Gene silencing

Abstract

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

Description

[Technical field]

[0001] The present invention relates to engineered transcriptional modulators (ETMs), such as engineered transcriptional repressors (ETRs), for gene editing and epigenetic modification. More specifically, the present invention relates to ETMs (e.g., ETRs) for use in multiplex methods, including during gene therapy applications, to modify expression of at least two target genes, where expression of a first target gene is modified by gene editing and expression of a second target gene is modified by epigenetic modification. [Background technology]

[0002] 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 transgenic T cell receptors (TCRs) or chimeric antigen receptors (CARs) that recognize tumor-associated antigens. TCRs and CARs can be introduced into ex vivo grown T cells by various means, including lentiviral and retroviral vectors. However, these vectors are prone to semi-random integration into the genome of T cells, raising safety concerns related to poor transcriptional control of tumor-promoting genes. To avoid this risk, genome editing with artificial nucleases such as CRISPR / Cas9 has been used to drive the insertion of CAR sequences into the endogenous TCR locus (J. Eyquem et al., Nature 2017 Mar 2;543(7643):113-117), an approach that also enhances the potency of T cells.

[0003] Genome editing has further been used to improve the efficiency and reduce toxicity of T cell therapy through knockout of additional important genes. The most common targets in this regard are the TCR genes (encoded by TRAC and TRBC, the latter present in two copies in cis on the same chromosome), the beta-2 microglobulin (B2M) gene, and the programmed cell death 1 (PDCD1, also called PD1) gene. Inactivation of TRAC and B2M is thought to reduce graft-versus-host reaction, while inactivation of PDCD1 is used to reduce the sensitivity of transplanted T cells to immune-damping signals caused by cancer cells / microenvironment.

[0004] Although these multiplexed gene editing approaches (i.e., disruption of multiple genes per cell) are promising, they come with two associated challenges. (i) Induction of a large number of DNA breaks per cell may overactivate the cellular DNA damage response, ultimately leading to apoptosis or poor function / fitness of the transplanted cells. In this regard, triple edits have been proposed as an upper limit for multiplexing, beyond which significant cytotoxicity may be observed. (ii) Chromosomal translocations can occur during or among multiple DNA breaks (including on- and off-target nuclease sites as well as spontaneous breaks, the latter occurring at a relatively high rate in cultured T cells), further compromising the safety of this approach. Alarming levels of genomic translocations (up to 5%) have been reported in multiplexed clinical and preclinical trials in CAR-T cell production, even with dual gene editing approaches (L. Poirot et al., Cancer Res. 2015 Sep 15;75(18):3853-64; W. Qasim et al., Sci Transl Med; 2017 Jan 25;9(374); E. Stadtaumer et al., Science 2020 Feb 28;367(6481)).

[0005] Targeted epigenetic modifications (e.g., episilencing) may represent a safer alternative to gene editing approaches for multiplexing in T cells. Episilencing utilizes epigenetics, e.g., DNA methylation at CpG sites, rather than DNA cleavage, to inactivate its intended target genes (A. Amabile et al., Cell. 2016 Sep 22;167(1):219-232).

[0006] Episilencing can be achieved by transient delivery of engineered transcriptional repressors (ETRs), i.e., proteins that contain catalytically disabled Cas9 (dCas9) or transcription activator-like effectors (TALEs) or zinc finger proteins (ZFPs) fused to epigenetic domains from naturally occurring epigenetic effector proteins (e.g., KRAB, DNMT3L, and DNMT3A). The application of ETRs in silencing individual and multicopy genes in cell lines and primary T lymphocytes has been reported by A. Amabile (supra) and T. Mlambo et al., Nucleic Acids Res. 2018 May 18;46(9):4456-4468. However, the activity of ETRs appears to occur preferentially in genes with CpG islands (CGIs), thus excluding some likely relevant targets (e.g., TCR genes and PD1, among others). Summary of the Invention [Problem to be solved by the invention]

[0007] Therefore, there remains a need to develop a technology that can modify multiple genes in the same cell.Technologies that reduce the number of multiple DNA breaks per cell are safer approaches compared to multiplexed gene editing strategies, and can avoid cellular DNA damage response and undesired chromosomal translocations. [Means for solving the problem]

[0008] The present invention relates to the development of a combined gene editing and epigenetic editing strategy for modifying multiple genes in the same cell.In particular, the present invention utilizes an engineered transcriptional modulator (ETM), such as an engineered transcriptional repressor (ETR), that comprises an epigenetic effector domain operably linked to an endonuclease (e.g., catalytically active Cas9) and a guide ribonucleic acid (gRNA) of various lengths to facilitate the permanent epigenetic editing (e.g., silencing) of one or more genes and the gene editing (e.g., inactivation) of another gene.

[0009] This orthogonal approach overcomes the risk of genotoxicity associated with the use of nuclease-mediated genome editing techniques to inactivate a large number of genes per cell.Advantageously, the present invention allows targeting of genes that are more difficult to achieve targeted epigenetic modification, and allows targeting of both genes with CpG islands (CGIs) and genes without CGIs in one multiplexing strategy.

[0010] Thus, the present invention provides a combination strategy of gene editing coupled with epigenetic modification, e.g., epigenetic silencing. This combination allows: (i) The burden of genomic translocation is reduced compared to multiplexed gene editing methods. The target selected for gene editing will typically lack CGIs. This gene is also used as a target site for the insertion of exogenous expression cassettes encoding tumor-restricted TCRs or CARs, for example, introduced by homologous recombination. (ii) Epigenetics is used to modify, e.g., silence, one or more CGI-containing genes. (iii) It allows the use of the same construct (ETM) to achieve silencing in two different ways, thereby reducing the amount of gene editor coding RNA that needs to be added to cells for proper silencing. An advantage of the present invention is that the number of constructs required for multiple modifications is reduced, thereby improving efficiency and reducing production costs.

[0011] Preferably, gene editing is limited to one gene (lacking a CGI) and at least one gene (e.g. at least two, or at least three, or more genes) that includes a CGI may be epigenetically modified.

[0012] Overall, the development of such combinatorial strategies will result in safer and more efficient T cell products for adoptive immunotherapy of cancer.

[0013] In one aspect, the present invention provides an engineered transcriptional modulator (ETM) comprising a) at least one epigenetic effector domain operably linked to b) an endonuclease.

[0014] In certain embodiments, the ETM is an engineered transcriptional repressor (ETR). In some embodiments, the ETM is an engineered transcriptional activator (ETA).

[0015] 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.

[0016] 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).

[0017] In some embodiments, the at least one epigenetic effector domain is selected from the group consisting of DNMT1, DNMT3A, DNMT3B, DNMT3L, and SETDB1.

[0018] 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 KRAB and DNMT3A; KRAB and DNMT3L; or KRAB, DNMT3A, and DNMT3L as epigenetic effector domains. 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 (eg, ETR) comprises a transcriptional repressor domain (eg, a Kruppel-associated box (KRAB) domain), a DNMT3A domain, and a DNMT3L domain.

[0019] In some embodiments, the endonuclease comprises an RNA binding domain.

[0020] In some embodiments, the endonuclease is a Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) / Cas system.

[0021] In some embodiments, the endonuclease is a Cas endonuclease.

[0022] In certain embodiments, the endonuclease is a Cas9 endonuclease. In certain embodiments, the endonuclease is a SpCas9 endonuclease.

[0023] In some embodiments, the ETM (e.g., ETR) comprises or consists of a fusion protein of Cas9-KRAB, Cas9-DNMT3A, or Cas9-DNMT3L, which can be used together.

[0024] In some embodiments, the ETM (eg, ETR) is a bipartite or tripartite fusion protein.

[0025] In another aspect, the present invention provides a gRNA comprising a spacer sequence comprising or consisting of any one of the sequences of SEQ ID NOs: 23 to 46, 562 to 1076, 2778 to 4478, and 4553 to 4565, or a homolog or fragment thereof. In another aspect, the present invention provides a gRNA comprising a spacer sequence comprising or consisting of any one of the sequences of SEQ ID NOs: 23 to 46, 562 to 1076, 2778 to 4478, and 4553 to 4565, or a homolog or fragment thereof.

[0026] In another embodiment, the spacer sequence is a fragment of any one of SEQ ID NOs: 23 to 46, 562 to 1076, or 2778 to 4478, for example, a 10-, 11-, 12-, 13-, 14-, 15-, 16-, 17-, 18-, 19-, or 20-nucleotide fragment of any one of SEQ ID NOs: 23 to 46, 562 to 1076, 2778 to 4478, or 4553 to 4565. In another embodiment, the spacer sequence is a fragment of any one of SEQ ID NOs: 23 to 46, 562 to 1076, 2778 to 4478, and 4553 to 4565, for example, a 10-, 11-, 12-, 13-, 14-, 15-, 16-, 17-, 18-, 19-, or 20-nucleotide fragment of any one of SEQ ID NOs: 23 to 46, 562 to 1076, 2778 to 4478, and 4553 to 4565. The fragment may be a truncation of the sequence from the 5' end.

[0027] In another aspect, the spacer sequence consists of a fragment of any one of SEQ ID NOs: 23 to 46, 562 to 1076, or 2778 to 4478, for example 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 consecutive nucleotides of any one of SEQ ID NOs: 23 to 46, 562 to 1076, or 2778 to 4478. In another aspect, the spacer sequence consists of a fragment of any one of SEQ ID NOs: 23 to 46, 562 to 1076, 2778 to 4478, and 4553 to 4565, for example 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 consecutive nucleotides of any one of SEQ ID NOs: 23 to 46, 562 to 1076, 2778 to 4478, and 4553 to 4565.

[0028] 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 can target the ETM (e.g., ETR) to one or more target genes. 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 a polynucleotide encoding the same and at least one gRNA or a polynucleotide encoding the same. The combination may comprise one or more ETMs (e.g., ETRs) according to the present invention, for example one, two, or three ETMs (e.g., ETRs) or polynucleotides encoding the same.

[0029] In some embodiments, each ETM is a fusion protein comprising a catalytically active CRISPR / Cas endonuclease domain.

[0030] In another aspect, the invention provides a combination for altering the transcription, expression, and / or activity of one or more (e.g., two or more) genes in a cell, the combination comprising: (A) one or more fusion proteins, or polynucleotides encoding the one or more fusion proteins, each comprising a catalytically active CRISPR / Cas endonuclease domain, collectively comprising a transcriptional repressor domain and a DNMT3L domain; (B) one or more guide RNAs (gRNAs) having a spacer sequence with a length that allows for epigenetic editing of a first gene and does not allow for gene editing, the first gene comprising a CpG island (CGI) in the cell; and (C) one or more gRNAs, or polynucleotides encoding the one or more gRNAs, having a spacer sequence with a length that allows for gene editing of a second gene in the cell.

[0031] 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.

[0032] In some embodiments, 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, 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.

[0033] In some embodiments, the spacer sequence is 16 nucleotides or less in length, hi some embodiments, the spacer sequence is 11-16 nucleotides in length, for example 12-16, 13-16, 14-16, or 15-16 nucleotides in length.

[0034] 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 17-30 nucleotides in length, such as 18-30, 19-30, or 20-30 nucleotides in length. In some embodiments, the spacer sequence is 17-25 nucleotides in length, such as 18-25, 19-25, or 20-25 nucleotides in length. In some embodiments, the spacer sequence is 17-20 nucleotides in length, such as 18-20 or 19-20 nucleotides in length.

[0035] In some embodiments, the spacer sequence is 17 nucleotides or less in length. In some embodiments, the spacer sequence is 11-17 nucleotides in length, e.g., 12-17, 13-17, 14-17, 15-17, 16-17, 12-16, 13-16, 14-16, or 15 nucleotides in length. In some embodiments, one or more gRNAs in (B) have a spacer sequence of 17 nucleotides or less. In some embodiments, one or more gRNAs in (B) have a spacer sequence of 11-17 nucleotides, e.g., 12-17, 13-17, 14-17, 15-17, 16-17, 12-16, 13-16, 14-16, or 15 nucleotides.

[0036] In some embodiments, the spacer sequence is 18 nucleotides or more in length, such as 19 or more or 20 or more nucleotides in length. In some embodiments, the spacer sequence is 18-30 nucleotides in length, such as 19-30 or 20-30 nucleotides in length. In some embodiments, the spacer sequence is 18-25 nucleotides in length, such as 19-25 or 20-25 nucleotides in length. In some embodiments, the spacer sequence is 18-21 nucleotides in length, such as 19-21 or 20-21 nucleotides in length. In some embodiments, the spacer sequence is 18-20 nucleotides in length, such as 19-20 nucleotides in length. In some embodiments, one or more gRNAs in (C) have a spacer sequence of 18 nucleotides or more, such as 19 or more, or 20 or more nucleotides in length. In some embodiments, one or more gRNAs in (C) have a spacer sequence of 18-30 nucleotides, such as 19-30 or 20-30 nucleotides in length. In some embodiments, one or more gRNAs in (C) have a spacer sequence of 18-25 nucleotides, e.g., 19-25 or 20-25 nucleotides. In some embodiments, one or more gRNAs in (C) have a spacer sequence of 18-21 nucleotides, e.g., 19-21 or 20-21 nucleotides. In some embodiments, one or more gRNAs in (C) have a spacer sequence of 18-20 nucleotides, e.g., 19-20 nucleotides.

[0037] 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.

[0038] At least two gRNAs can target different target genes to the ETM (e.g., ETR). For example, a first gRNA can target a first target gene to the ETM (e.g., ETR), and a second gRNA can target a second target gene to the ETM (e.g., ETR). A third gRNA can target a third target gene to the ETM (e.g., ETR), for example. An additional gRNA can target an additional target gene to the ETM (e.g., ETR).

[0039] In some embodiments, one target gene can be targeted by two or more gRNAs.For example, for optimal epigenetic modification, such as epigenetic silencing, it may be beneficial to target the same gene with several gRNAs.A second target gene can be targeted by another gRNA.

[0040] In certain embodiments, at least two gRNAs comprise spacer sequences of different lengths.

[0041] 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 for epigenetic editing of a target gene by an ETM, and / or at least one gRNA may have a spacer sequence with a length that allows for genetic editing of a target gene by an ETM.

[0042] In some embodiments, the first gRNA may have a spacer sequence having a length that allows epigenetic editing of a first target gene by an ETM, and the second gRNA may have a spacer sequence having a length that allows genetic editing of a second target gene by an ETM.

[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 ETM but does not allow gene editing, and / or at least one gRNA may have a spacer sequence with a length that allows gene editing of another target gene by ETM.

[0044] In some embodiments, the first gRNA may have a spacer sequence having a length that allows epigenetic editing of a first target gene by ETM but does not allow gene editing, and the second gRNA may have a spacer sequence having a length that allows gene editing of a second target gene by ETM.

[0045] Suitably, at least one gRNA may comprise a spacer sequence of 15, 16, 17, 18, 19, or 20 nucleotides in length.

[0046] Suitably, one of the at least two gRNAs may comprise a spacer sequence of 17 nucleotides or less (e.g., 16 or less) in length.

[0047] In some embodiments, the combination is: (a) a first gRNA comprising a spacer sequence that is 16 nucleotides or less in length, e.g., 15 nucleotides or less, 14 nucleotides or less, 13 nucleotides or less, or 12 nucleotides or less in length; and / or (b) a second gRNA comprising a spacer sequence of 17 nucleotides or more in length, e.g., 18 nucleotides or more in length, 19 nucleotides or more in length, or 20 nucleotides or more in length; Includes.

[0048] In some embodiments, the combination is: (a) a first gRNA comprising a spacer sequence of 11 to 16 nucleotides in length, e.g., 12 to 16, 13 to 16, 14 to 16, or 15 to 16 nucleotides in length; and / or (b) a second gRNA comprising a spacer sequence of 17 to 30 nucleotides in length, e.g., 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. Includes.

[0049] In some embodiments, the combination is: (a) a first gRNA comprising a spacer sequence that is 17 nucleotides or less in length, e.g., 16 nucleotides or less, 15 nucleotides or less, 14 nucleotides or less, 13 nucleotides or less, 12 nucleotides or less, or 11 nucleotides in length; and / or (b) a second gRNA comprising a spacer sequence of 18 nucleotides or more in length, e.g., 19 nucleotides or more in length, or 20 nucleotides or more in length; Includes.

[0050] In some embodiments, the combination is: (a) a first gRNA comprising a spacer sequence of 11 to 17 nucleotides in length, e.g., 12 to 17 (e.g., 12 to 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 comprising a spacer sequence of 18 to 30 nucleotides in length, e.g., 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; Includes.

[0051] In some embodiments, one or more guide RNAs (gRNAs) having a spacer sequence having a length that allows for epigenetic editing of a first gene in a cell and does not allow for gene editing are (a) 17 nucleotides or less (e.g., 16 nucleotides or less), e.g., 15 nucleotides or less, 14 nucleotides or less, 13 nucleotides or less, 12 nucleotides or less, or 11 nucleotides or less, or (b) 11 to 17 nucleotides (e.g., 11 to 16 nucleotides), e.g., 12 to 17 (e.g., 12 to 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. It has a spacer sequence of:

[0052] In some embodiments, the one or more gRNAs having a spacer sequence with a length that allows for gene editing of a second gene in a cell include: (a) 17 or more nucleotides (e.g., 18 or more nucleotides), e.g., 19 or more, or 20 or more nucleotides, or (b) 17 to 30 nucleotides, e.g., 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). It has a spacer sequence of:

[0053] In some embodiments, at least one target gene is selected from genes without CpG islands (CGIs), such as TRAC, TRBC, PDCD1, TIM-3, TIGIT, LAG3, CTLA4, AAVS1, and CCR5, and / or genes with CGIs, 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, and FAS. For example, the target gene may include one or more of B2M, TRAC, TET2, and TGFBR2. In some embodiments, the target gene may include, for example, B2M and TRAC. In some embodiments, the target gene may include, for example, B2M, TRAC, TET2, and TGFBR2. In some embodiments, the target genes may include 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.

[0054] 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, and FAS, and / or the second gene is selected from TRAC, TRBC, PDCD1, TIM-3, TIGIT, LAG3, CTLA4, AAVS1, and CCR5.

[0055] In some embodiments, the second gene is a TRAC gene, and optionally, the one or more gRNAs targeting the TRAC gene include a spacer having one of SEQ ID NOs: 562-611, and optionally SEQ ID NO: 604.

[0056] In some embodiments, the first gene is a B2M gene, and optionally one or more gRNAs targeting the B2M gene each have one of the sequences of SEQ ID NOs: 28-33 and 39-44, or one of the sequences of SEQ ID NOs: 2778-2878 truncated by 3-9 nucleotides at the 5' end, or one of the sequences of SEQ ID NOs: 2778, 2780, 2801, and 2863, optionally truncated by 3-9 nucleotides at the 5' end, and include a spacer selected from SEQ ID NOs: 4486-4492, 4497-4503, 4508-4514, and 4519-4525.

[0057] In some embodiments, the first gene is a TGFBR2 gene, and optionally, one or more gRNAs targeting the TGFBR2 gene each include a spacer having one of the sequences of SEQ ID NOs: 2929-2978 and 4553-4559 truncated by 3-9 nucleotides at the 5' end.

[0058] In some embodiments, the first gene is a TET2 gene, and optionally, one or more gRNAs targeting the TET2 gene each include a spacer having one of SEQ ID NOs: 4429-4478 and 4560-4565 truncated by 3-9 nucleotides at the 5' end.

[0059] In some embodiments, the combination is for altering the transcription, expression, and / or activity of one or more (e.g., two or more) genes in a cell, where the cell is a mammalian cell, optionally a human cell, and optionally the cell is a human immune cell or a human T cell.

[0060] In some embodiments, the combination further includes a donor DNA that includes 5' and 3' arms that are homologous to a sequence in a second gene.

[0061] In some embodiments, the combination is: i) an agent that promotes the survival, proliferation, and / or activity of a cell, e.g., a cell with or without the combination; and / or ii) agents that are detrimental to the survival, proliferation, activity, chemoresistance, and / or chemotaxis of cells, e.g., cells containing the combination or cells not containing the combination; and / or iii) Agents that allow for the selection of cells, e.g., cells that contain the combination or cells that do not contain the combination. In some embodiments, the agent is a CAR or a transgenic TCR. In some embodiments, the agent is FIX.

[0062] In another aspect, the present invention provides a combination for regulating one or more genes in a human cell, optionally an immune cell or a T cell, the combination comprising: one or more (e.g., 1-3) fusion proteins each comprising a catalytically inactive Cas9, optionally an endonuclease domain of SpCas9, collectively comprising a transcriptional repressor and a DNMT3L domain, or a polynucleotide encoding one or more (e.g., 1-3) fusion proteins, wherein the gene comprises a CpG island (CGI); (i) the gene is a B2M gene, and the combination further comprises two or more gRNAs each having a spacer having one of the sequences of SEQ ID NOs: 2778 to 2878, optionally truncated by 1 to 9 nucleotides at the 5' end, or a polynucleotide encoding a gRNA; (ii) the gene is a TGFBR2 gene, and the combination further comprises a gRNA having a spacer having any one of SEQ ID NOs: 2929 to 2978 and 4553 to 4559, optionally truncated by 1 to 9 nucleotides at the 5' end, or a polynucleotide encoding a gRNA; or (iii) The gene is a TET2 gene, and the combination optionally further includes a gRNA having a spacer having any one of SEQ ID NOs: 4429 to 4478 and 4560 to 4565, in which 1 to 9 nucleotides at the 5' end have been shortened, or includes a polynucleotide encoding the gRNA.

[0063] 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.

[0064] In some embodiments, the gene containing the CGI is the 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 truncated by 1 to 9 nucleotides, and optionally 3 to 9 nucleotides, at the 5' end.

[0065] In some embodiments, gRNAs targeting B2M include a gRNA comprising a spacer having the sequence of F4, optionally truncated by 1-9 nucleotides, optionally 3-9 nucleotides, at its 5' end; a gRNA comprising a spacer having the sequence of H8, optionally truncated by 1-9 nucleotides, optionally 3-9 nucleotides, at its 5' end; and a gRNA comprising a spacer having the sequence of H10, optionally truncated by 1-9 nucleotides, optionally 3-9 nucleotides, at its 5' end.

[0066] In some embodiments, gRNAs targeting B2M include a gRNA comprising a spacer having the sequence of C8, optionally truncated by 1-9 nucleotides, optionally 3-9 nucleotides, at its 5' end; a gRNA comprising a spacer having the sequence of H8, optionally truncated by 1-9 nucleotides, optionally 3-9 nucleotides, at its 5' end; and a gRNA comprising a spacer having the sequence of H10, optionally truncated by 1-9 nucleotides, optionally 3-9 nucleotides, at its 5' end.

