQ ID NO: 4449),TE13 (SEQ ID NO: 4433),TE14 (SEQ ID NO: 4442),TE15 (SEQ ID NO: 4430),TE16 (SEQ ID NO: 4431),TE17 (SEQ ID NO: 4474),TE18 (SEQ ID NO: 4432),TE19 (SEQ ID NO: 4565), orTE20 (SEQ ID NO: 4478),optionally with a 1 to 9, optionally 3 to 9, nucleotide truncation at the 5’ end.

18. The combination of claim 17, wherein the TET2-targeting gRNAs comprise (i) a gRNA comprising a spacer having the sequence of TE13 optionally with a 1 to 9, optionally 3 to 9, nucleotide truncation at the 5’ end, anda gRNA comprising a spacer having the sequence of TE14 optionally with a 1 to 9, optionally 3 to 9, nucleotide truncation at the 5’ end; or(ii) a gRNA comprising a spacer having the sequence of TE19 optionally with a 1 to 9, optionally 3 to 9, nucleotide truncation at the 5’ end, anda gRNA comprising a spacer having the sequence of TE20 optionally with a 1 to 9, optionally 3 to 9, nucleotide truncation at the 5’ end.219#14533290vl19. The combination of any one of claims 1 -18, further comprising gRNAs for targeting one or more additional genes in the cell, optionally wherein the combination comprises gRNAs targeting the following genes, or comprises polynucleotides coding for the gRNAs:(i) B2M and TRAC,(ii) B2M, TRAC, and TGFBR2,(iii) B2M, TRAC, and TET2,(iv) B2M, TGFBR2, and TET2, or(v) B2M, TGFBR2, TET2, and TRAC20. The combination of any one of claims 1 -19, wherein the one or more fusion proteins collectively further comprise a DNMT1, DNMT3A, DNMT3B, or SETDB1 domain, optionally DNMT3A.

21. The combination of any one of claims 1 -20, wherein the combination comprises(i) a first fusion protein comprising a transcriptional repressor domain and a Cas endonuclease domain, and a second fusion protein comprising a DNMT3L domain and a Cas endonuclease domain, or(ii) a fusion protein comprising, optionally from N-terminus to C-terminus, a transcriptional repressor domain, a Cas endonuclease domain, and a DNMT3L domain.

22. The combination of any one of claims 1 -20, wherein the combination comprises(i) a first fusion protein comprising a transcriptional repressor domain and a Cas endonuclease domain, a second fusion protein comprising a DNMT3L domain and a Cas endonuclease domain, and a third fusion protein comprising a DNMT3A domain and a Cas endonuclease domain, or(ii) a fusion protein comprising a transcriptional repressor domain, a Cas endonuclease domain, a DNMT3L domain, and a DNMT3A domain.220#14533290vl23. The combination of any one of claims 1 -22, wherein the transcriptional repressor domain is a Kruppel-associated box (KRAB) domain, optionally derived from human Kox1 or ZIM3.

24. The combination of any one of claims 12-23, wherein the combination comprises a fusion protein comprising, optionally from N terminus to C terminus, a KRAB domain derived from ZIM3, a dCas9 domain, and a DNMT3L domain, optionally comprising an amino acid sequence of SEQ ID NO: 4481.

25. The combination of any one of claims 1 -11 and 13-23, wherein the combination comprises a fusion protein comprising, optionally from N terminus to C terminus, a KRAB domain derived from ZIM3, a catalytically active Cas9 domain, and a DNMT3L domain, optionally comprising an amino acid sequence of SEQ ID NO: 4482.

26. The combination of any one of the claims 1 -25, wherein the gRNA(s) are chemically modified, optionally wherein the chemically modified gRNA(s) comprise phosphorothioate internucleoside linkages at the 5’ and / or 3’ ends, and / or 2’-O-methyl nucleotides.