[0067] In some embodiments, gRNAs targeting B2M include a gRNA comprising a spacer having the sequence of F4, optionally truncated by 1-9 nucleotides, optionally 3-9 nucleotides, at the 5' end, and a gRNA comprising a spacer having the sequence of H8, optionally truncated by 1-9 nucleotides, optionally 3-9 nucleotides, at the 5' end.

[0068] In some embodiments, a gRNA targeting B2M includes a gRNA comprising a spacer having the sequence of F4, optionally truncated by 1-9 nucleotides, optionally 3-9 nucleotides, at its 5' end, and a gRNA comprising a spacer having the sequence of H10, optionally truncated by 1-9 nucleotides, optionally 3-9 nucleotides, at its 5' end.

[0069] In some embodiments, gRNAs targeting B2M include a gRNA comprising a spacer having the sequence of H8, optionally truncated by 1-9 nucleotides, optionally 3-9 nucleotides, at the 5' end, and a gRNA comprising a spacer having the sequence of H10, optionally truncated by 1-9 nucleotides, optionally 3-9 nucleotides, at the 5' end.

[0070] In some embodiments, the gene comprising the CGI is a TGFBR2 gene, and the combination comprises one or more gRNAs or coding sequences of one or more gRNAs targeting the TGFBR2 gene, the one or more gRNAs being optionally truncated by 1-9 nucleotides, optionally 3-9 nucleotides, at the 5' end. TG1 (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 (sequence number 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 (sequence number 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), or TG20 (SEQ ID NO: 2932) Each of the above independently comprises a spacer having the sequence:

[0071] In some embodiments, the gRNA targeting TGFBR2 is (i) a gRNA comprising a spacer having a sequence of TG7, optionally shortened by 1 to 9 nucleotides, and optionally 3 to 9 nucleotides, at the 5' end; and A gRNA comprising a spacer having a TG8 sequence optionally shortened by 1 to 9 nucleotides, optionally 3 to 9 nucleotides, at the 5' end; or (ii) a gRNA comprising a spacer having a sequence of TG19, optionally shortened by 1 to 9 nucleotides, and optionally 3 to 9 nucleotides, at the 5' end; and gRNA comprising a spacer having a sequence of TG20, optionally shortened by 1 to 9 nucleotides, and optionally 3 to 9 nucleotides, at the 5' end Includes.

[0072] In some embodiments, the gene comprising the CGI is a TET2 gene, and the combination comprises one or more gRNAs or coding sequences of one or more gRNAs targeting the TET2 gene, wherein the one or more gRNAs are optionally truncated by 1-9 nucleotides, optionally 3-9 nucleotides, at the 5' end. TE1 (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), or TE20 (SEQ ID NO: 4478) Each of the above independently comprises a spacer having the sequence:

[0073] In some embodiments, the gRNA targeting TET2 is (i) a gRNA comprising a spacer having a sequence of TE13, optionally shortened by 1 to 9 nucleotides, and optionally 3 to 9 nucleotides, at the 5' end; and A gRNA comprising a spacer having a sequence of TE14, optionally shortened by 1 to 9 nucleotides, optionally 3 to 9 nucleotides, at the 5' end; or (ii) a gRNA comprising a spacer having a sequence of TE19, optionally shortened by 1 to 9 nucleotides, and optionally 3 to 9 nucleotides, at the 5' end; and gRNA comprising a spacer having a sequence of TE20, optionally shortened by 1 to 9 nucleotides, and optionally 3 to 9 nucleotides, at the 5' end Includes.

[0074] In some embodiments, the ETM (eg, one or more fusion proteins) further comprises a DNMT1, DNMT3A, DNMT3B, or SETDB1 domain, optionally collectively DNMT3A.

[0075] In some embodiments, 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 optionally comprising, from N-terminus to C-terminus, a transcriptional repressor domain, a Cas endonuclease domain, and a DNMT3L domain.

[0076] In some embodiments, 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.

[0077] In some embodiments, the epigenetic effector domain (eg, transcriptional repressor domain) is a Kruppel-associated box (KRAB) domain, optionally derived from human Kox1 or ZIM3.

[0078] In some embodiments, the combination comprises a fusion protein comprising, optionally from N-terminus to C-terminus, a KRAB domain from ZIM3, a catalytically active Cas9 domain, and a DNMT3L domain, and optionally an amino acid sequence of SEQ ID NO:4482.

[0079] In some embodiments, the combination further comprises a gRNA for targeting one or more additional genes in the cell, optionally comprising a gRNA or a polynucleotide encoding a gRNA targeting the following genes: (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.

[0080] In some embodiments, the gRNA is chemically modified, and optionally the chemically modified gRNA comprises phosphorothioate internucleoside linkages and / or 2'-O-methyl nucleotides at the 5' and / or 3' ends.

[0081] In a further aspect, the present invention provides a polynucleotide encoding at least one ETM (eg, an ETR) according to the present invention.

[0082] In another aspect, the present invention provides a nucleic acid construct comprising a nucleic acid sequence encoding at least one ETM (eg, an ETR) according to the present invention.

[0083] In some embodiments, the nucleic acid construct comprises: i) a nucleic acid sequence that promotes the survival, proliferation, and / or activity of a cell, e.g. a cell expressing said nucleic acid construct or a cell not expressing said nucleic acid construct, and / or ii) a nucleic acid sequence that is deleterious to the survival, proliferation, activity, chemoresistance, and / or chemotaxis of a cell, e.g. a cell expressing said nucleic acid construct or a cell not expressing said nucleic acid construct; and / or iii) further comprises a nucleic acid sequence that allows for the selection of cells, e.g., cells that contain the nucleic acid construct or cells that do not contain the construct.

[0084] In one aspect, the present invention provides a vector comprising a polynucleotide according to the invention or a nucleic acid construct according to the invention.

[0085] In another aspect, the present invention provides a method for producing a composition comprising: a) at least one polynucleotide encoding at least one ETM (e.g., ETR) according to the invention, and b) a polynucleotide providing at least one gRNA as disclosed herein, and optionally c) i) an agent that promotes the survival, proliferation, and / or activity of a cell, e.g., a cell that contains the polynucleotide or a cell that does not contain the polynucleotide; and / or ii) an agent that is detrimental to the survival, proliferation, activity, chemoresistance, and / or chemotaxis of a cell, e.g., a cell that contains the polynucleotide or a cell that does not contain the polynucleotide; and / or iii) An agent that allows for the selection of cells, e.g., cells that contain the polynucleotide or cells that do not contain the polynucleotide. Further polynucleotides comprising nucleic acid sequences encoding The present invention provides a kit of polynucleotides comprising:

[0086] In another aspect, the present invention provides a cell (e.g. an engineered cell) comprising an ETM (e.g. an ETR) according to the invention, at least one gRNA according to the invention, a combination according to the invention, a polynucleotide according to the invention, a nucleic acid construct according to the invention, a vector according to the invention, or a kit of polynucleotides according to the invention. In another aspect, the present invention provides a progeny (progeny) of the cell.

[0087] In another aspect, the present invention provides a cell obtained by the use or method of the present invention, or a progeny thereof.

[0088] In some embodiments, the cells are human T cells, optionally engineered to express a recombinant antigen receptor selected from a recombinant T cell receptor (TCR) or a chimeric antigen receptor (CAR).

[0089] In a further aspect, the present invention provides a composition comprising an ETM (e.g. an ETR) according to the invention, at least one gRNA according to the invention, a combination according to the invention, a polynucleotide according to the invention, a nucleic acid construct according to the invention, a vector according to the invention, a kit of polynucleotides according to the invention, or a cell according to the invention.

[0090] In another aspect, the present invention provides a pharmaceutical composition comprising an ETM (e.g. an ETR) according to the invention, at least one gRNA according to the invention, a combination according to the invention, a polynucleotide according to the invention, a nucleic acid construct according to the invention, a vector according to the invention, a kit of polynucleotides according to the invention, or a cell according to the invention.

[0091] In a further aspect, the present invention provides the use of an ETM (e.g. ETR) according to the invention, at least one gRNA according to the invention, a combination according to the invention, a polynucleotide according to the invention, a nucleic acid construct according to the invention, a vector according to the invention, a kit of polynucleotides according to the invention, or a cell according to the invention, for modifying the transcription, expression and / or activity of at least one target gene. The use may for example be an in vitro or ex vivo use.

[0092] In another aspect, the present invention provides a method for modifying the transcription, expression and / or activity of at least one target gene in a cell, comprising administering to a cell an ETM (e.g. an 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. The cell may for example be a T cell.

[0093] In some embodiments, altering the transcription, expression, and / or activity is inhibiting, e.g. silencing, the transcription, expression, and / or activity.

[0094] In some embodiments, the methods comprise suppressing the transcription and / or expression of at least two different target genes in a cell.

[0095] In some embodiments, the method comprises silencing at least two different target genes in a cell.

[0096] Preferably, the transcription and / or expression of at least one of the at least two target genes is epigenetically suppressed (e.g., silenced), at least one of the at least two target genes is suppressed (e.g., silenced) by gene editing, and at least one ETM (e.g., ETR) and at least two gRNAs are administered to the cell simultaneously, sequentially, or separately.

[0097] In one aspect, the ETM (e.g. ETR) according to the present invention, at least one gRNA according to the present invention, the combination according to the present invention, the polynucleotide according to the present invention, the nucleic acid construct according to the present invention, the vector according to the present invention, the kit of polynucleotides according to the present invention, the cell according to the present invention, or the pharmaceutical composition according to the present invention may be for use in therapy.

[0098] In another aspect, the present invention provides the use of an ETM (e.g. ETR) according to the invention, at least one gRNA according to the invention, a combination according to the invention, a polynucleotide according to the invention, a nucleic acid construct according to the invention, a vector according to the invention, a kit of polynucleotides according to the invention, a cell according to the invention, or a pharmaceutical composition according to the invention in the manufacture of a medicament for treating a human in need thereof.

[0099] Suitably, at least one ETM (eg, ETR) and at least two gRNAs may be administered to a subject simultaneously, sequentially, or separately.

[0100] In another aspect, the present invention provides a method for treating (therapeutic) and / or preventing (prophylactic) a disease comprising the step of administering an ETM (e.g. ETR) according to the invention, at least one gRNA according to the invention, a combination according to the invention, a polynucleotide according to the invention, a nucleic acid construct according to the invention, a vector according to the invention, a kit of polynucleotides according to the invention, a cell according to the invention, or a pharmaceutical composition according to the invention to a subject in need thereof.

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

[0102] In one aspect, the present invention provides a method for producing a method for treating a cancer cell comprising: (i) isolation of a sample comprising cells; (ii) introducing an ETM (e.g. ETR) according to the invention, at least one gRNA according to the invention, a polynucleotide according to the invention, a nucleic acid construct according to the invention, a vector according to the invention, or a kit of polynucleotides according to the invention into a cell, and (iii) administering the cells from step (ii) to a subject. The present invention provides a method for gene therapy comprising the steps of:

[0103] The polynucleotide, nucleic acid construct, or vector may be introduced, for example, by transduction or transfection.

[0104] In some embodiments, the cells are autologous. In some embodiments, the cells are allogeneic.

[0105] It is understood that an ETM (e.g. an ETR) according to the invention, at least one gRNA according to the invention, a combination according to the invention, a polynucleotide according to the invention, a nucleic acid construct according to the invention, a vector according to the invention, a kit of polynucleotides according to the invention, a cell according to the invention, or a pharmaceutical composition according to the invention may be used in the methods of treatment described herein, may be for use in the treatments described herein, or may be used in the manufacture of a medicament for the treatments described herein.

[0106] Other features, objects, and advantages of the present invention will become apparent in the following detailed description. It should be understood, however, that the detailed description, while illustrating embodiments and aspects of the present invention, is given for purposes of illustration only and not limitation. Various changes and modifications within the scope of the present invention will become apparent to those skilled in the art from the detailed description. [Brief description of the drawings]

[0107] [Figure 1] (A) Shown are the sequences in the B2M gene targeted by Cas9 or dCas9-ETR and targeted in the Examples herein (SEQ ID NOs: 21 and 22 for the sense and antisense strands, respectively) (protospacer adjacent motif (PAM) sequences are underlined), and (B) the sequences of the spacers used in gRNAs targeting B2M and used in the Examples herein (SEQ ID NOs: 23-34 in order of appearance). [Diagram 2]Histograms illustrating the percentage of mutated B2M alleles in cells transfected with Cas9 and the indicated gRNA are shown. Data are expressed as % of non-homologous end joining (NHEJ) at B2M (n=3; mean±sd). UT: untreated. [Diagram 3] Histograms illustrating the percentage of tdTomato negative cells 44 days after transfection with triple combinations of dCas9-based ETR and the indicated gRNAs are shown (n=3; mean±sd). [Figure 4] Histograms illustrating the percentage of tdTomato negative cells 20 days after transfection with triple combinations of dCas9-based ETRs (left panel) or Cas9 (right panel) and the indicated gRNAs, i.e., H8, C8, and H10, 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 by 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 nucleotides, respectively, at the 5' end of the full-length sequence. [Diagram 5] Histograms illustrating the percentage of mutated TRAC alleles in cells transfected with Cas9 and TRAC gRNA are shown. Data are expressed as % of NHEJ in TRAC (n=3; mean±sd). [Figure 6] Histograms illustrating the percentage of B2M-negative cells (B2M- cells) 25 days after transfection with the indicated Cas9 constructs (i.e., Cas9, dCas9-ETR, Cas9-ETR (i.e., ETM)) and gRNA combinations are shown (n=3; mean ± sd). [Figure 7] Representative flow cytometry dot plot analysis of cells treated with 16 nt B2M gRNA and Cas9-ETR (i.e., ETM) or dCas9-ETR is shown, performed 25 days after treatment. [Figure 8]Figure 1 shows a time course flow cytometry analysis of cells treated as indicated. Data are presented as % of B2M negative cells normalized to untreated (UT) cells. Analysis was performed 25 days after treatment (n=3; mean±sd). [Figure 9] Histograms illustrating the percentage of gene editing in the B2M or TRAC genes for the indicated treatment conditions are shown (n=3; mean±sd). [Figure 10] Polymerase chain reaction (PCR) analysis of the indicated treatment conditions for reciprocal chromosomal translocation between the B2M and TRAC loci. Top: Schematic diagram of the PCR strategy showing the primers (arrows) used in the analysis. Bottom: Photograph of an agarose stained gel loaded with PCR products from the indicated treatment conditions (each in triplicate). Translocation was only detected in samples treated with Cas9 or Cas9-ETR (i.e. ETM) in combination with the 20 nt B2M gRNA. MW: molecular weight. [Figure 11] Schematic diagram of the B2M gene showing the distribution of CpG islands (CGIs) and gRNAs, namely 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). [Figure 12] Shown are the percentages of B2M silencing by triple combinations of dCas9-based ETRs at days 12 and 25 after treatment with the indicated gRNAs alone (column 1 of each table) or in combination (columns 2 and 3 of each table). Data are presented as heat maps. [Figure 13] Representative flow cytometry analysis of T cells treated with the indicated gRNA combinations (i.e., C8+F4, C8+H8, or H8+F4) and triple combinations of dCas9-based ETR at days 12 and 25 post-treatment is shown. The fold increase in the efficiency of episilencing of B2M between C8+F4 and H8+F4 conditions is shown. [Figure 14]Figure 1 shows a time course flow cytometry analysis of T cells treated with the triple combination of dCas9-based ETR and the indicated gRNA combinations. Data are shown as % of B2M negative cells. UT: untreated T cells. Vertical dotted red lines indicate the day T cells were restimulated. [Figure 15] 1 shows a histogram illustrating the fold change in the percentage of B2M-negative T cells between days 25 and 12 after treatment, calculated based on the data shown in Figure 14. Data are expressed as the fold reduction in B2M-negative cells. [Figure 16] Figure 1 shows a time course flow cytometry analysis of T cells treated with the triple combination of dCas9-based ETR and the indicated gRNA combinations. Data are shown as % of B2M negative cells. UT: untreated T cells. Vertical dotted red lines indicate the day T cells were restimulated. [Figure 17] 1 shows a histogram illustrating the fold change in the percentage of B2M-negative T cells between days 25 and 12 after treatment, calculated based on the data shown in Figure 16. Data are expressed as the fold reduction in B2M-negative cells. [Figure 18A] Figure 1 shows a time course flow cytometry analysis of T cells treated with the indicated ETR combinations and 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 with DNMT3A and DNMT3L; K+3A: double ETR combination with KRAB and DNMT3A; K+3L: double ETR combination with KRAB and DNMT3L; vertical red dotted lines indicate the days when T cells were restimulated. [Figure 18B] Representative flow cytometry analysis of T cells in Figure 18A treated with the indicated ETR combinations and gRNA combination C8+F4. K+3A+3L: standard triple ETR combination; K: KRAB-based ETR alone; 3A+3L: double ETR combination with DNMT3A and DNMT3L; K+3A: double ETR combination with KRAB and DNMT3A; K+3L: double ETR combination with KRAB and DNMT3L. [Figure 19] FIG. 1 shows a time course flow cytometry analysis of T cells treated with a dual ETR combination containing KRAB and DNMT3L and the indicated gRNA combination. Data are presented as % of B2M negative cells. UT: untreated T cells. Vertical dotted red lines indicate the day T cells were restimulated. [Figure 20A] A schematic of the ZIM3:dCas9:3L fusion ETR is shown on the left, and a time course flow cytometry analysis of T cells co-treated with either a dual ETR combination containing DNMT3A and DNMT3L or ZIM3:dCas9:3L and the indicated gRNA combination is shown on the right. Data are shown as % of B2M negative cells. UT: untreated T cells. The vertical red dotted line indicates the day when T cells were restimulated. [Figure 20B] Representative flow cytometry analysis of T cells from Figure 20A treated with the indicated ETR and gRNA combinations is shown. Also shown is the fold change increase in episilencing efficiency between samples treated with dual ETR combinations and ETR fusions. [Figure 21] Representative flow cytometry analysis of T cells treated with decreasing doses (micrograms) of mRNA encoding the ZIM3:dCas9:3L fusion ETR and the indicated gRNA combinations are shown. [Figure 22] Representative flow cytometry analysis of T cells treated with or without Cas9, gRNA against TRAC (see FIG. 5), and transduced with AAV6 for targeted integration of the NY-ESO engineered TCR into TRAC. The top left quadrant shows wild type unedited cells. The bottom left quadrant shows cells containing a genetically disrupted TCR. The top right quadrant shows T cells with targeted integration of the NY-ESO TCR. [Figure 23]A schematic of the dual ETM combination including catalytically active Cas9 and KRAB and DNMT3L effectors is shown on the left, and a representative flow cytometry analysis of these ETMs and the indicated truncated gRNAs for B2M and full-length gRNAs for TRAC and TRAC-treated T cells for targeted integration of the NY-ESO TCR into TRAC is shown on the right. The flow cytometry dot plot on the left shows the expression levels of B2M. The flow cytometry dot plot in the middle shows the expression levels of the endogenous TCR and the target NY-ESO. The flow cytometry dot plot on the right shows the expression levels of NY-ESO and B2M. SSCH: side scatter height. [Figure 24-1] A schematic of the ETM containing catalytically active Cas9 and ZIM3 and DNMT3L effectors (i.e., ZIM3:Cas9:3L) is shown on the left, and a representative flow cytometry analysis of the ETM and the indicated truncated gRNA for B2M and full-length gRNA for TRAC and TRAC-treated T cells for targeting of the NY-ESO TCR to TRAC is shown on the right. The top left flow cytometry dot plot shows the expression level of B2M. The top middle flow cytometry dot plot shows the expression level of the endogenous TCR and target NY-ESO. The bottom flow cytometry dot plot shows the expression level of NY-ESO and B2M. The top right flow cytometry dot plot shows the expression level of B2M in NY-ESO positive cells. The bottom right flow cytometry dot plot shows the expression level of B2M in endogenous TCR negative cells. [Figure 24-2] This is a continuation of Figure 24-1. [Diagram 25]Polymerase chain reaction (PCR) analysis of the indicated treatment conditions for reciprocal chromosomal translocation between B2M and TRAC. Top: A schematic diagram of the PCR strategy showing the primers (arrows) used for the analysis. Bottom: A photograph of an agarose stained gel loaded with PCR products from the indicated treatment conditions is shown. The expected position of the B2M-TRAC translocation band is indicated with an asterisk. MW: molecular weight. The translocation was only detected in samples treated with ETM in combination with 20 nt gRNA for B2M and TRAC. [Figure 26] Schematic diagrams of the TGFBR2 (top) and TET2 (bottom) genes are shown, with the relative positions of the respective gRNAs and their pairings (P). The CpG islands (CGIs) of each gene are also shown. [Figure 27] Percentage of TGFBR2 episilencing for the indicated combinations of gRNA pairs is shown. Percentages are shown in the boxes. Boxes without labels indicate combinations that were already present in the matrix. np: not performed. [Figure 28] Percentage of TET episilencing for the indicated combinations of gRNA pairs is shown. Percentages are shown in the boxes. Unlabeled boxes indicate combinations that were already present in the matrix. Negative data indicate upregulation of TET2. np: not done. [Figure 29] Histograms illustrating the percentage of episilencing of TGFBR2 (left) and TET2 (right) in T cells treated with the indicated pairs of triple ETR combinations and gRNAs (P), alone or in combination, are shown. The pairs used in these studies correspond to those described in Figures 27 and 28. [Diagram 30] Histograms illustrating the percentage of epigenetic silencing of the indicated genes as measured by ddPCR are shown. [Diagram 31] Representative flow cytometry analysis of B2M (left plot) and TRAC (right plot) expression by T cells treated as indicated is shown on the left, and histograms illustrating the percentage of epigenetic silencing of TGFBR2 are shown on the right. [Diagram 32] Representative flow cytometry analysis of B2M (left plot) and TRAC (right plot) expression by T cells treated as indicated is shown on the left, and histograms illustrating the percentage of epigenetic silencing of TGFBR2 are shown on the right. [Diagram 33] Polymerase chain reaction (PCR) analysis of the indicated treatment conditions for reciprocal chromosomal translocations between B2M, TGFBR2, and TRAC. Top: Schematic diagram of the PCR strategy for two hypothetical genes (X and Y), where the arrows indicate the primers used in the analysis. Bottom: Photograph of an agarose stained gel loaded with PCR products from the indicated treatment conditions. The expected position of the translocation band is indicated with an asterisk. MW: molecular weight. Translocations were only detected in samples treated with ETM in combination with 20 nt gRNA for B2M, TGFBR2, and TRAC. [Diagram 34] Representative flow cytometry analysis of B2M (left plots) and TRAC (right plots) expression by T cells treated as indicated is shown on the left, and histograms illustrating the percentage of epigenetic silencing of TET2 are shown on the right. [Diagram 35] Representative flow cytometry analysis of B2M (left plots) and TRAC (right plots) expression by T cells treated as indicated is shown on the left, and histograms illustrating the percentage of epigenetic silencing of TET2 are shown on the right. [Diagram 36] Polymerase chain reaction (PCR) analysis of the indicated treatment conditions for reciprocal chromosomal translocations between B2M, TET2, and TRAC. Top: Schematic diagram of the PCR strategy for two hypothetical genes (X and Y), where the arrows indicate the primers used in the analysis. Bottom: Photograph of an agarose stained gel loaded with PCR products from the indicated treatment conditions. The expected position of the translocation band is indicated with an asterisk. MW: molecular weight. Translocations were only detected in samples treated with ETM in combination with 20 nt gRNA for B2M, TET2, and TRAC. [Figure 37]Representative flow cytometry analysis of B2M (left plot) and TRAC (right plot) expression by T cells treated as indicated is shown on the left, and histograms illustrating the percentage of epigenetic silencing of TGFBR2 and TET2 are shown on the right. [Figure 38] Representative flow cytometry analysis of B2M (left plot) and TRAC (right plot) expression by T cells treated as indicated is shown on the left, and histograms illustrating the percentage of epigenetic silencing of TGFBR2 and TET2 are shown on the right. [Figure 39] Polymerase chain reaction (PCR) analysis of the indicated treatment conditions for reciprocal chromosomal translocations between B2M, TGFBR2, TET2, and TRAC. Top: Schematic diagram of the PCR strategy for two hypothetical genes (X and Y), where the arrows indicate the primers used in the analysis. Bottom: Photograph of an agarose stained gel loaded with PCR products from the indicated treatment conditions. The expected position of the translocation bands is indicated with an asterisk. MW: molecular weight. Translocations were only detected in samples treated with ETM in combination with 20 nt gRNA for B2M, TGFBR2, TET2, and TRAC. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0108] Engineered transcriptional modulators (ETMs) In one aspect, the invention provides an engineered transcriptional modulator (ETM), e.g., an engineered transcriptional repressor (ETR), comprising a) at least one epigenetic effector domain operably linked to b) an endonuclease.

[0109] The ETM of the present invention may be an ETR. The ETR may repress the transcription and / or expression of a target gene.

[0110] The ETM (e.g., ETR) of the present invention is an agent that can enable multiplexing of gene editing and epigenetic editing of different target genes. For example, the ETM (e.g., ETR) of the present invention allows the suppression (e.g., silencing) of the transcription and / or expression of a number of different target genes, where one gene is suppressed (e.g., silenced) by gene editing and at least one gene is suppressed (e.g., silenced) by epigenetic suppression (e.g., silencing). The advantage of this multifunctional editing system is that there is no reciprocal translocation between the genes that are edited simultaneously, thus greatly improving the safety of multiple gene editing. Furthermore, the application of such a multifunctional editing approach allows orthogonal editing to be performed in one step without the need for sequential manipulation procedures, thereby greatly facilitating the production of products and reducing the associated costs and cytotoxicity. The target gene selected for gene editing is also used as a target site for the insertion of an exogenous expression cassette.

[0111] ETMs are also referred to as programmable multi-editors (ProMEs). For example, the design of gRNAs allows ETMs to be programmed to modify transcription, expression, and / or activity of multiple targets in the same cell. ETMs (e.g., ETRs) may be chimeric or fusion proteins consisting of at least one (e.g., one) endonuclease operably linked to at least one effector domain (e.g., KRAB domain, SETDB1 domain, DNMT3A domain, DNMT3B or DNMT1 domain, or DNMT3L domain, or homologs thereof, where the domains may be full-length proteins or functional fragments thereof, referred to herein as "KRAB", "SETDB1", "DNMT3A", "DNMT3B", "DNMT1", or "DNMT3L", respectively). The endonuclease allows cleavage of specific DNA sequences and may be selected or engineered to bind to selected nucleic acid sequences. The epigenetic effector domain may have catalytic activity that allows modification (e.g., repression) of transcription of a target gene. Alternatively or additionally, the effector domain may recruit additional agents within the cell to the target gene, resulting in altered (e.g., suppressed) transcription of the target gene. The present invention also contemplates engineered transcription activators (ETAs), or ETMs. ETAs may increase transcription and / or expression of the target gene.

[0112] "Operably linked" is understood to mean that the individual components are linked to each other in such a way that they can perform their functions (e.g., cleaving DNA, binding to DNA, catalyzing a reaction, or recruiting additional agents from inside the cell) substantially unhindered. For example, an endonuclease can be conjugated with an epigenetic effector domain to form, for example, a fusion protein. Methods for conjugating polypeptides are known in the art, and may be, for example, by providing a linker amino acid sequence (e.g., a linker comprising glycine and / or serine residues) that links the polypeptides. Alternative methods for conjugating polypeptides known in the art include chemical or light-induced conjugation methods (e.g., using chemical cross-linking agents). As an example, an endonuclease and an epigenetic effector domain of an ETM (e.g., a KRAB domain, a DNMT3A, DNMT3B, or DNMT1 domain, or a DNMT3L domain, or a homolog thereof) form a fusion protein.

[0113] In one embodiment, the ETM (e.g., ETR) comprises an RNA binding domain. The RNA binding domain may bind to a gRNA that is complementary to a genomic target site. That is, the RNA binding domain may allow the ETM (e.g., ETR) to target a target gene.

[0114] In one embodiment, the ETM (eg, ETR) is a fusion protein comprising a) at least one epigenetic effector domain, and b) an endonuclease.

[0115] In some embodiments, the ETM (e.g., ETR) is a bipartite fusion protein. For example, the ETM (e.g., ETR) may comprise two effector domains fused to the same endonuclease.

[0116] In some embodiments, the ETM (e.g., ETR) is a tripartite fusion. For example, the ETM (e.g., ETR) may comprise three effector domains fused to the same endonuclease.

[0117] In some embodiments, an ETM (eg, an ETR) may comprise four or five or six or more effector domains fused to the same endonuclease.

[0118] Suitably, where an ETM (e.g., an ETR) comprises multiple effector domains, the effector domains may be different. Suitably, where an ETM (e.g., an ETR) comprises multiple effector domains, the effector domains may be the same.

[0119] In one aspect, an ETM (e.g., ETR) according to the invention comprises or consists of a Cas9-KRAB, Cas9-DNMT3A, or Cas9-DNMT3L fusion protein.

[0120] Preferably, the ETM (e.g., ETR) according to the present invention may be a fusion protein comprising or consisting of an endonuclease, a KRAB, and a DNMT3A domain. Preferably, the ETM (e.g., ETR) according to the present invention may be a fusion protein comprising or consisting of an endonuclease, a DNMT3L, and a DNMT3A domain. Preferably, the ETM (e.g., ETR) according to the present invention may be a fusion protein comprising or consisting of an endonuclease, a DNMT3L, and a KRAB domain. Preferably, the ETM (e.g., ETR) according to the present invention may be a fusion protein comprising or consisting of an endonuclease, a DNMT3L, a KRAB, and a DNMT3A domain.

[0121] Preferably, the ETM (e.g., ETR) according to the present invention may be a fusion protein comprising or consisting of a Cas (e.g., Cas9), KRAB, and DNMT3A domain. Preferably, the ETM (e.g., ETR) according to the present invention may be a fusion protein comprising or consisting of a Cas (e.g., Cas9), DNMT3L, and DNMT3A domain. Preferably, the ETM (e.g., ETR) according to the present invention may be a fusion protein comprising or consisting of a Cas (e.g., Cas9), DNMT3L, and KRAB domain. Preferably, the ETM (e.g., ETR) according to the present invention may be a fusion protein comprising or consisting of a Cas (e.g., Cas9), DNMT3L, KRAB, and DNMT3A domain.

[0122] In one embodiment, the ETM (e.g., ETR) comprises or consists of an endonuclease-KRAB fusion protein, e.g., a Cas-KRAB, e.g., a Cas9-KRAB domain fusion protein.

[0123] An exemplary sequence of an ETM according to the present invention containing a KRAB domain (ETM-KRAB) is set forth below in SEQ ID NO:18. [ka]

[0124] In the above sequence, the Cas9 domain is in italics, the hemagglutinin (HA) tag is in bold, the linker domain is in bold and double underlined, the KRAB domain is in italics and underlined, and the nuclear localization signal (NLS) sequence is boxed.

[0125] It will be appreciated that alternatives to the HA tag and glycine-serine linker shown in these exemplary ETMs may be used, or may be omitted, in the ETMs according to the invention.

[0126] In one embodiment, the ETM (e.g., ETR) comprises or consists of an endonuclease-DNMT3A fusion protein, e.g., a Cas-DNMT3A, e.g., a Cas9-DNMT3A domain fusion protein.

[0127] 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. [ka] [ka]

[0128] In the above sequence, the Cas9 domain is in italics, the HA tag is in bold, the linker domain is in bold and double underlined, the DNMT3A domain is in italics and underlined, and the NLS sequence is boxed.

[0129] In one embodiment, the ETM (e.g., ETR) comprises or consists of an endonuclease-DNMT3L fusion protein, e.g., a Cas-DNMT3L, e.g., a Cas9-DNMT3L domain fusion protein.

[0130] 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. [ka] [ka]

[0131] The Cas9 domain is in italics, the HA tag is in bold, the linker domain is in bold and double underlined, the DNMT3L domain is in italics and underlined, and the NLS sequence is boxed.

[0132] The fusion protein may comprise, for example, an amino acid sequence having 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., the amino acid sequence substantially retains the native function of the protein represented by SEQ ID NO: 18, 19, 20, 4481, or 4482.

[0133] The fusion protein is encoded by a polynucleotide that includes a nucleic acid sequence encoding a protein, e.g., SEQ ID NO: 18, 19, 20, 4481, or 4482, or a protein having 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., the amino acid sequence substantially retains the native 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.

[0134] Epigenetic effector domains The term "epigenetic effector domain" should be understood to mean a part of an ETM that provides an epigenetic effect on a target gene, for example by catalyzing a reaction on DNA or chromatin (e.g., DNA methylation) or by recruiting additional agents from within the cell, resulting in, for example, the repression of transcription of the gene.

[0135] A "domain" in this context should be understood to mean a portion of an ETM that has a certain function. A domain may be an individual domain isolated from a naturally occurring protein (e.g., a catalytic domain) or may be the entire full-length naturally occurring protein. In other words, the full-length protein or a functional fragment thereof can be used as an effector domain. Thus, for example, a "Kruppel-associated box (KRAB) domain" or a "KRAB domain" refers to a portion of an ETM that includes an amino acid sequence that has the function of a KRAB domain.

[0136] Chromatin-remodeling enzymes known to be involved in permanent epigenetic silencing of endogenous retroviruses (ERVs; Feschotte, C. et al. (2012) Nat. Rev. Genet. 13: 283-96; Leung, DC et al. (2012) Trends Biochem. Sci. 37: 127-33) may provide suitable effector domains for use in the present invention.

[0137] In one embodiment, the epigenetic effector domain can suppress the transcription and / or expression of at least one target gene. The factor that can suppress the transcription of a gene is also called a transcription repressor. In one embodiment, the epigenetic effector domain is a repressor domain, for example a transcription repressor domain.

[0138] In one aspect, the epigenetic effector domain initiates chemical modification of chromatin and / or chromatin remodeling.

[0139] In one aspect, the epigenetic effector domain initiates DNA modification, such as DNA methylation, hi one aspect, the epigenetic effector domain is a DNA methyltransferase and / or is capable of recruiting a DNA methyltransferase.

[0140] In one embodiment, the epigenetic effector domain initiates histone modifications, such as histone methylation or histone acetylation, hi one embodiment, the epigenetic effector domain is a histone methyltransferase or a histone acetyltransferase.

[0141] In one embodiment, 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.

[0142] In one embodiment, at least one epigenetic effector domain is an antibody or derivative thereof, such as a nanobody, which binds to an epigenetic regulator, such as a chromatin regulator that can chemically modify and / or remodel chromatin.

[0143] See, e.g., Van et al., Nat Commun. 2021 Jan 22;12(1)537, which describes nanobody-mediated regulation of gene expression and epigenetic memory.

[0144] KRAB In some embodiments, at least one epigenetic effector domain comprises a KRAB domain. The family of Kruppel-associated box-containing zinc finger proteins (KRAB-ZFPs; Huntley, S. et al. (2006) Genome Res. 16: 669-77) plays an important role in silencing endogenous retroviruses. These transcription factors bind to specific ERV sequences through their ZFP DNA binding domains, which recruit KRAB-associated protein 1 (KAP1) with its conserved KRAB domain. KAP1 then binds to a number of effectors that promote the local formation of repressive chromatin (Iyengar, S. et al. (2011) J. Biol. Chem. 286: 26267-76).

[0145] The ETM of the present invention includes, for example, a KRAB domain. Various KRAB domains are known in the KRAB-ZFP protein family. For example, the ETM of the present invention includes the KRAB domain of human zinc finger protein 10 (ZNF10; Szulc, J. et al. (2006) Nat. Methods 3: 109-16): [ka]

[0146] Further examples of KRAB domains suitable for use in the present invention include: [ka] (KRAB domain of human ZIM3 protein; SEQ ID NO:2) [ka] (KRAB domain of ZNF350 protein; SEQ ID NO:3) [ka] (KRAB domain of ZNF197 protein; SEQ ID NO:4) [ka] (KRAB domain of RBAK protein; SEQ ID NO:5) [ka] (KRAB domain of ZKSCAN1 protein; SEQ ID NO:6) [ka] (KRAB domain of KRBOX4 protein; SEQ ID NO:7) [ka] (KRAB domain of ZNF274 protein; SEQ ID NO:8) Includes:

[0147] The above KRAB domain is only an example. Its functional variants are also contemplated herein. For example, the ZIM3 KRAB domain shown in SEQ ID NO: 4481 and 4482 (see Examples 3 and 4 below) can also be used. The ZIM3 KRAB domain has the following sequence: [ka]

[0148] DNMT In some embodiments, the epigenetic effector domain comprises a DNA methyltransferase (DNMT) domain. DNMTs catalyze the transfer of methyl groups to DNA. Examples of DNMTs include DNMT1, DNMT3A, and DNMT3B.

[0149] An ETM of the invention can include, for example, a domain, such as the catalytic domain, of human DNA methyltransferase 3A (DNMT3A; Law, JA et al. (2010) Nat. Rev. Genet. 11: 204-20). For example, an ETM of the invention can include, for example, the domain of human DNA methyltransferase 3A (DNMT3A; Law, JA et al. (2010) Nat. Rev. Genet. 11: 204-20). [ka] (catalytic domain of human DNMT3A; SEQ ID NO: 9).

[0150] DNA methyltransferases 3B and 1 (DNMT3B and DNMT1), like DNMT3A, are also involved in the deposition and maintenance of DNA methylation and are also used in the ETMs of the present invention. For example, the ETMs of the present invention have the sequence: [ka] (Catalytic domain of human DNMT3B; SEQ ID NO: 10) [ka] (Human DNMT3B: SEQ ID NO: 11) [ka] (Human DNMT1 catalytic domain; SEQ ID NO: 12) It may include any of the following.

[0151] Dear DNMT, In some embodiments, the epigenetic effector domain may be a DNMT-like domain. A "DNMT-like" domain refers to a protein or functional fragment thereof that is a member of the DNMT family but does not possess DNA methylation activity. DNMT-like proteins typically activate or recruit other epigenetic effector domains.

[0152] An ETM of the invention can include, for example, 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 invention can include a nucleotide sequence comprising the sequence: [ka] (human DNMT3L; SEQ ID NO: 13).

[0153] HMT In some aspects, the epigenetic effector domain can be a histone methyltransferase (HMT) domain, e.g., a catalytic domain. HMTs are histone modifying enzymes that catalyze the transfer of methyl groups to lysine and arginine residues of histone proteins.

[0154] The lysine-specific HMT may or may not contain a SET (Su(var)3-9, Enhancer of Zeste, Trithorax) domain.

[0155] An example of an HMT is divergent SET domain 1 (SETDB1).

[0156] 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. At the same time, KAP1 binds to heterochromatin protein 1 alpha (HP1α), which is read as H3K9me2 and H3K9me3, stabilizing KAP1-containing complexes. KAP1 can also interact with other well-known epigenetic silencers, such as lysine-specific histone demethylase 1 (LSD1), which inhibits transcription by removing histone H3 lysine-4 methylation, and the nucleosome remodeling and deacetylase complex (NURD), which removes acetyl groups from histones. Finally, KAP1-containing complexes contribute to the recruitment of de novo DNA methyltransferase 3A (DNMT3A), which methylates cytosines at CpG sites (Jones, PA (2012) Nat. Rev. Genet. 13: 484-92). Together, these data suggest a model in which the KAP1 complex ensures ERV silencing in preimplantation embryos through the concerted action of histone-modifying enzymes and DNA methylation. After implantation, DNA methylation already targeted to ERVs by KRAB-ZFPs is then stabilized (Reik, W. (2007) Nature 447: 425-32) and is inherited throughout mitosis and somatic differentiation without the need for continuous expression of ERV-specific KRAB-ZFPs. Unlike in embryonic stem cells, the KAP1 complex cannot efficiently induce DNA methylation in somatic cells, but can only deposit H3K9 methylation. However, this histone mark is not maintained without continued deposition at target sites by KRAB-ZFPs (Hathaway, NA et al. (2012) Cell 149: 1447-60).

[0157] In some embodiments, 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 that occurs in ERVs in embryonic stem cells) and the other based on, for example, DNMT3A (e.g., the final lock of the process). This approach allows recapitulating, on preselected target genes, the repressive chromatin state established in ERVs of preimplantation embryos and perpetuated throughout mammalian development and adult life.

[0158] An ETM of the invention may, for example, comprise a SETDB1 domain. For example, an ETM of the invention may comprise the sequence: [ka] [ka] (Human SETDB1; SEQ ID NO: 14) [ka] (Catalytic domain of human SETDB1; SEQ ID NO: 15) may include any of the following:

[0159] The ETMs of the present invention may comprise an amino acid sequence having at least 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity to, for example, SEQ ID NO:1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15, e.g., the amino acid sequence substantially retains the native 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.

[0160] The ETMs of the invention are encoded by a polynucleotide comprising a nucleic acid sequence encoding, for example, a protein of SEQ ID NO: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15, or a protein having at least 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% amino acid identity to SEQ ID NO: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15, e.g., the amino acid sequence substantially retains the native 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. The coding sequence may be codon optimized for optimal expression in human cells.

[0161] The ETMs of the present invention include, for example, an amino acid sequence having at least 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO:4637, e.g., the amino acid sequence substantially retains the native function of the protein represented by SEQ ID NO:4637.

[0162] The ETMs of the invention are encoded by a polynucleotide comprising a nucleic acid sequence encoding, for example, a protein of SEQ ID NO: 4637 or a protein having at least 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% amino acid identity to SEQ ID NO: 4637, e.g., the amino acid sequence substantially retains the native function of the protein represented by SEQ ID NO: 4637. The coding sequence may be codon optimized for optimal expression in human cells.

[0163] Endonucleases An ETM (eg, an ETR) of the present invention can comprise an endonuclease.

[0164] The endonuclease may be, for example, site-specific. As used herein, "site-specific endonuclease" may refer to an enzyme that induces site-directed double-stranded breaks in DNA. The site-specific endonuclease allows the activity of ETM (e.g., ETR) to be targeted to a specific site in a polynucleotide, e.g., the genome of a cell. For example, the endonuclease is site-specific when used in combination with gRNA, in other words, the endonuclease can induce site-specific DNA breaks when used in combination with gRNA.

[0165] In one aspect, the endonuclease has exonuclease activity in addition to endonuclease activity.

[0166] The endonuclease may, for example, bind to a binding site within the target gene or within a regulatory sequence of the target gene, such as a promoter sequence or enhancer sequence.

[0167] The endonuclease may bind to a binding site, for example, in a splice site. The splice variant of a given gene may be controlled by methylation / demethylation of DNA at the splice site. These modifications may then cause the exclusion / inclusion of exons in the mature transcript. This exclusion / inclusion may have therapeutic relevance. For example, in the case of Duchenne muscular dystrophy, exclusion (by gene ablation or exon skipping) of the exons harboring the most frequent pathogenic mutations from the mature mRNA has been proposed for treatment (Ousterout, DG et al. (2015) Mol. Ther. 23: 523-32; Ousterout, DG 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).

[0168] Several suitable endonucleases are known in the art. For example, the CRISPR / Cas system (Sander, JD et al. (2014) Nat. Biotechnol. 32: 347-55) may be employed as a suitable endonuclease in the ETM (e.g., ETR) of the present invention.

[0169] "CRISPR / Cas system" means the clustered regularly interspaced short palindromic repeats / CRISPR-associated nuclease system.

[0170] "Clustered Regularly Interspaced Short Palindromic Repeats" consist of short sequences that originate from viral genomes and are integrated into bacterial genomes. CRISPR-associated proteins (Cas) process these sequences and cut matching viral DNA sequences. By introducing Cas and specially constructed CRISPR into eukaryotic cells, the genome of the eukaryotic cell can be cut at any desired location.

[0171] The CRISPR / Cas system is an RNA-guided DNA binding system (van der Oost et al. (2014) Nat. Rev. Microbiol. 12: 479-92), and a guide RNA (gRNA) may be selected to enable the ETM (e.g., ETR) containing a Cas domain to target a specific sequence. That is, to employ the CRISPR / Cas system as an endonuclease of the present invention, it is understood that an epigenetic effector domain is operably linked to a Cas endonuclease, such as a Cas9 endonuclease. An ETM (e.g., ETR) containing a Cas endonuclease may be delivered to a target cell in combination with one or more gRNAs. The gRNA is designed to target the ETM (e.g., ETR) to a target gene of interest or a control element of the target gene (e.g., a promoter, enhancer, or splice site). Methods for designing gRNAs are known in the art. Additionally, fully orthogonal Cas9 proteins as well as structural / compositional modifications of the Cas9 / gRNA ribonucleoprotein complex and gRNA binding to various proteins have been developed to simultaneously and unidirectionally target various effector domains to desired genomic sites in cells (Esvelt et al. (2013) Nat. Methods 10: 1116-21; Zetsche, B. et al. (2015) Cell pii: S0092-8674(15)01200-3; Zalatan, JG et al. (2015) Cell 160: 339-50; Paix, A. et al. (2015) Genetics 201: 47-54) and are suitable for use in the present invention.