27. A combination for (1 ) regulating one or more genes, and (2) editing the sequence of a gene, in a human cell, the combination comprising:(A) a fusion protein, wherein the fusion protein comprises a catalytically active Cas9 endonuclease domain, a transcriptional repressor, and a DNMT domain, or a polynucleotide encoding the fusion protein, and(B)a guide RNA;wherein each of the one or more genes of (1 ) is characterized by the presence of a CpG island, andwherein the gene of (2) is a safe harbor genomic locus.

28. The combination of claim 27, wherein the human cell is a human lymphocyte, optionally, wherein the human lymphocyte is in vivo.221#14533290vl29. The combination of claim 28, wherein the human lymphocyte is a cytotoxic lymphocyte.

30. The combination of claim 28, wherein the human lymphocyte is a cytotoxic T-cell.

31. The combination of claim 28, wherein the human lymphocyte is an NK cell.

32. The combination of claim 28, wherein the human lymphocyte is a macrophage.

33. The combination of any one of claims 27-32, wherein the one or more genes of (1 ) is B2M, TET2, TGFBR2, A2AR, CISH, PTPN11, PTPN6, PTPA, PTPN2, JUNB, TOX, TOX2, NR4A1, NR4A2, NR4A3, MAP4K1, REL, IRF4, DGKA, PIK3CD, HLA-A, LISP16, DCK, CD3z, GUTA, or FAS, or a combination of two or more of these.

34. The combination of any one of claims 27-32, wherein the gene of (2) is TRAC, TRBC, PDCD1, TIM-3, TIGIT, LAG3, CTLA4, AAVS1, hRosa26, or CCR5.

35. The combination of any one of claims 27-34, wherein the one or more genes of (1) comprise a TGFBR2 gene, wherein the combination further comprises a gRNA that comprises a spacer having the sequence of any one of SEQ ID NOs: 2929-2978 and 4553-4559 optionally with a 1 to 9 nucleotide truncation at the 5’ end, or comprises polynucleotide(s) coding for the gRNA.

36. The combination of any one of claims 27-35, wherein one or more genes of (1 ) comprise a TET2 gene and the combination further comprises a gRNA that comprises a spacer having the sequence of any one of SEQ ID NOs: 4429-4478 and 4560-4565 optionally with a 1 to 9 nucleotide truncation at the 5’ end, or comprises polynucleotide(s) coding for the gRNA.222#14533290vl37. The combination of any one of claims 27-36, wherein the gene of (2) is a TRAC gene and the combination further comprises a gRNA comprising a spacer having the sequence of one of SEQ ID NOs: 562-611 or 4575, or comprises polynucleotide(s) coding for the gRNA.

38. The combination of any one of claims 27-37, wherein the one or more genes of (1) comprise (i) a TGFBR2 gene, wherein the combination further comprises a gRNA that comprises a spacer having the sequence of any one of SEQ ID NOs: 2929-2978 and 4553-4559 with 1 to 9 nucleotide truncation at the 5’ end, optionally with a 3 to 9 nucleotide truncation at the 5’ end, or comprises polynucleotide(s) coding for the gRNA; and (ii) a TET2 gene and the combination further comprises a gRNA that comprises a spacer having the sequence of any one of SEQ ID NOs: 4429-4478 and 4560-4565 with a 1 to 9 nucleotide truncation at the 5’ end, optionally with a 3 to 9 nucleotide truncation at the 5’ end, or comprises polynucleotide(s) coding for the gRNA; andthe gene of (2) is a TRAC gene and the combination further comprises a gRNA comprising a spacer having the sequence of one of SEQ ID NOs: 562-611, or comprises polynucleotide(s) coding for the gRNA.