[0172] In one embodiment, the ETM (e.g., ETR) comprises at least one endonuclease derived from a type II CRISPR bacterial immune system. In other words, the ET (e.g., ETR) can comprise a type II Cas.

[0173] Examples of Cas II type enzymes include Cas9, Csn2, and Cas4.

[0174] The Cas9 endonuclease typically contains RecI, RecII, a bridge helix, RuvC, HNH, and a PAM interaction domain.

[0175] The HNH and RuvC domains are nuclease domains. The RecI domain binds to the gRNA. The bridge helix initiates cleavage upon target DNA binding. The PAM-interacting domain confers PAM specificity and is responsible for initiating binding to target DNA.

[0176] The endonuclease may comprise or consist of a Cas endonuclease. That is, the endonuclease may have nuclease activity. For example, the endonuclease may be a catalytically active nuclease that binds to the gRNA and binds to the target DNA.

[0177] The endonuclease contained in the ETM (e.g., ETR) according to the present invention is a catalytically active endonuclease, in other words, the ETM (e.g., ETR) is capable of cleaving a target sequence, e.g., a target DNA.

[0178] In one embodiment, the endonuclease is a catalytically active Cas nuclease.

[0179] In one embodiment, the endonuclease is a modified or variant endonuclease, such as a modified Cas or modified Cas9 enzyme. For example, it will be appreciated that the enzyme may be modified to recognize a specific PAM site suitable for the target gene. The modified PAM may be different from the PAM naturally recognized by the enzyme.

[0180] In one embodiment, the ETM (e.g., ETR) according to the present invention does not include only catalytically inactive or catalytically dead (dCas) nucleases. In one embodiment, the ETM (e.g., ETR) according to the present invention does not include catalytically inactive or catalytically dead (dCas) nucleases, such as dCas9.

[0181] In one embodiment, the endonuclease is a catalytically active Cas9 nuclease.

[0182] In one embodiment, the endonuclease is a catalytically active Cas9 nuclease from Streptococcus pyogenes (SpCas9).

[0183] Methods for determining whether a protein is a catalytically active nuclease are known in the art, for example, gel assays, Kunitz assays, radiolabeled assays, and fluorescence-based methods are used. Gel assays can be performed using purified recombinant target DNA as a substrate in an assay buffer. The protein to be tested can be incubated with the substrate, for example, at 37° C. for 1 hour. The reaction products can be separated by electrophoresis on an agarose gel, for example, with the addition of ethidium bromide to visualize the products of the nuclease reaction. Other methods include fluorescent real-time quantification of DNA and RNA nuclease activity, for example, as reported in Sheppard, EC, et al. Sci Rep 9, 8853 (2019), and cell-free detection of Cas nucleases, as reported in J. Cox et al., Chem Sci. 2019 Mar 7; 10(9): 2653-2662.

[0184] For example, an ETM (e.g., an ETR) of the invention may comprise the following catalytically active Cas9 sequence: [ka] (SEQ ID NO:16)

[0185] An ETM (e.g., an ETR) of the present invention may comprise an amino acid sequence having at least 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity to, for example, SEQ ID NO:16, e.g., the amino acid sequence substantially retains a native function (e.g., an endonuclease function) of the protein represented by SEQ ID NO:16.

[0186] An ETM (e.g., an ETR) of the invention may be encoded by a polynucleotide comprising a nucleic acid sequence encoding, for example, the protein of SEQ ID NO: 16 or a protein having at least 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99% or 100% amino acid identity to SEQ ID NO: 16, e.g., the amino acid sequence substantially retains the native function of the protein represented by SEQ ID NO: 16. The coding sequence may be codon optimized for optimal expression in human cells.

[0187] For comparison, the sequence of catalytically dead Cas9 (dCas9) is [ka] (Catalytically dead Cas9; dCas9; SEQ ID NO: 17) It is.

[0188] The above sequence includes D9A and H839A substitutions relative to its catalytically active (i.e., live) counterpart (SEQ ID NO: 16). A catalytically dead Cas9 (e.g., dCas9 as described above) is used in an ETM for epigenetic editing of one or more target genes without simultaneous gene editing of another gene in a cell. For this use, the ETM (e.g., ETR) may include, for example, an amino acid sequence having at least 70%, 80%, 90%, 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO: 17, e.g., the amino acid sequence substantially retains the native function of the protein represented by SEQ ID NO: 16, except for the endonuclease function. An ETM (e.g., an ETR) may be encoded by a polynucleotide comprising a nucleic acid sequence encoding, for example, the protein of SEQ ID NO: 17 or a protein having at least 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% amino acid identity to SEQ ID NO: 17, e.g., the amino acid sequence substantially retains the native function of the protein represented by SEQ ID NO: 16, except for the endonuclease function. The coding sequence may be codon optimized for optimal expression in human cells.

[0189] gRNA In one aspect, the present invention provides a guide RNA (gRNA).

[0190] The gRNA targets the ETM (e.g., ETR) to a target gene. The gRNA can be, for example, an RNA sequence that recognizes a target DNA region of interest and directs an endonuclease within the ETM (e.g., ETR) to that region.

[0191] gRNAs typically consist of two parts: a) a spacer sequence (also called a targeting domain, guide sequence, or complementary region, which may constitute the CRISPR RNA (crRNA); and b) The scaffold sequence (also called tracrRNA in the CRISPR / Cas system). It consists of:

[0192] The spacer and scaffold sequences may, for example, be provided as separate molecules or may be linked, for example via a linker loop or other sequence, or may be fused to one another.

[0193] For example, the gRNA may be composed of two separate molecules, such as a spacer (crRNA) and a scaffold (tracrRNA), the 3' end of the spacer (crRNA) may be complementary to the 5' end of the scaffold (tracrRNA), and this complementarity may result in dimerization of the two molecules.

[0194] In another example, the spacer (crRNA) and the scaffold (tracrRNA) may be fused, for example, via a linker loop. This artificial construct may also be known as a single guide RNA (sgRNA).

[0195] In some embodiments, variants of the scaffold (tracrRNA) may be used. For example, the tetraloop and stem loop of the scaffold (tracrRNA) sequence are modified to include an RNA aptamer to which a domain of a specific protein can bind. In some embodiments, such modified gRNAs can be used to facilitate the recruitment of repressive or activating domains fused to the RNA aptamer that interacts with the protein.

[0196] Exemplary tracrRNA sequences include, but are not limited to, [ka] [ka] Includes.

[0197] "Spacer" or "spacer sequence" refers to a sequence that may be perfectly complementary to a target domain (ie, region) within a target sequence.

[0198] The 3' end of a genomic target sequence generally contains a protospacer adjacent motif (PAM) sequence. A "PAM" sequence is a DNA sequence of 2-6 base pairs that typically immediately follows the DNA sequence targeted by the nuclease. The PAM sequence is necessary for cleavage but is not part of the target gRNA sequence. PAM sequences vary depending on the species of 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 engineered nuclease enzymes may recognize a variety of PAM sequences.

[0199] For example, Francisella novicida Cas9 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. Francisella novicida Cas12a (or Cpf1) nuclease recognizes the PAM 5'-TTTN-3' or 5'-YTN-3'.

[0200] The nucleotides upstream of the PAM sequence (towards the 5' end of the target sequence) are a protospacer sequence.

[0201] The Cas9 nuclease will typically cleave approximately 3 bases upstream of the PAM.

[0202] It will be appreciated that a suitable nuclease for a particular context will be selected based on the specificity of the PAM and the genomic target.

[0203] A "scaffold" or "scaffold sequence" is a sequence necessary for the binding of an endonuclease, e.g., the binding of Cas.

[0204] In one embodiment, the present invention provides a single guide RNA (sgRNA). In one embodiment, the gRNA according to the present invention is a sgRNA. The sgRNA is a single RNA molecule that comprises a crRNA sequence fused to a scaffold tracrRNA sequence. In nature, the crRNA and tracrRNA exist as two separate RNA molecules, but the sgRNA has become the common format of CRISPR gRNA in research.

[0205] In one embodiment, the gRNA comprises a spacer sequence that is 10 nucleotides in length. In one embodiment, the gRNA comprises a spacer sequence that is 11 nucleotides in length. In one embodiment, the gRNA comprises a spacer sequence that is 12 nucleotides in length. In one embodiment, the gRNA comprises a spacer sequence that is 13 nucleotides in length. In one embodiment, the gRNA comprises a spacer sequence that is 14 nucleotides in length. In one embodiment, the gRNA comprises a spacer sequence that is 15 nucleotides in length. In one embodiment, the gRNA comprises a spacer sequence that is 16 nucleotides in length. In one embodiment, the gRNA comprises a spacer sequence that is 17 nucleotides in length. In one embodiment, the gRNA comprises a spacer sequence that is 18 nucleotides in length. In one embodiment, the gRNA comprises a spacer sequence that is 19 nucleotides in length. In one embodiment, the gRNA comprises a spacer sequence that is 20 nucleotides in length. In one embodiment, the gRNA comprises a spacer sequence that is 21 nucleotides in length.

[0206] While not wishing to be bound by theory, certain gRNAs (e.g., gRNAs containing a spacer sequence of about 20 nucleotides in length) are used to induce gene editing by ETM (e.g., ETR), while gRNAs containing a short spacer sequence (e.g., gRNAs containing a spacer sequence of about 16 nucleotides in length) may favor epigenetic editing such as episilencing by ETM (e.g., ETR). See, for example, FIG. 2. This shows that gRNAs containing a spacer sequence of about 18 to 20 nucleotides in length induce NHEJ, while gRNAs containing a spacer sequence of about 16 nucleotides or less in length do not induce NHEJ. FIG. 3 shows that gRNAs containing a spacer sequence of about 11 to 16 nucleotides in length can induce epigenetic modifications, such as episilencing of B2M.

[0207] In some embodiments, the gRNA comprises a spacer sequence that is 15, 16, or 17 nucleotides or less in length (e.g., 17 or 16 or less). In some embodiments, the gRNA comprises a spacer sequence that is 11-16 nucleotides in length, e.g., 12-16, 13-16, 14-16, 15-16, 12-17, 13-17, 14-17, 15-17, 16, or 17 nucleotides in length.

[0208] In some embodiments, the gRNA comprises a spacer sequence that is 16, 17, or 18 or more (e.g., 17 or 18 or more) nucleotides in length, such as 18 or more, or 19 or more, or 20 or more nucleotides in length. In some embodiments, the gRNA comprises a spacer sequence that is 17-30, such as 18-30, 19-30, or 20-30 nucleotides in length. In some embodiments, the gRNA comprises a spacer sequence that is 17-25 nucleotides in length, such as 18-25, 19-25, or 20-25 nucleotides in length. In some embodiments, the gRNA comprises a spacer sequence that is 17-20 nucleotides in length, such as 18-20 or 19-20 nucleotides in length.

[0209] The ETMs of the present invention can modify the transcription, expression, and / or activity (e.g., suppress the transcription and / or expression) of multiple target genes in the same cell through epigenetic editing and gene editing.

[0210] The present invention allows for the selection of gRNAs that promote targeted gene editing or epigenetic editing. In this way, gene editing can be performed on gene targets that are not amenable to epigenetic editing in a multiplexed approach, while simultaneously selecting to epigenetically target genes that are amenable to epigenetic editing.

[0211] In one embodiment, gRNA can promote epigenetic editing of target.Epigenetic editing can be measured by using methods known in the art.For example, as described in Example 2, the level of expression of reporter gene can be measured as a model of epigenetic editing.

[0212] In one embodiment, gRNA can promote targeted gene editing.Gene editing can be measured by using methods known in the art.For example, as described in Example 1, the level of non-homologous end joining can be measured as a model of gene editing.

[0213] Exemplary sequences of genomic target sites (i.e., protospacer and PAM) recognized by gRNAs for use in targeting the β2-microglobulin (B2M) gene include: [ka] The underlined nucleotides are the PAM.

[0214] In one aspect, the present invention provides a gRNA targeting the β2-microglobulin gene region described in SEQ ID NO:21 or SEQ ID NO:22 above.

[0215] Examples of spacer sequences used in gRNAs targeting the β2-microglobulin gene, particularly the target sites described above, include: [ka] [ka] - This spacer sequence is incorporated into the gRNA and is used for gene editing of B2M when used in combination with ETM as shown in Example 2. [ka] Includes:

[0216] In some embodiments, the spacer sequence comprises a "G" nucleotide at the 5' end, which may not be part of the target sequence, for example, and may be necessary when the promoter driving its expression is a U6 promoter.

[0217] For example, the "G" at the 5' end of SEQ ID NO:23 is used herein to drive expression from the U6 promoter, i.e., it will be understood that if the spacer sequence in SEQ ID NO:23 is not driven by the U6 promoter, the "G" at the 5' end may not be necessary.

[0218] In some aspects, a spacer sequence according to the invention comprises a "G" nucleotide at the 5' end.

[0219] Examples of gRNAs according to the present invention include: [ka] which contain the spacer sequence, SEQ ID NO:24 (underlined above).

[0220] Alternative gRNAs for episilencing of B2M can be found, for example, in Amabile et al., supra.

[0221] For example, alternative spacer sequences for use in the gRNAs according to the present invention include: [ka] There is.

[0222] Examples of gRNAs according to the present invention include: [ka] which contain the spacer sequence, SEQ ID NO:35 (underlined above).

[0223] Truncated spacer sequences based on SEQ ID NO: 35 suitable for use in gRNAs according to the present invention include: [ka] Includes:

[0224] Another spacer sequence (H8) used in the gRNA according to the present invention is [ka] There is. Examples of gRNAs with this spacer (underlined) include: [ka] Truncated spacer sequences based on SEQ ID NO: 2780 suitable for use in a gRNA according to the present invention include: [ka] Includes:

[0225] Another spacer sequence (H10) used in the gRNA according to the present invention is [ka] There is. Examples of gRNAs with this spacer (underlined) include: [ka] [ka] There is. Truncated spacer sequences based on SEQ ID NO: 2863 suitable for use in a gRNA according to the present invention include: [ka] Includes:

[0226] Another spacer sequence (H11) used in the gRNA according to the present invention is [ka] Examples of gRNAs with this spacer (underlined) include: [ka] Truncated spacer sequences based on SEQ ID NO: 2778 suitable for use in a gRNA according to the present invention include: [ka] Includes:

[0227] Another spacer sequence (H12) used in the gRNA according to the present invention is [ka] Examples of gRNAs with this spacer (underlined) include: [ka] Truncated spacer sequences based on SEQ ID NO: 2801 suitable for use in a gRNA according to the present invention include: [ka] [ka] Includes:

[0228] Examples of spacer sequences for use in gRNAs targeting the TRAC gene include: [ka] Includes: Examples of gRNAs with this spacer (underlined) include: [ka] There is.

[0229] The present disclosure also provides variations of the above exemplified gRNAs in which the spacer sequence (underlined) is truncated, e.g., by 1-9 (e.g., 3-9) nucleotides at the 5' end. The present disclosure also provides gRNAs in which a spacer as described herein (full-length or truncated version) is linked to the above exemplified tracrRNA (a portion of the gRNA above, e.g., SEQ ID NOs: 4574 and 4575, which are not underlined).

[0230] In one aspect, the present invention provides a gRNA comprising a spacer sequence comprising or consisting of any one of the sequences set forth in SEQ ID NOs: 23 to 46, 562 to 1076, 2778 to 4478, and 4553 to 4565, or homologs thereof.

[0231] In one aspect, the present invention provides a gRNA comprising a spacer sequence comprising or consisting of a sequence described in any one of SEQ ID NOs: 23-46, 562-1076, 2778-4478, and 4553-4565, which has one or more (e.g., two, three, four, or five) conservative substitutions. A spacer sequence comprising one or more conservative substitutions retains substantially the same activity as a spacer sequence having a sequence described in any one of SEQ ID NOs: 23-46, 562-1076, 2778-4478, and 4553-4565.

[0232] In one aspect, the present invention provides a gRNA comprising a spacer sequence comprising or consisting of a sequence set forth in any one of SEQ ID NOs: 23 to 46, 562 to 1076, 2778 to 4478, and 4553 to 4565, or a fragment thereof.

[0233] Suitably, the spacer sequence comprises or consists of a sequence set forth in any one of SEQ ID NOs: 23 to 46, 562 to 1076, 2778 to 4478, and 4553 to 4565, and is 21 nucleotides or less in length (e.g. 20 nucleotides, such as 19 nucleotides, for example 18 nucleotides, for example 17 nucleotides, for example 16 nucleotides, for example 15 nucleotides, for example 14 nucleotides, for example 13 nucleotides, for example 12 nucleotides, for example 11 nucleotides, or for example 10 nucleotides).

[0234] In one embodiment, the spacer sequence may include a sequence described in any one of SEQ ID NOs: 23 to 46, 562 to 1076, 2778 to 4478, and 4553 to 4565, or a fragment thereof that includes or consists of a sequence having a length of 21 or less consecutive nucleotides (e.g., 20 consecutive nucleotides, 19 consecutive nucleotides, 18 consecutive nucleotides, 17 consecutive nucleotides, 16 consecutive nucleotides, 15 consecutive nucleotides, 14 consecutive nucleotides, 13 consecutive nucleotides, 12 consecutive nucleotides, 11 consecutive nucleotides, or 10 consecutive nucleotides). The fragment may be, for example, a shortening of SEQ ID NOs: 23 to 46, 562 to 1076, 2778 to 4478, and 4553 to 4565 from the 5' end (i.e., the 5' end nucleotide has been removed).

[0235] In some embodiments, gRNA can be chemically modified. For example, chemical modifications can increase the stability of gRNA when administered to target cells, for example, as described in (Yin et al., Nat Biotechnol. 2017 Dec;35(12):1179-1187). Such chemical modifications are known in the literature and include, but are not limited to, locked nucleic acids (LNA), phosphorothioate modified oligonucleotides, 2'-O-methoxyethyl modified oligonucleotides, and 2' O-methyl modified oligonucleotides.

[0236] In some aspects, the first three nucleosides and the last three nucleosides of a gRNA are 2'-O-methyl modified nucleosides regardless of the length of the gRNA. In some aspects, the first three internucleoside linkages and the last three internucleoside linkages of a gRNA are phosphorothioate linkages regardless of the length of the gRNA.

[0237] For a gRNA sequence having a tracr RNA of SEQ ID NO: 4567 (which is 80 nucleotides in length), the tracr sequence portion of the full-length gRNA can be modified as follows (with nucleoside 1 at the 5' end of the tracr RNA sequence and nucleoside 80 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, and Nucleosides 49-80: 2'-O-Me modified nucleosides. 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 to the 5' end of this modified tracr sequence to form a full-length gRNA in which 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; The first three internucleoside linkages are phosphorothioate linkages. A general outline of this full-length gRNA is shown below, where lowercase letters represent 2'-O-Me nucleosides, uppercase letters represent unmodified RNA nucleosides, s represents a phosphorothioate linkage, each X independently represents an A, C, G, or U nucleoside, and each x represents a 2'-O-Me A, C, G, or U nucleoside. [ka]

[0238] More specifically, for a gRNA sequence having a full length spacer RNA (i.e., 20 nucleotides) and the tracr RNA of SEQ ID NO:4567 (which is 80 nucleotides in length and the gRNA is 100 nucleotides in length), the gRNA is modified as follows (with nucleoside 1 as the 5' end of the oligonucleotide and nucleotide 100 as the 3' end of the oligonucleotide): Nucleosides 1-3: 2'-O-Me modified nucleosides, Nucleosides 4 to 28: unmodified RNA nucleosides, Nucleosides 29-40: 2'-O-Me modified nucleosides, Nucleosides 41-68: Unmodified RNA nucleosides, and Nucleosides 79–100: 2′-O-Me modified nucleosides. In such modified gRNAs, 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) can be phosphorothioate linkages. The remainder of the internucleoside linkages are phosphate linkages.

[0239] Similar modifications may be made to a truncated gRNA (e.g., a gRNA with a spacer of 11-19 nucleotides). For example, the first three and 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.

[0240] For example, the sequence of SEQ ID NO: 4568 can be modified as follows: [ka] where N: RNA nucleoside, n: 2'-O-methyl nucleoside, s: phosphorothioate backbone modification between the two nucleosides.

[0241] Another example is modification of the sequence of SEQ ID NO:4569. [ka]

[0242] An exemplary full-length modified gRNA targeting B2M is shown below. [ka] [ka]

[0243] Exemplary truncated modified gRNAs targeting B2M are shown below. [ka]

[0244] An exemplary full-length modified gRNA targeting TRAC is shown below. [ka]

[0245] Exemplary truncated modified gRNAs targeting TET2 are shown below. [ka]

[0246] Exemplary truncated modified gRNAs targeting TGFBR2 are shown below. [ka]

[0247] An exemplary full-length modified gRNA targeting TET2 is shown below. [ka] [ka]

[0248] An exemplary full-length modified gRNA targeting TGFBR2 is shown below. [ka] [ka]

[0249] An exemplary full-length modified gRNA targeting GFP is shown below. [ka]

[0250] In some embodiments, the present invention utilizes two or more gRNAs.

[0251] Preferably, the two or more gRNAs can make ETM (e.g., ETR) target different target genes.Preferably, the two or more gRNAs can comprise spacer sequences of different lengths.For example, spacer sequences of different lengths can make ETM (e.g., ETR) endonuclease target different target genes.

[0252] In some embodiments, two or more gRNAs may target the same target gene. For example, for optimal epigenetic modification, such as epigenetic silencing, it may be beneficial to target the same gene by two gRNAs.

[0253] In one embodiment, at least one of the at least two gRNAs comprises a spacer sequence 18, 19, or 20 nucleotides in length.

[0254] In one embodiment, at least one of the at least two gRNAs comprises a spacer sequence that is 17 nucleotides or less in length, such as 16 nucleotides in length, such as 15 nucleotides in length, such as 14 nucleotides in length, such as 13 nucleotides in length, such as 12 nucleotides in length, such as 11 nucleotides in length, or such as 10 nucleotides in length.

[0255] Multiplexing - modifying multiple genes in the same cell The present invention relates to the development of a combination of gene editing and epigenetic editing strategies to modify the expression and / or activity of multiple target genes in the same cell. In particular, the present invention may utilize ETMs (e.g., ETRs) that contain epigenetic effector domains and endonucleases, and gRNAs that contain spacer sequences of different lengths to facilitate epigenetic editing of one or more genes and gene editing of another gene.

[0256] As used herein, "modifying expression and / or activity" means increasing or decreasing (eg, decreasing) the expression and / or activity of a target gene.