39. The combination of any one of claims 27-37, wherein the one or more genes of (1) comprise (i) a TGFBR2 gene, wherein the combination further comprises a gRNA comprising a spacer having the sequence of SEQ ID NO: 4645, and / or a gRNA comprising a spacer having the sequence of SEQ ID NO 4646; each individually with a 3 to 9 nucleotide truncation at 5’ end, or comprises polynucleotide(s) coding for the gRNA; and (ii) a TET2 gene and the combination further comprises a gRNA that comprises a spacer having the sequence of SEQ ID NO: 4643, and / or a gRNA that comprises a spacer having the sequence of SEQ ID NO: 4644; each individually with a 3 to 9 nucleotide truncation at the 5’ end, or comprises polynucleotide(s) coding for the gRNA;40. The combination of any one of claims 27-39, wherein the gene of (2) is a TRAC gene, wherein the combination further comprises a gRNA comprising a spacer having the sequence of SEQ ID NO: 4575.223#14533290vl41. The combination of any one of claims 27-40, wherein the one or more genes of (1) comprise (i) a TGFBR2 gene, wherein the combination further comprises a gRNA comprising a spacer having the sequence of SEQ ID NO: 4645, and / or a gRNA comprising a spacer having the sequence of SEQ ID NO 4646; each individually with a 3 to 9 nucleotide truncation at 5’ end, or comprises polynucleotide(s) coding for the gRNA; and (ii) a TET2 gene and the combination further comprises a gRNA that comprises a spacer having the sequence of SEQ ID NO: 4643, and / or a gRNA that comprises a spacer having the sequence of SEQ ID NO: 4644; each individually with a 3 to 9 nucleotide truncation at the 5’ end, or comprises polynucleotide(s) coding for the gRNA; and the gene of (2) is a TRAC gene, wherein the combination further comprises a gRNA comprising a spacer having the sequence of SEQ ID NO: 4575.

42. The combination of any one of claims 27-41, wherein the fusion protein further comprises a DNMT1, DNMT3A, DNMT3B, or SETDB1 domain, optionally a DNMT3A domain.

43. The combination of any one of claims 27-42, wherein the transcriptional repressor domain is a Kruppel-associated box (KRAB) domain, optionally derived from human Kox1 orZIM3.

44. The combination of any one of claims 27-43, wherein the combination comprises a fusion protein comprising, optionally from N terminus to C terminus, a KRAB domain derived from ZIM3, a dCas9 domain, and a DNMT3L domain.

45. The combination of any one of claims 27-43, wherein the combination comprises a fusion protein comprising, optionally from C terminus to N terminus, a KRAB domain derived from ZIM3, a dCas9 domain, and a DNMT3L domain.

46. The combination of any one of claims 27-43, wherein the combination comprises a fusion protein comprising, optionally from N terminus to C terminus, a224#14533290vlKRAB domain derived from ZIM3, a catalytically active Cas9 domain, and a DNMT3L domain.

47. The combination of any one of claims 27-43, wherein the combination comprises a fusion protein comprising, optionally from C terminus to N terminus, a KRAB domain derived from ZIM3, a catalytically active Cas9 domain, and a DNMT3L domain.

48. The combination of any one of claims 27-43, wherein the combination comprises a fusion protein comprising an amino acid sequence of SEQ ID NO: 4481.

49. The combination of any one of claims 27-43, wherein the combination comprises a fusion protein comprising an amino acid sequence of SEQ ID NO: 4482.

50. The combination of any one of claims 27-49, wherein the gRNA(s) are chemically modified, optionally wherein the chemically modified gRNA(s) comprise phosphorothioate internucleoside linkages at the 5’ and / or 3’ ends, and / or 2’-O-methyl nucleotides.

51. The combination of any one of claims 27-50, wherein the combination further comprises a donor template nucleic acid.

52. The combination of claim 51, wherein the donor template nucleic acid comprises, from 5’ to 3’, a 5’ homology arm, a transgene, and a 3’ homology arm, optionally wherein each homology arm, individually, comprises a sequence of 30-300 nucleotides that are homologous to a sequence of the gene of (2).

53. The combination of claim 52, wherein the donor template nucleic acid comprises, from 5’ to 3’, a 5’ TRAC homology arm, a transgene, and a 3’ TRAC homology arm, wherein each homology arm, individually, comprises a sequence of 30-300 nucleotides that are homologous to a sequence of a TRAC gene.