[0257] In one aspect, the transcription and / or expression of a target gene may be inhibited.

[0258] In one aspect, a target gene can be silenced.

[0259] In one aspect, the target gene can be enhanced. In other words, the expression of the target gene can be increased. For example, the expression of an endogenous target gene can be increased.

[0260] In another example, an endogenous target (e.g., a gene) can be modified (e.g., mutated) by gene editing to increase expression of the modified target (e.g., gene).

[0261] The effect of an ETM or combination of ETMs can be studied by comparing the transcription or expression of a target gene, e.g., a gene endogenous to a cell, in the presence and absence of an ETM or combination of ETMs. Methods for analyzing gene transcription or expression are known in the art.

[0262] The effect of the ETM or combination of ETMs and gRNAs may be studied using a model system that monitors the expression of a reporter gene, such as a gene encoding a fluorescent protein. Suitable methods for monitoring the expression of such reporter genes include flow cytometry, fluorescence-activated cell sorting (FACS), and fluorescence microscopy.

[0263] For example, a vector carrying a reporter gene may be transfected into a population of cells. The vector may be constructed such that the reporter gene is expressed when the vector is transfected into bacteria. Suitable reporter genes include genes encoding fluorescent proteins, such as green, yellow, cherry, cyan, or orange fluorescent proteins. Additionally, the population of cells may be transfected with a vector encoding an ETM of interest and / or a gRNA. The number of cells expressing and not expressing the reporter gene, as well as the level of expression of the reporter gene, may then be quantified using a suitable technique, such as FACS. The level of expression of the reporter gene may then be compared in the presence and absence of the ETM and / or gRNA.

[0264] Methods for determining the transcription of genes, such as the target of ETM, are known in the art. Suitable methods include reverse transcription PCR and Northern blot-based approaches. In addition to methods for determining the transcription of genes, methods for determining the expression of genes are known in the art. Suitable further methods include Western blot-based or flow cytometry approaches.

[0265] Target gene transcription and expression In some aspects, the products (e.g., ETMs and / or gRNAs) according to the invention are used in methods to suppress the transcription and / or expression of at least one target gene. Suitably, the target gene may be an endogenous gene.

[0266] In one aspect, the transcription and / or expression of the target gene is suppressed by epigenetic editing. In one aspect, the transcription and / or expression of the target gene is suppressed by gene editing.

[0267] In some aspects, the products according to the invention (e.g., ETMs and / or gRNAs) are used in a method to suppress the transcription and / or expression of at least two target genes. Suitably, at least one or both of the target genes may be endogenous genes.

[0268] In one embodiment, the transcription and / or expression of a single gene is silenced by gene editing.

[0269] Following administration of an ETM (e.g., an ETR) of the invention (together with a suitable gRNA), the level of transcription or expression of the target gene may be reduced, for example, by 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., an ETR).

[0270] In some embodiments, the product (e.g., ETM and / or gRNA) according to the present invention is used in a method for silencing at least one target gene.Preferably, the target gene may be an endogenous gene.Preferably, the target gene may be an exogenous gene, such as a viral gene.

[0271] In one aspect, the target gene is silenced by epigenetic editing. In one aspect, the target gene is silenced by gene editing.

[0272] In some aspects, the products (e.g., ETMs and / or gRNAs) according to the invention are used in a method of silencing at least two target genes. Suitably, at least one or both of the target genes may be endogenous genes.

[0273] In one embodiment, a single gene is silenced by gene editing.

[0274] Without wishing to be bound by theory, restricting gene editing activity to a single gene may reduce the likelihood of unwanted genomic translocations.

[0275] By "silencing a target gene", it is understood that expression of the target gene is reduced to a sufficient degree to achieve a desired effect. The reduction in expression may be sufficient to achieve a therapeutically relevant effect, such as prevention or treatment of a disease. For example, a dysfunctional target gene that leads to a disease may be suppressed to the point where expression of the target gene is eliminated or the remaining level of expression of the target gene is low enough to ameliorate or prevent the disease state. Furthermore, the reduction in expression allows the purification of cells with gene silencing.

[0276] The reduction in expression may be sufficient to allow investigation of the function of the gene to be carried out by studying cells in which that function is reduced or absent.

[0277] Repression of a target gene can occur, for example, following transient delivery or expression of an ETM (eg, an ETR) of the invention (eg, with a suitable gRNA) to or in a cell.

[0278] Target gene enhancement It should be understood that "enhancing a target gene" means that the expression of the target gene is increased to a sufficient degree 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 that leads to a disease may be increased until there is sufficient expression of the target gene to ameliorate or prevent the disease state. Alternatively, the increased expression of the target gene may compensate for the dysfunctional activity of a disease-related gene. Furthermore, the increased expression of a target gene allows for the selection of cells that express that particular target gene de novo.

[0279] Following administration of an ETM of the invention (e.g., with a suitable gRNA), the level of transcription or expression of the target gene may be increased, for example, by at least 50%, 60%, 70%, 80%, 90%, 95%, 99%, 100%, 200%, 300%, 400%, or 500% compared to the level of transcription or expression in the absence of the ETM.

[0280] Enhancement of a target gene can occur, for example, following transient delivery or expression of an ETM of the invention (eg, with a suitable gRNA) to or in a cell.

[0281] Transient expression "Transient expression" is understood to mean that expression of the ETM (e.g., ETR) is not stable over time. For example, the polynucleotide encoding the ETM (e.g., ETR) may not be integrated into the host genome. More specifically, transient expression can be expression that is substantially lost within 20 weeks after introduction of the polynucleotide encoding the ETM (e.g., ETR) into a cell. For example, expression can be substantially lost within 12, 6, 4, or 2 weeks after introduction of the polynucleotide encoding the ETM (e.g., ETR) into a cell.

[0282] Similarly, "transient delivery" should be understood to mean that the ETM (e.g., ETR) does not substantially remain in the cell (i.e., is substantially lost by the cell) over an extended period of time. More specifically, transient delivery can 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) can be substantially lost within 12, 6, 4, or 2 weeks following introduction of the ETM (e.g., ETR) into the cell.

[0283] In one embodiment, the ETM and / or gRNA may be delivered transiently. Transient delivery may result in, for example, a permanent change. Transient delivery of the ETM and / or gRNA may result in DNA methylation of the repressive regulatory element, which may then result in gene activation (e.g., given this epigenetic modification, permanent gene activation).

[0284] The target gene may be, for example, permanently suppressed, silenced, or enhanced. It is to be understood that the "permanent suppression", "permanent silencing", or "permanent enhancement" of a target gene means that the transcription or expression of the target gene is reduced or increased (e.g., at least 60%, at least 70%, at least 80%, at least 90%, or 100% reduced or increased) for at least 2 months, 6 months, 1 year, 2 years, or the entire life of the cell / organism, compared to the level of transcription or expression in the absence of the ETM (e.g., ETR). For example, a permanently suppressed, silenced, or enhanced target gene may remain suppressed, silenced, or enhanced for the remainder of the cell's life.

[0285] In one embodiment, the ETM and / or gRNA are stably expressed. For example, stable expression may be necessary to achieve permanent gene activation of some targets. The target gene may remain suppressed, silenced, or enhanced, for example, in the descendants of the cells to which the product of the invention is administered (i.e., the suppression, silencing, or enhancement of the target gene is inherited by the descendants of the cells). For example, the ETM (e.g., ETR) and gRNA of the invention may be administered to stem cells (e.g., hematopoietic stem cells) to suppress or silence the target gene in the descendants of the stem cells, which may include stem cells and cells differentiated from the stem cells.

[0286] Target Gene The target gene may, for example, produce a therapeutic effect when altered, e.g., repressed or silenced.

[0287] The products of the invention may be used to modify, e.g., suppress or silence, genes that do not have CpG islands (CGIs), including TRAC, TRBC, PDCD1, TIM-3, TIGIT, LAG3, CTLA4, AAVS1, and CCR5.

[0288] For example, a target gene, e.g., a gene that does not have a CGI, can produce allogeneic products (e.g., by targeting TRAC and / or TRABC); altering resistance to the immunosuppressive tumor microenvironment (e.g., by targeting PDCD1, TIM-3, TIGIT, LAG3, and / or CTLA4); and / or It may allow integration of the CAR / transgenic TCR at a safe site (e.g., by targeting AAVS1 and / or CCR5).

[0289] In one aspect, the present invention provides a gRNA targeting a sequence set forth in any one of SEQ ID NOs: 47 to 561.

[0290] By way of example, target genes without CGI islands and exemplary gRNAs suitable for targeting said genes are provided in Table 1 below (SEQ: sequence number).

[0291] [Table 1-1] [Table 1-2] [Table 1-3] [Table 1-4] [Table 1-5] [Table 1-6] [Table 1-7] [Table 1-8] [Table 1-9] [Table 1-10] [Table 1-11] [Table 1-12]

[0292] The products of the invention may be used to modify, e.g., suppress or silence, genes with CpG islands (CGIs), including 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.

[0293] For example, a target gene, e.g., a gene having a CGI, can produce allogeneic products (e.g., by targeting B2M and / or HLA-A); Altering resistance to the immunosuppressive tumor microenvironment (e.g., by targeting TGFBR2, A2AR, PTPN11, PTPN6, PTPN2, and / or DGKA); May allow integration of CAR / transgenic TCR at a safe site (e.g., by targeting AAVS1 and / or CCR5); may provide resistance to attrition (e.g., by targeting FAS, CISH, PTPA, PIK3CD, MAP4K1, NR4A1, NR4A2, NR4A3, JUNB, REL, TOX, TOX2, IRF4, and / or TET2); and / or T cell senescence may be delayed (eg, by targeting USP16).

[0294] Silencing of the TCR genes, namely PDCD1 and CTLA4, may be used to improve the efficacy of cancer immunotherapy approaches.

[0295] Silencing of B2M may be used to generate allogeneic HSPCs, T cells, or mesenchymal cells for use in transplantation.

[0296] In one aspect, the present invention provides a gRNA targeting a sequence set forth in any one of SEQ ID NOs: 1077 to 2777.

[0297] By way of example, target genes with CGI islands and exemplary gRNAs suitable for targeting said genes are provided in Table 2 below (SEQ: sequence number).

[0298] [Table 2-1] [Table 2-2] [Table 2-3] [Table 2-4] [Table 2-5] [Table 2-6] [Table 2-7] [Table 2-8] [Table 2-9] [Table 2-10] [Table 2-11] [Table 2-12] [Table 2-13] [Table 2-14] [Table 2-15]

Table 2-16

Table 2-17

Table 2-18

Table 2-19

Table 2-20

Table 2-21

Table 2-22

Table 2-23

Table 2-24

Table 2-25

Table 2-26

Table 2-27

Table 2-28

Table 2-29

Table 2-30

Table 2-31

Table 2-32

[0299] It will be appreciated that it may be beneficial to increase the expression of certain targets, for example c-jun is a beneficial gene when activated and increasing expression in, for example, T cells may increase cell survival.

[0300] cell In one aspect, the present invention provides a cell comprising an ETM (e.g. an 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.

[0301] The cell may be any cell that can be used to express the product of the invention.

[0302] The cell may be an immune effector cell. An "immune effector cell" is a cell that has differentiated into a form capable of modulating or effecting a particular 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. Preferably, the cell may be an alpha / beta T cell. Preferably, the cell may be a B cell. Preferably, the cell may be a gamma / delta T cell. Preferably, the cell may be a T cell, such as a cytotoxic T cell, such as a CD8+ T cell. Preferably, the cell may be a NK cell, such as a cytotoxic NK cell. Preferably, the cell may be a macrophage.

[0303] In one aspect, the cell may be a stem cell. "Stem cell" refers to an undifferentiated cell that can give rise indefinitely to more stem cells of the same type, from which other specialized cells arise by differentiation. Adult stem cells are usually pluripotent, whereas induced or embryo-derived stem cells are totipotent.

[0304] In another embodiment, the cell can be a progenitor cell, which refers to a cell that can differentiate to form one or more cell types, but has limited self-renewal in vitro and in vivo.

[0305] Suitably, the cells are capable of differentiating into T cells. Suitably, the cells are capable of differentiating into NK cells. Suitably, the cells are capable of differentiating into macrophages. Suitably, the cells may be embryonic stem cells (ESCs). Suitably, the cells may be hematopoietic stem cells or hematopoietic progenitor cells. Suitably, the cells may be induced pluripotent stem cells (iPSCs). Suitably, the cells may be obtained from umbilical cord blood. Suitably, the cells may be obtained from adult peripheral blood or mobilized from bone marrow.

[0306] "Hematopoietic stem and progenitor cells" or "HSPCs" refer to cells that express the antigenic marker CD34 (CD34+) and populations of such cells. In certain embodiments, the term "HSPCs" refers to cells that are identified by the presence of the antigenic marker CD34 (CD34+) and the absence of lineage (lin) markers. Populations of cells that include CD34+ and / or Lin(-) cells include hematopoietic stem and progenitor cells.

[0307] HSPCs can be obtained or isolated from adult bone marrow, including femur, hip bone, rib, sternum, and other bones. Bone marrow aspirate containing HSPCs can be obtained or isolated directly from hip bone using 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 certain embodiments, in order to harvest sufficient amounts of HSPCs for use in therapeutic applications, it is necessary to mobilize stem and progenitor cells in a subject.

[0308] As used herein, the term "induced pluripotent stem cells" or "iPSCs" refers to non-pluripotent cells that have been reprogrammed to a pluripotent state. Once a subject's cells have been reprogrammed to a pluripotent state, the cells can be programmed into a desired cell type, such as a hematopoietic stem cell or hematopoietic progenitor cell (HSC and HPC, respectively).

[0309] As used herein, the term "reprogramming" refers to a method of increasing the potential of a cell towards a less differentiated state, and "programming" refers to a method of decreasing the potential of a cell or differentiating a cell towards a more differentiated state.

[0310] Suitably, the cells may be matched to the subject or may be autologous to the subject. The cells may be generated ex vivo from the patient's own peripheral blood, or from a donor's peripheral blood.

[0311] Suitably, the cells may be autologous to the subject, hi some aspects, the cells may be derived from ex vivo differentiation of inducible progenitor cells or embryonic progenitor cells into immune cells.

[0312] In these examples, the cells are generated by introducing DNA or RNA encoding the ETM (e.g., ETR) of the invention by any one of a number of means, including transduction with a viral vector or transfection with DNA or RNA.

[0313] In some aspects, the cells are i) an agent that promotes the survival, proliferation, and / or activity of a cell, e.g., a cell that contains a polynucleotide or a cell that does not contain a polynucleotide; and / or ii) an agent that is detrimental to the survival, proliferation, activity, chemoresistance, and / or chemotaxis of a cell, e.g., a cell containing the polynucleotide or a cell not containing the polynucleotide; and / or iii) An agent that allows for the selection of cells, e.g., cells that contain the polynucleotide or cells that do not contain the polynucleotide. The present invention further includes a polynucleotide encoding the polypeptide, e.g., an integrating vector.

[0314] combination In one aspect, the invention provides a combination (e.g., a system) comprising an ETM (e.g., ETR) according to the invention and at least one gRNA that targets the endonuclease of the ETM (e.g., ETR) to a target gene.

[0315] The combination may include at least two gRNAs (e.g., at least three, at least four, at least five, at least six, at least seven, or at least eight gRNAs).

[0316] The combination may include gRNAs that target the endonuclease to at least two different target genes.

[0317] In some embodiments, one target gene may be targeted by more than one gRNA. For example, for optimal epigenetic modification, such as epigenetic silencing, it may be beneficial to target the same gene with several gRNAs.

[0318] The combination may include at least two gRNAs that include spacer sequences of different lengths. Preferably, at least one gRNA includes a spacer sequence that is 15, 16, 17, 18, 19, or 20 nucleotides in length. Preferably, at least one of the at least two gRNAs includes a spacer sequence that is 17 nucleotides or less in length (e.g., 16 nucleotides or less in length). Preferably, at least one of the at least two gRNAs includes a spacer sequence that is 17 nucleotides or less in length (e.g., 16 nucleotides or less in length) and at least one of the at least two gRNAs includes a spacer sequence that is more than 17 nucleotides in length.

[0319] Without wishing to be bound by theory, gRNAs containing spacer sequences of different lengths may cause the ETM (e.g., ETR) to target different target genes, with a first target gene being modified by gene editing and at least a second target gene being modified by epigenetic editing.

[0320] In one embodiment, 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.

[0321] Suitably, the combination may comprise a first gRNA and a second gRNA having the sequences of C8 and F4, respectively, and optionally, the combination further comprises a third gRNA having the sequence of H8, H10, H11, or H12.

[0322] Suitably, the combination may comprise a first gRNA and a second gRNA having the sequences of C8 and H8, respectively, and optionally, the combination further comprises a third gRNA having the sequences of F4, H10, H11, or H12.

[0323] Suitably, the combination may comprise a first gRNA and a second gRNA having the sequences of C8 and H10, respectively, and optionally, the combination further comprises a third gRNA having the sequences of F4, H8, H11, or H12.

[0324] Suitably, the combination may comprise a first gRNA and a second gRNA having the sequences of C8 and H11, respectively, and optionally, the combination further comprises a third gRNA having the sequences of F4, H8, H10, or H12.

[0325] Suitably, the combination may comprise a first gRNA and a second gRNA having the sequences of C8 and H12, respectively, and optionally, the combination further comprises a third gRNA having the sequences of F4, H8, H10, or H11.

[0326] Suitably, the combination may comprise a first gRNA and a second gRNA having the sequences of F4 and H8, respectively, and optionally, the combination further comprises a third gRNA having the sequences of C8, H10, H11, or H12.

[0327] Suitably, the combination may comprise a first gRNA and a second gRNA having the sequences of F4 and H10, respectively, and optionally, the combination further comprises a third gRNA having the sequences of C8, H8, H11, or H12.

[0328] Suitably, the combination may comprise a first gRNA and a second gRNA having the sequences of F4 and H11, respectively, and optionally, the combination further comprises a third gRNA having the sequences of C8, H8, H10, or H12.

[0329] Suitably, the combination may comprise a first gRNA and a second gRNA having the sequences of F4 and H12, respectively, and optionally, the combination further comprises a third gRNA having the sequences of C8, H8, H10, or H11.

[0330] Suitably, the combination may comprise a first gRNA and a second gRNA having the sequences of H8 and H10, respectively, and optionally, the combination further comprises a third gRNA having the sequences of C8, F4, H11, or H12.

[0331] Suitably, the combination may comprise a first gRNA and a second gRNA having the sequences of H10 and H11, respectively, and optionally, the combination further comprises a third gRNA having the sequences of C8, F4, H8, or H12.

[0332] Suitably, the combination may comprise a first gRNA and a second gRNA having the sequences of H10 and H12, respectively, and optionally, the combination further comprises a third gRNA having the sequences of C8, F4, H8, or H11.

[0333] Suitably, the combination may comprise a first gRNA and a second gRNA having the sequences of H11 and H12, respectively, and optionally, the combination further comprises a third gRNA having the sequences of C8, F4, H8, or H10.

[0334] A combination may have a gRNA that comprises or consists of, for example, H8+F4, H8+H10, C8+H10, F4+H10, F4+H8+H10, or C8+F4+H10. In certain cases, the gRNA may comprise or consist of F4+H8+H10.

[0335] In one aspect, the combination is i) an agent that promotes the survival, proliferation, and / or activity of a cell, e.g., a cell with or without the combination; and / or ii) an agent that is detrimental to the survival, proliferation, activity, chemoresistance, and / or chemotaxis of a cell, e.g., a cell containing the combination or a cell not containing the combination; and / or iii) Agents that allow for the selection of cells, e.g., cells that contain the combination or cells that do not contain the combination. Further includes:

[0336] The combination may further include an agent that modifies the tissue microenvironment.

[0337] The agent may be a protein, such as a cytokine or chemokine, that promotes the survival, proliferation, and / or activity of cells according to the invention.

[0338] As used herein, an "agent that promotes cell survival, proliferation, and / or activity" means that in the presence of the agent, survival, proliferation, and / or activity of a cell, including a product according to the invention, is increased.

[0339] An agent may, for example, be beneficial to some cells and detrimental to other cells.

[0340] The agent may play a role in homeostasis, for example blood clotting. An example of a suitable agent may be clotting factor IX or FVIII.

[0341] The agent may, for example, allow for the selection of the cells. An example of a suitable agent is delta low affinity nerve growth factor (LNGFR).

[0342] The agent may, for example, be detrimental to the cell. The agent may be a thymidine kinase (TK) or a caspase, such as CASP9. Activation of these agents may be used to eliminate cells containing the agent in vivo, for example when it is desired to eliminate engineered T cells from a subject.

[0343] Suitably, in the presence of the agent, the survival, proliferation and / or activity of a cell comprising a product according to the invention (e.g. a cell according to the 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%.

[0344] The combination may include agents that are detrimental to the survival, proliferation, activity, chemoresistance, and / or chemotaxis of cells, such as tumor cells.

[0345] As used herein, an "agent harmful to" means that in the presence of the agent, the survival, growth or activity of cells (e.g., tumor cells) that do not contain the product according to the invention is impaired, reduced or completely abolished.

[0346] Suitably, in the presence of a deleterious agent, the survival, proliferation or activity of cells (e.g. tumour cells) that do not contain a product according to the invention 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%.

[0347] 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 tumor-specific marker or a marker specific for the engineered cells, such as a CAR or transgenic TCR), performing cell cycle analysis using 5-bromo-2'-deoxyuridine (BrdU) and / or propidium iodide (PI) incorporated into newly generated DNA and analyzed by flow cytometry in combination with a cell population specific marker, and / or measuring the number of viable cells by measuring apoptosis, for example, by 7AAD and / or Annexin V staining using flow cytometry.

[0348] In one embodiment, the combination further comprises a CAR. In one embodiment, the combination further comprises a transgenic CAR.

[0349] Agents that promote the survival, proliferation, and / or activity of a cell (or population of cells), e.g., a cell (or population of cells) expressing an ETM (e.g., ETR), or allow the selection of cells, and / or agents that are detrimental to the survival, proliferation, activity, chemoresistance, and / or chemotaxis of cells that do not express an ETM (e.g., ETR), may be introduced into the genome of a cell by any method, which may include, for example, using an integrating vector (a procedure independent of the multiplexing strategy implemented by the ETM (e.g., ETR) according to the invention), or targeting an agent (e.g., a CAR or transgenic TCR) in a site recognized by a nuclease (a procedure that relies on the nuclease activity of the ETM (e.g., ETR) according to the invention).