54. The combination of claim 53, wherein the donor template nucleic acid comprises a 5’ TRAC homology arm and a 3’ TRAC homology arm, wherein each 225#14533290vlhomology arm, individually, comprises a sequence of 30-300 nucleotides that are homologous to a sequence of a TRAC gene immediately adjacent to the sequence of SEQ ID NO: 4575 comprised in the TRAC gene.

55. The combination of any one of claims 51 -54, wherein the transgene comprised in the donor sequence comprises a nucleic acid sequence encoding a CAR.

56. The combination of any one of claims 51 -54, wherein the transgene comprised in the donor sequence comprises a nucleic acid sequence encoding a CAR targeting CD19.

57. The combination of any one of claims 27-56, wherein the combination is formulated in an LNP.

58. The combination of any one of claims 27-56, wherein the combination is formulated in a T-cell targeting LNP.

59. The combination of any one of claims 27-56, wherein the combination is formulated in a VLP.

60. The combination of any one of claims 27-56, wherein the combination is formulated in a lymphocyte targeting VLP.

61. A pharmaceutical composition comprising the combination of any one of claims 1-60.

62. A cell comprising the combination of any one of claims 1 -61, or a progeny of the cell.

63. The cell or the progeny thereof of claim 62, wherein the cell or the progeny thereof is characterized by expressing the transgene and by a reduction of the expression of TGFBR2 and TET2 as compared to a cell of the same cell type but not comprising the combination.226#14533290vl64. A method of modifying transcription, expression and / or activity of one or more gene in a human T cell, comprising introducing the combination of any one of claim 1 -60 into the cell in vitro or ex vivo.

65. A method of modifying transcription, expression and / or activity of one or more gene in a human T cell, comprising introducing the combination of any one of claim 1-60 into the cell in vivo.

66. The method of claim 64 or 65, wherein the method further comprises expressing the transgene of any one of claims 52-56 in the human T cell.

67. A cell obtained by the method of any one of claims 64-66, or a progeny of the cell.

68. The cell of any one of claims 62, 63, and 67, wherein the cell is a human T cell, optionally engineered to express a recombinant antigen receptor, optionally selected from a recombinant T cell receptor (TCR) or a chimeric antigen receptor (CAR).

69. The cell of any one of claims 62, 63, 67 and 68 for use in treating a human in need thereof.

70. Use of the cell of any one of claims 62, 63, 67 and 68 in the manufacture of a medicament for treating a human in need thereof.

71. A method of treating a human in need thereof, comprising administering to the human the cell of any one of claims 62, 63, 67 and 68.

72. The cell for use according to claim 69, the use of claim 70, or the method of claim 71, wherein the cell is allogeneic or autologous to the human.227#14533290vl73. A method, comprising administering to a subject in need thereof a pharmaceutical composition encapsulated in an LNP, wherein the LNP comprises a lipid bilayer comprising a T-cell targeting ligand or moiety on its external layer, the pharmaceutical composition comprises(A) a fusion protein, wherein the fusion protein comprises a catalytically active Cas9 endonuclease domain, a transcriptional repressor, and a DNMT domain, or a polynucleotide encoding the fusion protein, and(B) one or more guide RNAs each individually having a spacer sequence that is identical to a target sequence of a gene characterized by the presence of a CGI, or a polynucleotide encoding the guide RNA;(C)one or more guide RNAs having a spacer sequence that is identical to a target sequence of a gene that is a safe harbor genomic locus; and (D) a donor template nucleic acid that comprises, from 5’ to 3’, a 5’-homology arm, a transgene, and a 3’ homology arm, wherein the 5’ and the 3’ homology arms comprise a sequence of at least 30 nucleotides that is identical with a sequence immediately adjacent to the target sequence of the safe harbor genomic locus.

74. The method of claim 73, wherein the gene characterized by the presence of a CGI is B2M, TET2, TGFBR2, A2AR, CISH, PTPN11, PTPN6, PTPA, PTPN2, JUNB, TOX, TOX2, NR4A1, NR4A2, NR4A3, MAP4K1, REL, IRF4, DGKA, PIK3CD, HLA-A, LISP16, DCK, CD3z, CIITA, or FAS, or a combination of two or more of these.

75. The method of claim 73 or 74, wherein the gene that is a safe harbor genomic locus is TRAC, TRBC, PDCD1, TIM-3, TIGIT, LAG3, CTLA4, AAVS1, hRosa26, or CCR5.

76. The method of any one of claims 73-75, wherein the gene characterized by the presence of a CGI comprises a TGFBR2 gene, wherein the combination further comprises a gRNA that comprises a spacer having the sequence of any one of SEQ ID NOs: 2929-2978 and 4553-4559 optionally with a 1 to 9 nucleotide truncation at the 5’ end, or comprises polynucleotide(s) coding for the gRNA.228#14533290vl77. The method of any one of claims 73-76, wherein the gene characterized by the presence of a CGI comprises a TET2 gene and the combination further comprises a gRNA that comprises a spacer having the sequence of any one of SEQ ID NOs: 4429-4478 and 4560-4565 optionally with a 1 to 9 nucleotide truncation at the 5’ end, or comprises polynucleotide(s) coding for the gRNA.

78. The method of any one of claims 73-77, wherein the gene that is a safe harbor genomic locus is a TRAC gene and the combination further comprises a gRNA comprising a spacer having the sequence of one of SEQ ID NOs: 562-611 or 4575, or comprises polynucleotide(s) coding for the gRNA.

79. The method of any one of claims 73-78, wherein the gene characterized by the presence of a CGI comprises (i) a TGFBR2 gene, wherein the combination further comprises a gRNA that comprises a spacer having the sequence of any one of SEQ ID NOs: 2929-2978 and 4553-4559 with 1 to 9 nucleotide truncation at the 5’ end, optionally with a 3 to 9 nucleotide truncation at the 5’ end, or comprises polynucleotide(s) coding for the gRNA; and (ii) a TET2 gene and the combination further comprises a gRNA that comprises a spacer having the sequence of any one of SEQ ID NOs: 4429-4478 and 4560-4565 with a 1 to 9 nucleotide truncation at the 5’ end, optionally with a 3 to 9 nucleotide truncation at the 5’ end, or comprises polynucleotide(s) coding for the gRNA; andthe gene that is a safe harbor genomic locus is a TRAC gene and the combination further comprises a gRNA comprising a spacer having the sequence of one of SEQ ID NOs: 562-611, or comprises polynucleotide(s) coding for the gRNA.

80. The method of any one of claims 73-79, wherein the gene characterized by the presence of a CGI comprises (i) a TGFBR2 gene, wherein the combination further comprises a gRNA comprising a spacer having the sequence of SEQ ID NO: 4645, and / or a gRNA comprising a spacer having the sequence of SEQ ID NO 4646; each individually with a 3 to 9 nucleotide truncation at 5’ end, or comprises polynucleotide(s) coding for the gRNA; and (ii) a TET2 gene and the combination further comprises a gRNA that comprises a spacer having the sequence of SEQ ID NO: 4643, and / or a gRNA that comprises a spacer having the sequence of SEQ ID 229#14533290vlNO: 4644; each individually with a 3 to 9 nucleotide truncation at the 5’ end, or comprises polynucleotide(s) coding for the gRNA;81. The method of any one of claims 73-80, wherein the gene that is a safe harbor genomic locus is a TRAC gene, wherein the combination further comprises a gRNA comprising a spacer having the sequence of SEQ ID NO: 4575.