[0350] That is, in some aspects, the combination further comprises a polynucleotide, e.g., an integration vector, that encodes an agent that allows selection of or promotes the survival, proliferation, and / or activity of a cell (or population of cells), e.g., a cell (or population of cells) that comprises the polynucleotide, and / or an agent that is detrimental to the survival, proliferation, activity, chemoresistance, and / or chemotaxis of cells that do not comprise the polynucleotide, and / or an agent that is beneficial to the survival, proliferation, and / or activity of a cell, tissue, or organ, e.g., a cell, tissue, or organ that does not comprise the combination.

[0351] Polynucleotides In one aspect, the invention provides a polynucleotide encoding at least one ETM (eg, an ETR) according to the invention.

[0352] The polynucleotides of the present invention may comprise DNA or RNA. They may be single-stranded or double-stranded. As a result of the degeneracy of the genetic code, it will be understood by those skilled in the art that many different polynucleotides can code for the same polypeptide. Furthermore, it will be understood by those skilled in the art that nucleotide substitutions can be made using routine techniques to reflect the codon usage of any particular host organism in which the polypeptide of the present invention is expressed without affecting the sequence of the polypeptide encoded by the polynucleotide of the present invention.

[0353] 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 life span of polynucleotides of the invention.

[0354] Polynucleotides, such as DNA polynucleotides, may be produced recombinantly, synthetically, or by any means available to those of skill in the art. They may be cloned by standard techniques.

[0355] Long polynucleotides are generally produced using recombinant means, e.g., using PCR cloning techniques. This involves creating a pair of primers (e.g., of about 15-30 nucleotides) that flank the target sequence one wishes to clone, contacting the primers with mRNA or cDNA from an animal or human cell, performing the polymerase chain reaction under conditions that result in amplification of the desired region, isolating the amplified fragment (e.g., by purifying the reaction mixture on 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.

[0356] Constructs In one aspect, the invention provides a nucleic acid construct comprising a nucleic acid sequence encoding at least one ETM (eg, ETR) according to the invention.

[0357] The nucleic acid construct is i) an agent that promotes the survival, proliferation, and / or activity of a cell, e.g., a cell expressing the nucleic acid construct or a cell not expressing the nucleic acid construct; and / or ii) an agent that is detrimental to the survival, proliferation, activity, chemoresistance, and / or chemotaxis of a cell, e.g., a cell expressing the nucleic acid construct or a cell not expressing the nucleic acid construct; and / or iii) Agents that allow for the selection of cells, e.g., cells that contain the nucleic acid construct or cells that do not contain the construct. It may further comprise a nucleic acid sequence encoding:

[0358] protein As used herein, the term "protein" includes single-chain polypeptide molecules as well as multi-polypeptide complexes in which individual component 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 linked by peptide or disulfide bonds.

[0359] Variants, Derivatives, Analogs, Homologs, and Fragments In addition to the specific proteins and nucleotides mentioned herein, the present invention also encompasses the use of variants, derivatives, analogs, homologs, and fragments thereof.

[0360] In the context of the present invention, a variant of any given sequence is a sequence in which a 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 alteration of at least one residue present in the naturally occurring protein.

[0361] The term "derivative" as used herein with respect to a protein or polypeptide of the invention includes any substitution, variation, modification, replacement, deletion, and / or addition of one (or more) amino acid residues from or to the sequence, provided that the resulting protein or polypeptide substantially retains at least one of its endogenous functions.

[0362] The term "analog" as used herein with respect to a polypeptide or polynucleotide includes any mimetic, i.e., a chemical compound that retains at least one of the endogenous functions of the polypeptide or polynucleotide that it mimics.

[0363] Typically, amino acid substitutions may be made, for example from 1, 2 or 3 to 10 or 20 substitutions, provided that the altered sequence substantially retains the required activity or ability. Amino acid substitutions may include the use of non-naturally occurring analogues.

[0364] The proteins used in the present invention may have deletions, insertions, or substitutions of amino acid residues that produce silent changes and result in functionally equivalent proteins. Deliberate amino acid substitutions may be made based on similarity in polarity, charge, solubility, hydrophobicity, hydrophilicity, and / or amphipathic nature of the residues, so long as the intrinsic 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 with similar hydrophilicity values ​​include asparagine, glutamine, serine, threonine, and tyrosine.

[0365] Conservative substitutions may be made, for example, according to the following table: Amino acids in the same block in the second column, and in a specific example, in the same line in the third column, may be substituted for each other.

[0366] [Table 3A]

[0367] The term "homolog" as used herein means an entity that has a certain homology with a wild-type amino acid sequence or a wild-type nucleotide sequence. The term "homology" may be equivalent to "identity".

[0368] A homologous sequence may comprise an amino acid sequence which may be at least 50%, 55%, 65%, 75%, 85% or 90% identical to the subject sequence, for example at least 95% or 97% or 99% identical. Typically, a homolog will comprise an identical active site etc. as the subject amino acid sequence. Although homology can also be considered in relation to 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 relation to sequence identity.

[0369] A homologous sequence may comprise a nucleotide sequence that is at least 50%, 55%, 65%, 75%, 85% or 90% identical to the subject sequence, for example at least 95% or 97% or 99% identical. Although homology can also be considered in relation to similarity, in the context of the present invention it is preferred to express homology in relation to sequence identity.

[0370] Reference to a sequence having a percent identity with any one of the SEQ ID NOs detailed herein may mean, for example, a sequence having the stated percent identity over the entire length of the referenced SEQ ID NO.

[0371] 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 the percent homology or identity between two or more sequences.

[0372] Percent homology may be calculated over a contiguous sequence, i.e., one sequence is aligned with the other sequence and each amino acid in one sequence is directly compared, one residue at a time, to the corresponding amino acid in the other sequence. This is called an "ungapped" alignment. Typically, such ungapped alignments are performed only over a relatively short number of residues.

[0373] Although this is a very simple and consistent method, it fails to take into account that, for example, in a pair of otherwise identical sequences, a single insertion or deletion in a nucleotide sequence may exclude subsequent codons from the alignment, thereby resulting in a large reduction in the percentage of homology when a global alignment is performed. As a result, most sequence comparison methods are designed to produce optimal alignments that take into account possible insertions and deletions without unduly penalizing the overall homology score. This is achieved by inserting "gaps" in the sequence alignment to try to maximize local homology.

[0374] However, these more complex methods assign a "gap penalty" 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 a high degree of relatedness between the two sequences being compared, will achieve a higher score than an alignment with many gaps. Typically, an "affine gap cost" is used, which imposes a relatively high cost for the presence 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 result in an optimized alignment with fewer gaps. In most alignment programs, it is possible to modify the gap penalties. However, it is preferable to use the default values ​​when using such software to compare sequences. For example, when using the GCG Wisconsin Bestfit package, the default gap penalty for amino acid sequences is -12 per gap and -4 for each extension.

[0375] Thus, the calculation of maximum percent homology first requires the production of an optimal alignment, taking into account gap penalties. A suitable computer program for performing such an alignment is the GCG Wisconsin Bestfit package (University of Wisconsin, USA; Devereux et al. (1984) Nucleic Acids Res. 12: 387). Examples of other software capable of performing sequence comparisons include, but are not limited to, the BLAST package (see Ausubel et al. (1999) supra - Ch. 18), FASTA (Atschul et al. (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 et al. (1999) supra, 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).

[0376] Although the final percentage of homology can be measured in terms of identity, the alignment process itself is typically not based on an all-or-nothing pairwise comparison. Instead, a scaled similarity score matrix is ​​commonly used that assigns a score 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 custom symbol comparison tables, if supplied (see user manual for further details). For some applications, it is preferable to use the public default values ​​for the GCG package, or in the case of other software, a default matrix such as BLOSUM62.

[0377] Once the software has produced an optimal alignment, it is possible to calculate the percentage of homology, e.g. percentage sequence identity. The software typically does this as part of the sequence comparison and generates a numerical result.

[0378] A "fragment" is also a variant, and the term typically refers to a selected region of a polypeptide or polynucleotide that is of interest functionally or, for example, in an assay, i.e., a "fragment" refers to an amino acid or nucleic acid sequence that is a portion of a full-length polypeptide or polynucleotide.

[0379] Such variants may be prepared using standard recombinant DNA techniques, such as site-directed mutagenesis. If an insertion is to be made, synthetic DNA encoding the insertion may be made with 5' and 3' flanking regions corresponding to the naturally occurring sequence on either side of the insertion site. The flanking regions will contain convenient restriction sites corresponding to sites in the naturally occurring sequence, so that the sequence can be cleaved with the appropriate enzyme and the synthetic DNA ligated to the cleavage site. The DNA is then expressed according to the invention to produce the encoded protein. These methods are merely illustrative of the many standard techniques known in the art for manipulating DNA sequences, and other known techniques may also be used.

[0380] Codon Optimization The polynucleotides used in the present invention may be codon-optimized. Codon optimization has already been described in WO1999 / 41397 and WO2001 / 79518. Different cells differ in their use of certain codons. This codon bias corresponds to the bias in the relative abundance of certain tRNAs in a cell type. Expression can be increased by changing the codons in the sequence and adjusting the codons to match the relative abundance of the corresponding tRNA. Similarly, expression can be decreased by deliberately selecting codons whose corresponding tRNAs are known to be rare in a particular cell type. That is, an additional degree of translational control is available.

[0381] vector In one aspect, the present invention provides a vector comprising a polynucleotide according to the invention or a nucleic acid construct according to the invention.

[0382] A vector is a tool that allows or facilitates the transfer of an entity from one environment to another. According to the present invention and by way of example, some vectors used in recombinant nucleic acid techniques allow the transfer of an entity, such as a segment of nucleic acid (e.g., a heterologous DNA segment, e.g., a heterologous cDNA segment), into a target cell. A vector may serve the purpose of maintaining a heterologous nucleic acid (DNA or RNA) in a cell, facilitating the replication of a vector containing a segment of nucleic acid, or facilitating the expression of a protein encoded by a segment of nucleic acid. A vector 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 mRNA), chromosomes, artificial chromosomes, and viruses. A vector may be, for example, a naked nucleic acid (e.g., DNA). In its simplest form, a vector may itself be the nucleotide of interest.

[0383] The vector used in the present invention may be for example a plasmid, mRNA or viral vector and may comprise a promoter for the expression of the polynucleotide and optionally a regulator of the promoter.

[0384] Vectors containing polynucleotides used in the present 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, such as infection with recombinant viral vectors, such as retroviruses, lentiviruses (e.g., integration defective lentiviruses), adenoviruses, adeno-associated viruses, baculoviruses, and herpes simplex virus vectors, direct injection of nucleic acids, and biolistic transformation.

[0385] Non-viral delivery systems include, but are not limited to, DNA or RNA transfection methods. Here, transfection includes the process of using non-viral vectors to deliver genes to target cells. Exemplary transfection methods include electroporation, DNA biolistics, lipid-mediated transfection, compacted DNA-mediated transfection, liposomes, immunoliposomes, lipofection, cationic agent-mediated transfection, cationic facial amphiphiles (CFAs) (Nat. Biotechnol. (1996) 14: 556), and combinations thereof.

[0386] The term "transfection" should be understood to encompass delivery of a polynucleotide into a cell by both viral and non-viral delivery.

[0387] Protein transduction As an alternative to delivery of a polynucleotide to a cell, the products and ETMs (eg ETRs) of the invention may be delivered to a cell by protein transduction.

[0388] Protein transduction may be via vector delivery (Cai, Y. et al. (2014) Elife 3: e01911; Maetzig, T. et al. (2012) Curr. Gene Ther. 12: 389-409). Vector delivery involves engineering a viral particle (e.g., a lentiviral particle) to contain the protein to be delivered to a cell. Thus, when the engineered viral particle enters a cell as part of its natural life cycle, the protein contained within the particle is brought into the cell.

[0389] Protein transduction may be via protein delivery (Gaj, T. et al. (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 the cells.

[0390] composition 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 invention may be provided in compositions.

[0391] The products of the invention, such as combinations, ETMs (e.g., ETRs), gRNAs, polynucleotides, nucleic acid constructs, vectors, compositions, and cells of the invention, may be formulated for administration to a subject with a pharma- ceutically acceptable carrier, diluent, or excipient. Suitable carriers and diluents include isotonic saline solutions, such as phosphate buffered saline, possibly including human serum albumin.

[0392] Handling of cell therapy products may be performed in accordance with the international standards for cell therapy of the Foundation for the Accreditation of Cellular Therapy and the Joint Accreditation Committee-International Society Cell & Gene Therapy (ISCT) and the European Society for Blood and Marrow Transplantation (EBMT) (FACT-JACIE).

[0393] In one aspect, a combination of a chemically modified mRNA encoding an ETM or ETR and a chemically modified gRNA is provided.

[0394] In another embodiment, a protein-RNA ribonucleic acid complex is provided that comprises an ETR protein bound to a chemically modified gRNA.

[0395] kit In one aspect, the present invention provides a method for producing a method for treating a cancer cell comprising: a) at least one polynucleotide encoding at least one ETM (e.g., ETR) according to the invention, and b) Polynucleotides providing at least one gRNA as described herein Including, in some cases c) i) an agent that promotes the survival, proliferation, and / or activity of a cell, e.g., a cell that contains the polynucleotide or a cell that does not contain the polynucleotide; and / or ii) an agent that is detrimental to the survival, proliferation, activity, chemoresistance, and / or chemotaxis of a cell, e.g., a cell that contains the polynucleotide or a cell that does not contain the polynucleotide; and / or iii) An agent that allows for the selection of cells, e.g., cells that contain the polynucleotide or cells that do not contain the polynucleotide. The present invention provides a kit of polynucleotides further comprising a nucleic acid sequence encoding

[0396] The kit may include instructions for use, such as instructions for simultaneous, sequential, or separate administration of at least one ETM (e.g., ETR) and at least two gRNAs to a subject in need thereof.

[0397] use In one 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. the use is an in vitro or ex vivo use.

[0398] Advantageously, the use can suppress (e.g., silence) the transcription and / or expression of at least one target gene. Advantageously, the use can suppress (e.g., silence) the transcription and / or expression of at least two target genes. For example, the transcription and / or expression of a first gene can be suppressed (e.g., silenced) by gene editing, and the transcription and / or expression of a second target gene can be suppressed (e.g., silenced) by epigenetic editing.

[0399] Suitably, the use may enhance at least one target gene.

[0400] In another aspect, the present invention provides a method for inhibiting (e.g. silencing) the transcription and / or expression of at least one target gene in a cell, comprising the step of administering to a cell 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.

[0401] Preferably, transcription and / or expression of at least two target genes is suppressed (e.g., silenced), at least one of the at least two target genes is epigenetically suppressed (e.g., silenced), at least one of the at least two target genes is suppressed (e.g., silenced) by gene editing, and at least one ETM (e.g., ETR) and at least two gRNAs are administered to the cell simultaneously, sequentially, or separately.

[0402] In another aspect, the invention provides products, ETMs (e.g., ETRs), gRNAs, combinations, polynucleotides, nucleic acid constructs, vectors, kits of polynucleotides, cells, and pharmaceutical compositions of the invention for use in therapy.

[0403] Therapeutic uses may be, for example, for the preparation of "universally" allogeneic transplantable cells (e.g., by silencing β2-microglobulin, B2M), which may be applied, for example, to the preparation of hematopoietic stem and / or progenitor cells (HSPCs), whole organ transplantation, and immunotherapy of cancer.

[0404] The ETM (e.g., ETR) (or a polynucleotide, nucleic acid construct, or vector encoding same) and gRNA may be administered simultaneously, in combination, sequentially, or separately (as part of a dosing regimen).

[0405] It is to be understood that "concurrently" is used to mean that two or more agents are administered at the same time, and the term "in combination" is used to mean that they are administered "sequentially" in a time frame in which they are otherwise available to act therapeutically in the same time frame. That is, "sequential" administration may allow one agent to be administered within 5 minutes, 10 minutes, or a matter of hours after the other, provided that the circulating half-life of the first administered agent is such that the two or more agents are present simultaneously in therapeutically effective amounts. The time delay between administration of the components will vary depending on the exact nature of the components, the interaction between them, and their respective half-lives.

[0406] "Individually," as opposed to "in combination" or "sequentially," should be understood to mean that there is a significant gap between the administration of one agent and the other, i.e., the agent administered first may no longer be present in the bloodstream in therapeutically effective amounts when the second agent is administered.

[0407] In another aspect, the invention provides a method of treating and / or preventing a disease or condition comprising administering any of the products of the invention (e.g., an ETM (e.g., an ETR), a gRNA, a combination, a polynucleotide, a nucleic acid construct, a vector, a kit of polynucleotides, a cell, or a pharmaceutical composition according to the invention) to a subject in need thereof.

[0408] Suitably, the ETM (e.g., ETR) and the gRNA may be administered to the subject simultaneously, sequentially, or separately.

[0409] In one aspect, the present invention provides a method for producing a method for treating a cancer cell comprising: (i) isolation of a sample comprising cells; (ii) introducing into a cell a polynucleotide according to the invention, a nucleic acid construct according to the invention, at least one gRNA according to the invention, an ETM (e.g. an ETR) according to the invention, a vector according to the invention, or a kit of polynucleotides according to the invention, and (iii) administering the cells of step (ii) to a subject. The present invention provides a method for gene therapy comprising the steps of:

[0410] The nucleic acid construct or vector may be introduced by transduction or transfection.

[0411] The cells can be, for example, autologous. The cells can be, for example, allogeneic.

[0412] All references herein to treatment include curative, palliative and prophylactic treatment, although it will be appreciated that in the context of the present invention references to prevention relate more generally to prophylactic treatment. Treatment of mammals, particularly humans, is preferred. Both human and veterinary treatments are within the scope of the present invention.

[0413] Diseases and Conditions By way of example, the products, ETMs (e.g., ETRs), polynucleotides, and cells of the invention may be used in the treatment of, for example, Huntington's disease, spinocerebellar ataxias, collagenopathies, hemoglobinopathies, and diseases caused by trinucleotide expansions. Additionally, the products of the invention may be used in the treatment or prevention of certain infectious diseases (e.g., CCR5-tropic HIV infections) by inactivating pathogen-associated gene products or host genes required for the pathogen life cycle.

[0414] Additionally or alternatively, the products, ETMs (e.g. ETRs), polynucleotides and cells of the invention are useful for the treatment of disorders listed in WO1998 / 005635, a partial list of which is provided here for ease of reference: cancer, inflammation or inflammatory diseases, skin disorders, fever, cardiovascular effects, bleeding, coagulation and acute phase response, cachexia, anorexia, acute infections, HIV infection, shock states, graft versus host reaction, autoimmune diseases, reperfusion injury, meningitis, migraine and aspirin-dependent antithrombosis, tumor growth, invasion and spread, angiogenesis, metastasis, malignancy, ascites and malignant pleural effusion, cerebral ischemia, ischemic 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 ulcers, retinopathy and surgical wound healing, rhinitis, allergic conjunctivitis, eczema, anaphylaxis, restenosis, congestive heart failure, endometriosis, atherosclerosis, or endosclerosis.

[0415] Additionally or alternatively, the products, ETMs (e.g. ETRs), polynucleotides and cells of the invention may be useful in the treatment of disorders listed in WO1998 / 007859, a partial list of which is provided here for ease of reference. For example in human or veterinary medicine, cytokine and cell proliferation / differentiation activity, immunosuppressive or immunostimulatory activity (e.g. to treat immune deficiencies including infection with the human immunodeficiency virus), control of lymphocyte proliferation, treatment of cancer and many autoimmune diseases, as well as prevention of transplant rejection and induction of tumor immunity), control of hematopoiesis, e.g. treatment of bone marrow or lymphatic diseases, promotion of growth of bone, cartilage, tendon, ligament and nerve tissue, e.g. wound healing, treatment of burns, ulcers, and periodontal disease and nerve regeneration, inhibition or activation of follicle stimulating hormone (regulation of conception), chemotropic / chemokinetic activity (e.g. to recruit specific cell types to the site of injury or infection), hemostatic and thrombolytic activity (e.g. to treat hemophilia and stroke), anti-inflammatory activity (e.g. to treat septic shock or Crohn's disease), as antimicrobial agents, e.g. metabolism or behavior regulators, as analgesics, treatment of certain deficiency disorders, e.g. treatment of psoriasis.

[0416] Additionally or alternatively, the products, ETMs (e.g. ETRs), polynucleotides and cells of the invention are useful for the treatment of disorders listed in WO1998 / 009985, a partial list of which is provided here for ease of reference: macrophage inhibitory and / or T cell inhibitory activity and therefore anti-inflammatory activity, anti-immune activity, i.e. inhibitory effects on cellular and / or humoral immune responses including responses not associated with inflammation, inhibition of the ability of macrophages and T cells to adhere to extracellular matrix components and fibronectin and upregulated expression of fas receptors in T cells, unwanted immune responses and inflammation including arthritis including rheumatoid arthritis, hypersensitivity, allergic reactions, asthma, systemic lupus erythematosus, collagenosis, and other autoimmune diseases, inflammation associated with atherosclerosis, arteriosclerosis, atherosclerotic heart disease, reperfusion injury, heart failure, myocardial infarction, vascular inflammatory disorders, respiratory distress syndrome, or other cardiopulmonary diseases, peptic ulcer, ulcerative colitis, and other diseases of the gastrointestinal tract, liver fibrosis, liver cirrhosis, and other liver diseases, thyroiditis or other glandular diseases, glomerulonephritis or other renal and urinary diseases, otitis or other ear, nose, and throat diseases, dermatitis or other skin diseases, periodontal disease or other dental diseases, orchitis or epididymitis, infertility, testicular trauma or other immune-related testicular diseases, placental insufficiency, habitual abortion, eclampsia, pre-eclampsia and other immune and / or inflammatory related gynecological diseases, posterior uveitis, intermediate uveitis, anterior uveitis, conjunctivitis, chorioretinitis, uveoretinitis, optic neuritis, intraocular inflammation, e.g. retinitis or cystoid macular oedema, sympathetic ophthalmia, scleritis, inflammation associated with retinitis pigmentosa, immune and inflammatory properties of degenerative fundus disease components, ocular inflammation caused by ocular trauma, infection, proliferative vitreoretinopathy, acute ischemic optic neuropathy, the inflammatory component of excessive scarring after glaucoma filtration surgery, inflammation associated with immune and / or inflammatory responses to ocular implants and other immune and inflammatory related ophthalmic diseases, inflammation associated with autoimmune diseases or conditions or disorders in the central nervous system (CNS) or any other organ where immune and / or inflammatory suppression would be beneficial, Parkinson's disease, complications and / or side effects from treatment of Parkinson's disease,AIDS-related dementia complex HIV-associated encephalopathy, Devic's disease, Sydenham's correa, Alzheimer's disease and other degenerative diseases, conditions or disorders of the CNS, inflammatory components of stroke, post-polio syndrome, immune and inflammatory components of psychiatric disorders, myelitis, encephalitis, subacute sclerosing panencephalitis, encephalomyelitis, acute neuritis, subacute neuritis, chronic neuritis, Guillain-Barre syndrome, Sydenham's correa, myasthenia gravis, pseudotumor cerebri, Down's syndrome, Huntington's disease, amyotrophic lateral sclerosis, inflammatory components of CNS compression or CNS trauma or infection of the CNS, inflammatory components of amyotrophic and dystrophic diseases, conditions, or disorders related to immunity and inflammation of the central and peripheral nervous system. , post-traumatic inflammation, septic shock, inflammatory diseases, surgery, inflammatory complications or side effects of bone marrow transplantation or other transplant complications and / or side effects, e.g. inflammatory and / or immune complications and side effects of gene therapy due to infection with a viral carrier, inhibition of inflammation associated with AIDS, suppression or inhibition of humoral and / or cellular immune responses, treatment or amelioration of monocyte or leukocyte proliferative disorders, e.g. leukemia, by reducing the amount of monocytes or lymphocytes, prevention and / or treatment of graft rejection in the case of transplantation of natural or synthetic cells, tissues and organs, e.g. cornea, bone marrow, organs, lenses, pacemakers, natural or artificial skin tissue,

[0417] For example, the present invention may be used to treat genetic diseases, such as β-hemoglobinopathies, by targeting hemoglobin F (HBF) or hemoglobin subunit beta (HBB), or to treat severe combined immunodeficiency (SCID), Wiskott-Aldrich syndrome protein (WASP), sickle cell disease (SCD), or adenosine deaminase deficiency (ADA).

[0418] Those skilled in the art will understand that they can combine any or all features of the invention disclosed herein without departing from the scope of the invention disclosed.

[0419] Further Aspects The invention also provides further aspects defined in the numbered paragraphs below. 1. An engineered transcriptional modulator (ETM) comprising (a) at least one epigenetic effector domain operably linked to (b) an endonuclease. 2. The ETM of paragraph 1, wherein 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. The ETM of paragraph 1 or paragraph 2, wherein at least one epigenetic effector domain is selected from the group consisting of DNMT1, DNMT3A, DNMT3B, DNMT3L, and SETDB1. 4. The ETM of any one of paragraphs 1 to 3, wherein the endonuclease comprises an RNA binding domain. 5. The ETM of any one of paragraphs 1 to 4, wherein the endonuclease is a Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) / Cas system. 6. The ETM of any one of paragraphs 1 to 5, wherein the endonuclease is a Cas9 endonuclease. 7. The ETM of any one of paragraphs 1 to 6, comprising or consisting of a fusion protein of Cas9-KRAB, Cas9-DNMT3A, or Cas9-DNMT3L. 8. The ETM of any one of paragraphs 1 to 7, which is a bipartite or tripartite fusion protein. 9. A gRNA comprising a spacer sequence comprising any one of the sequences of SEQ ID NOs: 23 to 46, 562 to 1076, 2778 to 4478, and 4553 to 4565, or fragments thereof, or consisting of these. 10. A combination comprising an ETM according to any one of paragraphs 1 to 8 and at least one guide RNA (gRNA). 11. The combination of paragraph 10, comprising one or more ETMs, each ETM being a fusion protein comprising a catalytically active CRISPR / Cas endonuclease domain. 12. A combination according to paragraph 10 or 11 comprising one to three ETMs. 13. The combination according to any one of paragraphs 10 to 12, wherein at least one epigenetic effector domain is a transcriptional repressor domain and / or at least one epigenetic effector domain is a DNMT3L domain. 14. The combination of any one of paragraphs 10 to 13, wherein one or more ETMs collectively comprise a transcriptional repressor domain and a DNMT3L domain. 15. A combination described in any one of paragraphs 10 to 14, comprising at least two gRNAs. 16. The combination of paragraph 15, wherein the gRNA targets the ETM to at least two different target genes. 17. The combination of paragraph 15 or paragraph 16, wherein at least two gRNAs comprise spacer sequences of different lengths. 18. The combination of any one of paragraphs 10 to 13, wherein at least one gRNA comprises a spacer sequence of 15, 16, 17, 18, 19, or 20 nucleotides in length. 19. The combination of any one of paragraphs 15 to 18, wherein at least one of the two gRNAs comprises a spacer sequence of 17 nucleotides or less (e.g., 16 or less) in length. 20. The combination according to any one of paragraphs 10 to 19, wherein at least one target gene is selected from genes without a CpG island (CGI), such as TRAC, TRBC, PDCD1, TIM-3, TIGIT, LAG3, CTLA4, AAVS1, and CCR5, and / or genes with a 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, and FAS. 21. The combination of any one of paragraphs 10 to 20, comprising one or more guide RNAs (gRNAs) having a spacer sequence with a length that allows epigenetic editing of a first gene in a cell and does not allow gene editing, optionally where the first gene comprises a CpG island (CPI), and one or more gRNAs having a spacer sequence with a length that allows gene editing of a second gene in the cell. 22. One or more guide RNAs (gRNAs) having a spacer sequence having a length that allows epigenetic editing of a first gene in a cell and does not allow gene editing, (a) 17 nucleotides or less (e.g., 16 nucleotides or less), or (b) 11 to 17 nucleotides (e.g., 11 to 16 nucleotides) 22. The combination according to paragraph 21, having a spacer sequence of 23. One or more gRNAs having a spacer sequence having a length that allows gene editing of a second gene in a cell, (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) 23. A combination according to paragraph 21 or paragraph 22, having a spacer sequence of 24. A combination comprising one or more polynucleotides encoding an ETM (e.g. a fusion protein) and / or a gRNA as defined in any one of paragraphs 10 to 23. 25. The combination of any one of paragraphs 21 to 24, further comprising donor DNA comprising 5' and 3' arms that are homologous to a sequence in a second gene. 26. The combination of any one of paragraphs 10 to 25, wherein the endonuclease domain is derived from a Cas9 protein, optionally SpCas9. 27. 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, and FAS; and / or 27. The combination of any one of paragraphs 21 to 26, wherein the second gene is selected from TRAC, TRBC, PDCD1, TIM-3, TIGIT, LAG3, CTLA4, AAVS1, and CCR5. 28. The combination of any one of paragraphs 21 to 27, wherein the second gene is a TRAC gene and optionally one or more gRNAs targeting the TRAC gene comprise a spacer having one of SEQ ID NOs: 562 to 611, and optionally SEQ ID NO: 604. 29. The combination of any one of paragraphs 21 to 28, wherein the first gene is a B2M gene and optionally one or more gRNAs targeting the B2M gene each have one of the sequences of SEQ ID NOs: 28 to 33 and 39 to 44, or one of the sequences of SEQ ID NOs: 2778 to 2878 truncated by 3 to 9 nucleotides at the 5' end, or one of the sequences of SEQ ID NOs: 2778, 2780, 2801, and 2863, optionally truncated by 3 to 9 nucleotides at the 5' end, and comprises a spacer selected from SEQ ID NOs: 4486 to 4492, 4497 ​​to 4503, 4508 to 4514, and 4519 to 4525. 30. The combination of any one of paragraphs 21 to 28, wherein the first gene is the TGFBR2 gene and optionally wherein one or more gRNAs targeting the TGFBR2 gene each comprise a spacer having one of the sequences of SEQ ID NOs: 2929-2978 and 4553-4559 truncated by 3-9 nucleotides at the 5' end. 31. The combination of any one of paragraphs 21 to 28, wherein the first gene is the TET2 gene and optionally one or more gRNAs targeting the TET2 gene each comprise a spacer having one of the sequences of SEQ ID NOs: 4429 to 4478 and 4560 to 4565 truncated by 3 to 9 nucleotides at the 5' end. 32. A combination according to any one of paragraphs 10 to 31, for modifying the transcription, expression, and / or activity of one or more (e.g. two or more) genes in a cell, wherein the cell is a mammalian cell, optionally a human cell, and optionally the cell is a human immune cell or a human T cell. 33. i) an agent that promotes the survival, proliferation, and / or activity of a cell, e.g., a cell that contains the combination or a cell that does not contain the combination; and / or ii) agents that are detrimental to the survival, proliferation, activity, chemoresistance, and / or chemotaxis of cells, e.g., cells containing the combination or cells not containing the combination; and / or iii) Agents that allow for the selection of cells, e.g., cells that contain the combination or cells that do not contain the combination. 33. The combination according to any one of paragraphs 10 to 32, further comprising: 34. A combination described in any one of paragraphs 10 to 33, comprising at least one gRNA described in paragraph 9. 35. The gene containing the CGI is the B2M gene, and the gRNA targeting this gene is optionally truncated by 1 to 9 nucleotides, or optionally 3 to 9 nucleotides, at the 5' end. C8 (SEQ ID NO: 35), F4 (SEQ ID NO: 24), H8 (sequence number 2780), H10 (sequence number 2863), H11 (SEQ ID NO: 2778), or H12 (sequence number 2801) 35. The combination of any one of paragraphs 20 to 34, wherein the gRNAs each independently comprise a spacer having the sequence: 36. gRNA targeting B2M (i) a gRNA comprising a spacer having a sequence of F4, optionally shortened by 1 to 9 nucleotides, and optionally 3 to 9 nucleotides, at the 5' end; A gRNA comprising a spacer having a sequence of H8, optionally shortened by 1 to 9 nucleotides, and optionally shortened by 3 to 9 nucleotides, at the 5' end; and A gRNA comprising a spacer having a sequence of H10, optionally shortened by 1 to 9 nucleotides, and optionally 3 to 9 nucleotides, at the 5' end; (ii) a gRNA comprising a spacer having a C8 sequence, optionally shortened by 1 to 9 nucleotides, and optionally 3 to 9 nucleotides, at the 5' end; A gRNA comprising a spacer having a sequence of H8, optionally shortened by 1 to 9 nucleotides, and optionally shortened by 3 to 9 nucleotides, at the 5' end; and A gRNA comprising a spacer having a sequence of H10, optionally shortened by 1 to 9 nucleotides, and optionally 3 to 9 nucleotides, at the 5' end; (iii) a gRNA comprising a spacer having a sequence of F4, optionally shortened by 1 to 9 nucleotides, and optionally 3 to 9 nucleotides, at the 5' end; and A gRNA comprising a spacer having a sequence of H8, optionally shortened by 1 to 9 nucleotides, and optionally 3 to 9 nucleotides, at the 5' end; (iv) a gRNA comprising a spacer having a sequence of F4, optionally shortened by 1 to 9 nucleotides, and optionally 3 to 9 nucleotides, at the 5' end; and A gRNA comprising a spacer having a sequence of H10, optionally shortened by 1 to 9 nucleotides, optionally 3 to 9 nucleotides, at the 5' end; or (v) a gRNA comprising a spacer having a sequence of H8, optionally shortened by 1 to 9 nucleotides, and optionally 3 to 9 nucleotides, at the 5' end; and A gRNA comprising a spacer having a sequence of H10, optionally shortened by 1 to 9 nucleotides, and optionally 3 to 9 nucleotides, at the 5' end. 36. The combination according to paragraph 35, comprising: 37. The combination of any one of paragraphs 21 to 36, wherein the ETMs (e.g., one or more fusion proteins) collectively comprise a DNMT1, DNMT3A, DNMT3B, or SETDB1 domain, optionally further comprising DNMT3A. 38. (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 optionally comprising, from N-terminus to C-terminus, a transcriptional repressor domain, a Cas endonuclease domain, and a DNMT3L domain. 38. The combination according to any one of paragraphs 10 to 37, comprising: 39. (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. 38. The combination according to any one of paragraphs 10 to 37, comprising: 40. The combination of any one of paragraphs 10 to 39, wherein the epigenetic effector domain (e.g. transcriptional repressor domain) is optionally a Kruppel-associated box (KRAB) domain derived from human Kox1 or ZIM3. 41. The combination of any one of paragraphs 10 to 40, optionally comprising a fusion protein comprising, from N-terminus to C-terminus, a KRAB domain derived from ZIM3, a catalytically active Cas9 domain, and a DNMT3L domain, and optionally comprising the amino acid sequence of SEQ ID NO: 4482. 42. The combination of any one of paragraphs 10 to 41, further comprising a gRNA for targeting one or more additional genes in the cell. 43. The combination of any one of paragraphs 10 to 42, wherein the gRNA is chemically modified, and optionally the chemically modified gRNA comprises phosphorothioate internucleoside linkages and / or 2'-O-methyl nucleotides at the 5' and / or 3' ends. 44. A polynucleotide encoding at least one ETM as defined in any one of paragraphs 1 to 8 or in any one of paragraphs 10 to 43. 45. A nucleic acid construct comprising a nucleic acid sequence encoding at least one ETM as defined in any one of paragraphs 1 to 8 or in any one of paragraphs 10 to 43. 46. ​​i) a nucleic acid sequence that promotes the survival, proliferation, and / or activity of a cell, e.g., a cell expressing the nucleic acid construct or a cell not expressing the nucleic acid construct, and / or ii) a nucleic acid sequence that is deleterious to the survival, proliferation, activity, chemoresistance, and / or chemotaxis of a cell, e.g. a cell expressing said nucleic acid construct or a cell not expressing said nucleic acid construct; and / or iii) A nucleic acid construct according to paragraph 45, further comprising a nucleic acid sequence that allows for the selection of cells, e.g. cells that contain the nucleic acid construct or cells that do not contain the construct. 47. A vector comprising a polynucleotide according to paragraph 44 or a nucleic acid construct according to paragraph 45 or 46. 48. a) at least one polynucleotide encoding at least one ETM as defined in any one of paragraphs 1 to 8 or in any one of paragraphs 10 to 43, and b) a polynucleotide providing at least one gRNA according to any one of paragraphs 9 or 10 to 32 or 35 to 43, and optionally c) i) an agent that promotes the survival, proliferation, and / or activity of a cell, e.g., a cell that contains the polynucleotide or a cell that does not contain the polynucleotide; and / or ii) an agent that is detrimental to the survival, proliferation, activity, chemoresistance, and / or chemotaxis of a cell, e.g., a cell that contains the polynucleotide or a cell that does not contain the polynucleotide; and / or iii) An agent that allows for the selection of cells, e.g., cells that contain the polynucleotide or cells that do not contain the polynucleotide. Further polynucleotides comprising nucleic acid sequences encoding A kit of polynucleotides comprising: 49. A cell comprising an ETM according to any one of paragraphs 1 to 8, at least one gRNA according to paragraph 9, a combination according to any one of paragraphs 10 to 43, a polynucleotide according to paragraph 44, a nucleic acid construct according to paragraph 45 or paragraph 46, a vector according to paragraph 47, or a kit of polynucleotides according to paragraph 48. 50. A cell that is a descendant of a cell under paragraph 49. 51. A composition comprising an ETM according to any one of paragraphs 1 to 8, at least one gRNA according to paragraph 9, a combination according to any one of paragraphs 10 to 43, a polynucleotide according to paragraph 44, a nucleic acid construct according to paragraph 45 or paragraph 46, a vector according to paragraph 47, a kit of polynucleotides according to paragraph 48, or a cell according to paragraph 49 or paragraph 50. 52. A pharmaceutical composition comprising an ETM according to any one of paragraphs 1 to 8, at least one gRNA according to paragraph 9, a combination according to any one of paragraphs 10 to 43, a polynucleotide according to paragraph 44, a nucleic acid construct according to paragraph 45 or paragraph 46, a vector according to paragraph 47, a kit of polynucleotides according to paragraph 48, or a cell according to paragraph 49 or paragraph 50. 53. Use of an ETM according to any one of paragraphs 1 to 8, at least one gRNA according to paragraph 9, a combination according to any one of paragraphs 10 to 43, a polynucleotide according to paragraph 44, a nucleic acid construct according to paragraph 45 or paragraph 46, a vector according to paragraph 47, a kit of polynucleotides according to paragraph 48, or a cell according to paragraph 49 or paragraph 50 for modifying (e.g. suppressing or silencing) the transcription, expression and / or activity of at least one target gene in a cell. 54. A method for modifying (e.g. suppressing or silencing) the transcription, expression and / or activity of at least one target gene in a cell, comprising the step of administering to a cell an ETM described in any one of paragraphs 1 to 8, at least one gRNA described in paragraph 9, a combination described in any one of paragraphs 10 to 43, a polynucleotide described in paragraph 44, a nucleic acid construct described in paragraph 45 or paragraph 46, a vector described in paragraph 47, or a kit of polynucleotides described in paragraph 48. 55. The use or method of paragraph 53 or 54, wherein the cell is a T cell. 56. The use or method of any one of paragraphs 53 to 55, wherein the ETM, at least one gRNA, combination, polynucleotide, nucleic acid construct, vector, or kit of polynucleotides is introduced into a cell in vitro or ex vivo. 57. The method of any one of paragraphs 54 to 56, wherein at least two target genes are silenced, at least one of the at least two target genes is epigenetically silenced, at least one of the at least two target genes is silenced by gene editing, and at least one ETM and at least two gRNAs are administered to the cell simultaneously, sequentially, or separately. 58. A cell or progeny of a cell obtained by the use or method of any one of paragraphs 53 to 57. 59. The cell of any one of paragraphs 49, 50, or 58, which is a human T cell, optionally engineered to express a recombinant antigen receptor selected from a recombinant T cell receptor (TCR) or a chimeric antigen receptor (CAR). 60. An ETM according to any one of paragraphs 1 to 8, at least one gRNA according to paragraph 9, a combination according to any one of paragraphs 10 to 43, a polynucleotide according to paragraph 44, a nucleic acid construct according to paragraph 45 or paragraph 46, a vector according to paragraph 47, a kit of polynucleotides according to paragraph 48, a cell according to paragraph 49, 50, 58 or 59, or a pharmaceutical composition according to paragraph 52, for use in therapy (e.g. for use in the treatment of a human in need thereof). 61. Use of an ETM according to any one of paragraphs 1 to 8, at least one gRNA according to paragraph 9, a combination according to any one of paragraphs 10 to 43, a polynucleotide according to paragraph 44, a nucleic acid construct according to paragraph 45 or paragraph 46, a vector according to paragraph 47, a kit of polynucleotides according to paragraph 48, a cell according to paragraph 49, 50, 58 or 59, or a pharmaceutical composition according to paragraph 52 in the manufacture of a 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 the use described in paragraph 60 or the use described in paragraph 61, wherein at least one ETM (e.g. a fusion protein) and at least two gRNAs are administered simultaneously, sequentially or separately to a cell or subject. 63. A method for treating and / or preventing a disease (e.g. in a human in need thereof), comprising administering to a subject in need thereof an ETM according to any one of paragraphs 1 to 8, at least one gRNA according to paragraph 9, a combination according to any one of paragraphs 10 to 43, a polynucleotide according to paragraph 44, a nucleic acid construct according to paragraph 45 or paragraph 46, a vector according to paragraph 47, a kit of polynucleotides according to paragraph 48, a cell according to paragraph 49, 50, 58 or 59, or a pharmaceutical composition according to paragraph 52. 64. A method for treating and / or preventing a disease according to paragraph 63, wherein at least one ETM (e.g. a fusion protein) and at least two gRNAs are administered simultaneously, sequentially or separately to a cell or subject. 65. (i) Isolation of a cell-containing substance; (ii) introducing into a cell an ETM according to any one of paragraphs 1 to 8, at least one gRNA according to paragraph 9, a combination according to any one of paragraphs 10 to 43, a polynucleotide as defined in paragraph 44, a nucleic acid construct according to paragraph 45 or paragraph 46, a vector according to paragraph 47, and / or a polynucleotide according to paragraph 48, and (iii) administering the cells of step (ii) to a subject. A method of gene therapy comprising the steps of: 66. The method of paragraph 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 the use according to paragraph 60 or 62, the use according to paragraph 61 or 62, or the method according to any one of paragraphs 63 to 66, wherein the cells are autologous. 68. An ETM, combination, polynucleotide, nucleic acid construct, vector, kit of polynucleotides, cell or pharmaceutical composition for the use according to paragraph 60 or 62, the use according to paragraph 61 or 62, or the method according to any one of paragraphs 63 to 66, wherein the cells are allogeneic.

[0420] Unless otherwise defined herein, scientific and technical terms used in connection with this disclosure shall have the meanings commonly understood 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 may also be used in the practice or testing of this disclosure. In the event of a conflict, the present specification, including definitions, will control. In general, the nomenclature and techniques used in connection with medicine, medicinal chemistry, 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 instructions, as generally established in the art or as described herein. Furthermore, unless otherwise required by context, singular terms include plurals and plural terms include the singular. Throughout this specification and the embodiments, the words "have" and "comprise" or variations such as "has, having", "comprises, comprising" are understood to mean 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 to include "and / or" within its meaning unless the content clearly dictates otherwise. As used herein, the term "about" refers to a numerical range that is plus or minus 10%, 5%, or 1% from the recited numerical value, within the context of the particular use. Additionally, the headings provided herein are for convenience only and do not interpret the scope or meaning of the claimed embodiments.

[0421] The practice of the present invention will employ, unless otherwise indicated, conventional techniques of chemistry, biochemistry, molecular biology, microbiology, and immunology that are within the capabilities of those skilled in the art, and such techniques are explained in the literature. See, for example, Sambrook, J., Fritsch, EF and Maniatis, T. (1989) Molecular Cloning: A Laboratory Manual, 2nd Edition, Cold Spring Harbor Laboratory Press; Ausubel, FM et al. (1995 and periodic appendices) 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, JM and McGee, J.O'D. (1990) In Situ Hybridization: Principles and Practice, Oxford University Press; Gait, MJ (1984) Oligonucleotide Synthesis: A Practical Approach, IRL Press; and Lilley, DM and Dahlberg, JE (1992) Methods in Enzymology: DNA Structures Part A: Synthesis and Physical Analysis of See DNA, Academic Press.

[0422] All publications and other references mentioned herein are incorporated by reference in their entirety. Although certain documents are cited in this specification, the citation does not constitute an admission that any of these documents form part of the common general knowledge in the art.

[0423] In order that this invention may be better understood, the following examples are set forth. These examples are for illustrative purposes only and should not be construed as limiting the scope of this invention in any manner.

[0424] [Example]

[0425] [Example 1] gRNAs containing truncated spacer sequences promote epigenetic silencing without causing mutagenesis. To evaluate the feasibility of using gRNAs containing truncated spacer sequences to promote ETR-mediated episilencing of B2M while protecting the gene from mutagenesis, we first designed a 20 nt long gRNA against B2M (designated F4; SEQ ID NO: 24) and a corresponding panel of 5' truncated B2M gRNAs with spacer sequences of various lengths, and then tested these against B2M tdTomato The K-562 cell line (Amabile et al., supra) was tested.