82. The method of any one of claims 73-81, wherein the gene characterized by the presence of a CGI comprises (i) a TGFBR2 gene, wherein the combination further comprises a gRNA comprising a spacer having the sequence of SEQ ID NO: 4645, and / or a gRNA comprising a spacer having the sequence of SEQ ID NO 4646; each individually with a 3 to 9 nucleotide truncation at 5’ end, or comprises polynucleotide(s) coding for the gRNA; and (ii) a TET2 gene and the combination further comprises a gRNA that comprises a spacer having the sequence of SEQ ID NO: 4643, and / or a gRNA that comprises a spacer having the sequence of SEQ ID NO: 4644; each individually with a 3 to 9 nucleotide truncation at the 5’ end, or comprises polynucleotide(s) coding for the gRNA; and the gene that is a safe harbor genomic locus is a TRAC gene, wherein the combination further comprises a gRNA comprising a spacer having the sequence of SEQ ID NO: 4575.

83. The method of any one of claims 73-82, wherein the fusion protein further comprises a DNMT1, DNMT3A, DNMT3B, or SETDB1 domain, optionally a DNMT3A domain.

84. The method of any one of claims 73-82, wherein the transcriptional repressor domain is a Kruppel-associated box (KRAB) domain, optionally derived from human Kox1 or ZIM3.

85. The method of any one of claims 73-84, wherein the combination comprises a fusion protein comprising, optionally from N terminus to C terminus, a KRAB domain derived from ZIM3, a dCas9 domain, and a DNMT3L domain.230#14533290vl86. The method of any one of claims 73-84, wherein the combination comprises a fusion protein comprising, optionally from C terminus to N terminus, a KRAB domain derived from ZIM3, a dCas9 domain, and a DNMT3L domain.

87. The method of any one of claims 73-84, wherein the combination comprises a fusion protein comprising, optionally from N terminus to C terminus, a KRAB domain derived from ZIM3, a catalytically active Cas9 domain, and a DNMT3L domain.

88. The method of any one of claims 73-84, wherein the combination comprises a fusion protein comprising, optionally from C terminus to N terminus, a KRAB domain derived from ZIM3, a catalytically active Cas9 domain, and a DNMT3L domain.

89. The method of any one of claims 73-84, wherein the combination comprises a fusion protein comprising an amino acid sequence of SEQ ID NO: 4481.

90. The method of any one of claims 73-84, wherein the combination comprises a fusion protein comprising an amino acid sequence of SEQ ID NO: 4482.

91. The method of any one of claims 73-90, wherein the gRNA(s) are chemically modified, optionally wherein the chemically modified gRNA(s) comprise phosphorothioate internucleoside linkages at the 5’ and / or 3’ ends, and / or 2’-O-methyl nucleotides.

92. The method of any one of claims 73-91, wherein each homology arm of the donor template nucleic acid, individually, comprises a sequence of 30-300 nucleotides.

93. The method of any one of claims 73-92, wherein the gene that is a safe harbor genomic locus is a TRAC gene, and wherein the donor template comprises, from 5’ to 3’, a 5’ TRAC homology arm, a transgene, and a 3’ TRAC homology arm.

94. The method of claim 93, wherein the donor template comprises a 5’ TRAC homology arm and a 3’ TRAC homology arm, wherein each homology arm, individually, comprises a sequence of 30-300 nucleotides that are homologous to a 231#14533290vlsequence of a TRAC gene immediately adjacent to the sequence of SEQ ID NO: 4575 comprised in the TRAC gene.

95. The method of any one of claims 73-94, wherein the transgene comprised in the donor template sequence comprises a nucleic acid sequence encoding a CAR.

96. The method of any one of claims 73-94, wherein the transgene comprised in the donor template sequence comprises a nucleic acid sequence encoding a CAR targeting CD19.

97. The method of any one of claims 73-96, wherein the subject has been diagnosed with a malignancy.

98. The method of any one of claims 73-96, wherein the subject has been diagnosed with a hematological malignancy.

99. The method of any one of claims 73-96, wherein the subject has been diagnosed with a B-cell leukemia or lymphoma.232#14533290vl

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