[0426] Specifically, gRNAs containing spacer sequences ranging from 21 to 10 nt in length and containing the same seed and PAM sequences (Figure 1) were individually delivered into cells along with Cas9 or dCas9-based ETR combinations containing KRAB, DNMT3A, or DNMT3L. Cells were then analyzed for genetic signatures of Cas9 activity in the B2M gene or expression of tdTomato, the latter of which was used as a proxy for epigenetic silencing of B2M.

[0427] gRNAs containing the standard 20-nt-long B2M spacer sequence and Cas9 or dCas9-ETR were included as positive controls for gene disruption or epigenetic silencing, respectively. Molecular analysis of B2M target sites in Cas9-treated cells demonstrated a threshold effect. gRNAs containing spacer sequences 17 nt or longer in length mediated high and comparable B2M editing levels (approximately 30%), whereas gRNAs containing spacer sequences 16 nt or shorter resulted in undetectable gene editing (Figure 2). Flow cytometry analysis of ETR-treated cells showed a different trend. All gRNAs, except for those containing a 10-nt-long spacer sequence, were able to induce efficient epigenetic silencing of B2M, albeit at different levels (30–48% of tdTomato-negative cells, Figure 3). Importantly, gRNAs containing truncated spacer sequences (i.e., 16 nt or shorter), which were ineffective in promoting gene editing by Cas9, were highly effective in mediating epigenetic silencing by dCas9-ETR. To assess whether these findings could be applied to other gRNAs, we performed similar truncation experiments with three other gRNAs (designated 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 loses its activity ranged from 15 nt to 17 nt in length, depending on the specific gRNA used (Figure 4, left panel). Consistent with the results shown above, ETR was able to induce episilencing of B2M even with truncated gRNAs (Figure 4, right panel).

[0428] Overall, these data indicate that gRNAs containing a truncated spacer sequence of 17 nt or less promote epigenetic silencing of B2M while protecting the gene from mutagenesis induced by Cas9-based ETR. Moreover, these data provide the first demonstration that episilencing can be achieved using gRNAs containing truncated spacer sequences. In parallel with these experiments, we also generated gRNAs containing a 20 nt spacer sequence that can induce gene editing at the TRAC locus (Figure 5).

[0429] [Example 2] The combination of ETR and gRNA allows for the simultaneous inactivation of two genes without inducing chromosomal translocations. Based on these data, we constructed an ETR equipped with a catalytically active Cas9 (hereafter referred to as Cas9-ETR containing KRAB, DNMT3A, or DNMT3L) and used it to tdTomato Their multiplexing efficiency with gRNAs containing truncated or full-length spacer sequences in K-562 cells was evaluated. In particular, cells were co-transfected with the triple Cas9-ETR combination and the F4-derived B2M gRNA containing a 16-nt-long spacer sequence (see Figures 2 and 3) and the TRAC gRNA with a 20-nt-long spacer. The following controls were also included in the experiment: (i) cells co-transfected with the gRNA combinations mentioned above and Cas9 or standard triple dCas9-ETR, used as positive controls for gene disruption of TRAC or episilencing of B2M and disruption of TRAC, respectively; (ii) cells co-transfected with two gRNAs containing 20-nt-long spacer sequences, one against B2M and the other against TRAC, and Cas9 or Cas9-ETR, used here as positive controls for co-disruption of B2M and TRAC. The latter condition was also included to assess whether simultaneous gene editing of two loci would result in a reciprocal chromosomal translocation.Upon transfection, cells were monitored longitudinally for tdTomato expression by flow cytometry for up to 25 days.

[0430] As shown in Figures 6 and 7, when delivered with gRNAs containing a 16-nt spacer sequence, Cas9 and dCas9-based ETRs performed equally in episilencing B2M (14% vs. 21%, respectively). Cas9 promoted inactivation of B2M only when linked to gRNAs containing a 20-nt-long spacer sequence, and not its truncated counterpart (24% vs. 1.7% of tdTomato-negative cells), further confirming the results in Figures 2 and 3. Using gRNAs containing the 20-nt-long B2M spacer sequence with Cas9-ETR resulted in a higher percentage of tdTomato-negative cells (up to 44%) than found in all other conditions, a finding expected given the additive effects of gene editing and epigenetic editing at this locus. Remarkably, silencing was stable over time under all conditions analyzed (Figure 8), indicating that Cas9-ETR with a gRNA containing a 16-nt spacer sequence can also direct epigenetic modifications that are inherited mitotically.

[0431] Next, we analyzed the cells for gene editing (Figure 9) and found that both Cas9 and Cas9-ETR induced efficient editing of TRAC (up to 37%). On the other hand, gene editing of B2M was limited to conditions where Cas9 or Cas9-ETR was co-delivered with a gRNA containing a 20-nt long B2M spacer sequence. Finally, we performed PCR analysis using primers specific for reciprocal chromosomal translocations between B2M and TRAC and found the occurrence of these events only in conditions where they were co-treated with two gRNAs containing a 20-nt long spacer sequence, but not when a gRNA containing a 16-nt spacer sequence was used (Figure 10).

[0432] Overall, these data indicate that Cas9-ETR functions similarly to their dCas9-based counterparts in terms of silencing efficiency and stability. Furthermore, the employment of Cas9-ETR in combination with gRNAs containing truncated and full-length spacer sequences can be safely used to simultaneously inactivate two genes without inducing chromosomal translocations.

[0433] [Example 3] Optimization of B2M episilencing procedures in human primary T lymphocytes Inactivation of B2M has emerged as a promising approach to generate allogeneic T cell products. To evaluate the feasibility of B2M episilencing in human primary T cells, we first expanded the repertoire of gRNAs for this gene to include two other guides: H11_20 (spacer sequence number 2778; gRNA sequence number 4572) and H12_20 (spacer sequence number 2801; gRNA sequence number 4573) (Figure 11). Each of these six gRNAs was then delivered to T cells together with mRNA encoding the triple ETR combination. Time-lapse flow cytometry analysis of treated cells was then used to evaluate the efficiency and stability of B2M episilencing. Unexpectedly, at 12 days after treatment, all but one of the gRNAs tested failed to induce episilencing of B2M (Figure 12). The only gRNA that worked (i.e., gRNA C8) resulted in up to 2% B2M-negative cells, which were lost upon T cell restimulation (analyzed on day 25).

[0434] We then tested whether combinatorial delivery of gRNAs improves the efficiency of episilencing. For this purpose, gRNA C8 or H8 was combined with all other gRNAs and these double gRNA combinations were delivered to T cells together with the triple ETR combination. Flow cytometry analysis 12 days after treatment revealed that all gRNA combinations were able to induce episilencing of B2M, albeit at different levels (Figure 12). For example, gRNA combination H12+H8 induced limited silencing, while the combination F4+H8 resulted in up to 28% B2M-negative cells. Importantly, for some of these gRNA combinations, episilencing resisted the T cell restimulation process, with long-term stable B2M-negative cells ranging from 11% to 20% (Figures 12 and 13). Extended time course flow cytometry analysis over two rounds of T cell restimulation during a 37 day time frame showed that the majority of gRNA combinations tested induced an initial wave of B2M episilencing, which then decayed after the first T cell restimulation (day 12) and approached stability by the second round of T cell restimulation (day 25) (Figure 14). The percentage of B2M then increased until the end of the experiment (day 37). Notably, the efficiency of episilencing depended on the gRNA combination used, with H8+F4 being the most effective in the long term (up to 30% B2M negative cells), while H8+H11 and H8+H12 showed little, if any, detectable episilencing.

[0435] The stability of episilencing also depended on the gRNA combination (Figure 15). Indeed, by comparing the percentage of B2M-negative cells on day 25 (just before the second round of T cell restimulation) and day 12 (just before the first round of T cell restimulation), it was found that some gRNA combinations were less tolerant (<0.5 fold reduction in B2M-negative cells), while others were more tolerant (>0.5 fold reduction in B2M-negative cells), although none of them could provide completely stable gene silencing. Among the most stable combinations, the combinations H8+F4 and H8+H10 were the best performing combinations.

[0436] Similar experiments were then performed in which the ineffective gRNA combinations H8+H11 and H8+H12 were excluded and a new double gRNA combination F4+H10 was included. Additionally, triple gRNA combinations (i.e., C8+F4+H8, C8+F4+H10, C8+H8+H10, and F4+H8+H10) were included to evaluate whether they could further improve the efficacy and stability of episilencing. Among the double gRNA combinations tested, F4+H10 was the most effective in the long term (32 days), reaching 36.5% B2M-negative cells. Among the triple gRNA combinations tested, F4+H8+H10 outperformed the others by 1.6-fold, reaching up to 66% B2M-negative cells at the end of the experiment (Figure 16). As observed in previous experiments, the first round of T cell restimulation elicited a significant reduction in the percentage of B2M-negative cells for most of the gRNA combinations (Figure 16). Notable exceptions to this were the gRNA combinations containing F4+H10 (including triple C8+F4+H10 and F4+H8+H10), for which the percentage of B2M-negative cells at day 28 and day 14 was nearly similar (fold reduction of B2M-negative cells of about 1; Figure 17). Similar findings were obtained with the double C8+H10 gRNA combination (Figure 17). Overall, these data suggest that the efficiency and persistence of episilencing of B2M depended on which gRNA combination was used, with the triple combination based on F4+H8+H10 being the best-functioning combination.

[0437] Aiming to reduce the molecular complexity of the technique, we investigated whether all components of the triple ETR combination are necessary for episilencing B2M. For this purpose, we transiently delivered a double gRNA combination containing C8 and F4 into T cells together with mRNA encoding (i) the triple ETR combination featured here as a reference for the episilencing efficiency of B2M, (ii) the double ETR combination containing KRAB and DNMT3L effector domains, (iii) the double ETR combination containing DNMT3A and DNMT3L effector domains, or (iv) the double ETR combination containing KRAB and DNMT3L effector domains. T cells were then analyzed by flow cytometry for the expression of B2M up to 37 days after treatment (Figures 18A and 18B). This experiment showed that, among all double ETR combinations tested, only the combination based on KRAB and DNMT3L effector domains induced long-term silencing (up to 14% of B2M-negative cells) with an efficiency comparable to that observed with the triple ETR combination. The double ETR combination based on KRAB and DNMT3A induced only a transient B2M suppression, which returned to the level observed in naive T cells after the first round of T cell restimulation. Unexpectedly, the double ETR combination based on DNMT3A and DNMT3L failed to induce any B2M silencing, even at early time points after treatment.

[0438] Based on these results, we performed an experiment similar to that shown in Figure 16, but using a double ETR combination containing KRAB and DNMT3L, and confirmed that this combination works as efficiently as the triple combination for all gRNA combinations tested (Figure 19). For the triple ETR combination, the condition in which gRNAs F4 and H10 coexisted was the most resistant to T cell restimulation (Figure 19). Overall, these data indicate that the double ETR combination containing KRAB and DNMT3L works as efficiently as the canonical triple ETR combination in silencing B2M in T cells.

[0439] Based on these results, we compared the efficiency of B2M episilencing between a dual ETR combination containing KRAB and DNMT3L and an integrated bipartite ETR with DNMT3L and the KRAB domain homolog of the zinc finger imprinted 3 (ZIM3) protein (Alerasool et al., Nat Methods (2020) 17(11):1093-6), hereafter referred to as the ZIM:dCas9:DNMT3L fusion (Figure 20A, left schematic). The amino acid sequence of this fusion protein is shown below, with the SV40 nuclear localization signal (NLS) in a box, the ZIM3 KRAB repressor domain in bold, the flexible linker in underlined and bold, dCas9 in underline, and the DNMT3L domain in italics (only). [ka]

[0440] In these experiments, T cells were co-transfected with mRNA encoding ETR and (i) gRNA F4 or C8, (ii) double gRNA combination C8+F4, or (iii) the best performing triple gRNA combination F4+H8+H10 to evaluate whether the bipartite ETR can restore the episilencing efficiency of the individual gRNAs. Cells were then analyzed by flow cytometry until day 55. To avoid confounding effects due to the delivery of different amounts of mRNA encoding ETR, these experiments were performed by using 1.5 μg of each ETR for the double combination, and 1.5 μg of ZIM:dCas9:DNMT3L fusion. Thus, matching amounts of epigenetic effectors were used. Consistent with our previous data, individual gRNAs were ineffective in the double ETR combination, and the adoption of ZIM:dCas9:DNMT3L fusion only slightly increased the episilencing efficiency of B2M for gRNA C8 (Figure 20A, right graph). On the other hand, when comparing the double ETR combination and ZIM:dCas9:DNMT3L fusion for the double gRNA combination C8+F4, a significant increase in episilencing of B2M was found (11% to 70%, respectively, Figure 20A-B). Similar results were obtained for the triple gRNA combination F4+H8+H10, but the difference between the double ETR combination and ZIM:dCas9:DNMT3L fusion was less pronounced (86% and 95% B2M-negative cells for the double ETR combination and ZIM:dCas9:DNMT3L fusion, respectively, Figure 20A-B). This effect may be due to the already high episilencing efficiency of the dual ETR combination. For all conditions of the triple gRNA combination, episilencing of B2M was durable and survived two rounds of T cell restimulation. The stability observed with the ZIM:dCas9:DNMT3L fusion and triple gRNA combination is noteworthy, reaching 95% of B2M-negative cells 8 days after treatment and remaining stable up to day 55.Finally, to assess whether the dose of ZIM:dCas9:DNMT3L fusion mRNA was saturating, we performed a dose titration experiment in T cells and found that one-third of the standard dose (1 vs. 1.5 μg) was already sufficient to obtain efficient episilencing of B2M (Figure 21).

[0441] Overall, these data show that the adoption of the fusion protein ZIM:dCas9:DNMT3L improves episilencing in T cells, achieving up to 95% B2M negative cells. Interesting features of this fusion protein include reduced production costs compared to triple or double ETR combinations and the fact that it can show efficient silencing at a dose that is one-third that of the double ETR combination.

[0442] [Example 4] Orthogonal editing of B2M and TRAC in human primary T cells does not induce reciprocal chromosomal translocations Based on the above data, we tested whether co-delivery of a Cas9-based ETR with a truncated gRNA for B2M and a full-length gRNA for TRAC (SEQ ID NO: 4575) could induce orthogonal editing (i.e., episilencing of B2M and targeting integration into the TRAC gene) in human primary T cells without inducing reciprocal chromosomal translocations. To mediate episilencing of B2M, we used several of the truncated gRNAs described above (see Figures 2-4), namely, truncated C8 (C8_16; 16 nt spacer; gRNA SEQ ID NO: 4578), truncated F4 (F4_16; 16 nt spacer; gRNA SEQ ID NO: 4579), and truncated H8 (H8_15; 15 nt spacer; gRNA SEQ ID NO: 4577), which were co-delivered to T cells as triple combinations, where all truncations start from the 5' end of the full-length sequence. In these experiments, we also used the truncated ETR combinations / architectures identified above, i.e., dual ETRs containing KRAB and DNMT3L, or related integrated fusion proteins containing the ZIM3 KRAB domain and DNMT3L, both of which were engineered to contain catalytically active Cas9. The integrated fusion containing ZIM3 KRAB, active Cas9, and DNMT3L domains has the following amino acid sequence, where the SV40 NLS is in frame, the ZIM3 KRAB repressor domain in bold, the flexible linker in underlined and bold, Cas9 in underline, and the DNMT3L domain in italics (only). [ka] [ka]

[0443] For Cas9-mediated targeted integration into the TRAC locus, we utilized a previously developed AAV6-based donor template that contains sequences encoding a transgenic TCR against the tumor antigen NY-ESO embedded within the TRAC homology arms (Roth et al., Nature (2018) 559(7714):405-9). Upon targeted integration, the transgenic TCR is expressed from the endogenous TRAC locus and can be measured by flow cytometry using specific pentamers (Figure 22). For T cells transfected with the dual Cas9-based ETR combination, this treatment unexpectedly resulted in little, if any, episilencing of B2M, while editing of TRAC was highly efficient, resulting in up to 70% NY-ESO positive cells and up to 6% endogenous TCR-destroyed cells (Figure 23). Notably, the use of ZIM3:Cas9:DNMT3L fusion protein efficiently restored episilencing of B2M, resulting in up to 70% B2M-negative T cells (Figure 24). High levels of editing of the TRAC locus were also measured in these conditions. Further analysis of NY-ESO-positive T cells showed that 65% of them were also B2M-negative. A similar analysis focusing on TCR-disrupted cells showed a similar efficiency of co-editing. Analysis 34 days after treatment showed that the orthogonal editing survived restimulation of T cells.

[0444] The presence of a reciprocal chromosomal translocation between B2M and TRAC was then assessed by PCR analysis. Notably, no evidence of a reciprocal translocation was found (Figure 25), indicating that (i) the B2M gene was silenced by an epigenetic mechanism rather than by gene inactivation, and (ii) the truncated gRNA abolished Cas9 cleavage, indicating that B2M tdTomato Our previous findings in the K-562 cell line (see Example 2) were confirmed. Contrary to these data, T cells co-transfected with the Cas9-based ETR and full-length gRNAs against both B2M and TRAC showed high levels of co-editing and clear signs of reciprocal chromosomal translocations (Figure 25).

[0445] Overall, these data indicate that by employing different length gRNAs together with a Cas9-based ETR, it is possible to promote orthogonal editing (i.e., episilencing and target integration or episilencing and gene disruption) with high efficiency in primary human T cells without inducing reciprocal chromosomal translocations.

[0446] [Example 5] Identification of gRNAs that mediate high-level episilencing of TET2 and TGFBR2 in human primary T lymphocytes To extend the orthogonal editing approach to more than two genes, we designed a panel of gRNAs targeting TET2 and TGFBR2. Inactivation of these genes represents a possible therapeutic approach to increase the persistence of T cell products or protect them from immune-attenuating signals originating from the tumor microenvironment (see, for example, Fraietta et al., Nature (2018) 558(7709):307-12; Nobles et al., J Clin Invest (2020) 130(2):673-85; Li et al., Nature (2020) 587(7832):121-5; Alishah et al., J Transl Med (2021) 19(1):482). For each of these genes, we designed 20 gRNAs in a genomic window of 1 Kb around their transcription start sites (Figure 26). We then designed to test the episilencing efficacy of these gRNAs directly in T cells using standard triple ETR combinations that contain KRAB, DNMT3A, and DNMT3L effector domains. We pooled consecutive gRNAs and linked each of these pairs with the others to obtain possible pair combinations, if any. The pairs tested are shown in Table 3 below (SEQ: sequence number). The gRNAs used in this experiment contained a 20 nucleotide (full-length) spacer sequence.

[0447] [Table 3B-1] [Table 3B-2] [Table 3B-3]

[0448] These new pools were then delivered individually to T cells together with mRNAs encoding the triple ETR combinations. The two genes were expressed at low levels, and the detection of their protein products by flow cytometry was complicated by the nuclear localization of TET2 and the inducibility of TGFBR2. Thus, to quantify the efficiency of episilencing, we used digital droplet PCR (ddPCR), a highly sensitive technique for measuring the expression profile of selected genes. Finally, the cells were analyzed 28 days after treatment. Figure 27 shows the percentage of episilencing of TGFBR2 for each pair combination normalized to the expression level of TGFBR2 in mock-treated cells. This analysis shows that almost all pair combinations are able to induce episilencing of TGFBR2 with high efficiency (more than 60%). On the other hand, a similar analysis performed for TET2 showed that episilencing of this gene was more variable, with some pair combinations exhibiting no activity, while others were highly effective (Figure 28). In this regard, combinations including pair number 7 (P7) or number 10 (P10) were the most effective combinations, resulting in a reduction of TET2 by up to 92% when combined together.

[0449] The episilencing efficiency of selected gRNA pairs was evaluated with the aim of reducing the number of gRNAs required to silence each of these genes to two. In this regard, pairs no. 4 (gRNA IDs: TG7_20 and TG8_20) and 10 (gRNA IDs: TG19_20 and TG20_20) for TGFBR2 and pairs no. 7 (gRNAs: TE13_20 and TE14_20) and 10 (gRNAs: TE19_20 and TE20_20) for TET2 were selected. Individual co-delivery of these pairs with the triple ETR combination followed by analysis by ddPCR 22 days after treatment showed that pairs no. 4 and no. 10 were the most effective in promoting episilencing of TGFBR2 and TET2, respectively, resulting in a reduction of the two transcripts by 35% and 90% (Figure 29). Notably, in contrast to TGFBR2, for which the pair combination resulted in an 89% reduction, the episilencing efficiency of pair number 10 for TET2 was comparable to that observed when delivering the parental pair combination. Overall, these data indicate that TET2 and TGFBR2 can be efficiently silenced by ETR technology.

[0450] We then tested the multiplexed epigenetic silencing of B2M, TET2, and TGFBR2. For this purpose, human primary T cells were co-treated with (i) mRNA encoding ETR ZIM3:dCas9:DNMT3L fusion, (ii) F4+H8+H10 combination of full-length gRNAs for B2M (i.e., gRNA IDs: F4_20, H8_20, and H10_20), (iii) pair number 10 of full-length gRNAs for TET2, and (iv) pair number 4 and 10 of full-length gRNAs for TGFBR2. We then measured the expression levels of these genes by ddPCR and found that they were all significantly downregulated, resulting in up to 47%, 92%, and 67% episilencing of B2M, TET2, and TGFBR2, respectively (Figure 30). Overall, these data indicate that B2M, TET2, and TGFBR2 can be co-silenced by ETR technology.

[0451] [Example 6] Multifunctional orthogonal editing of multiple genes by ETM does not cause reciprocal chromosomal translocations in human primary T lymphocytes We decided to combine the orthogonal editing of B2M and TRAC with episilencing of TGFBR2 or TET2. For this purpose, we first shortened the gRNAs for TET2 and TGFBR2 in Figure 30 by 15 nt in length. The gRNAs with the truncated spacers are shown in the table below. Primary human T cells were then transfected with mRNA encoding the ETM ZIM3:Cas9:DNMT3L fusion together with (i) truncated gRNAs against B2M, namely F4 (gRNA ID: F4_16; spacer 16 nt long; gRNA SEQ ID NO: 4579), H8 (H8_15; spacer 15 nt long; gRNA SEQ ID NO: 4577), and H10 (H10_14; spacer 14 nt long; gRNA: SEQ ID NO: 4576), (ii) full-length gRNA against TRAC (SEQ ID NO: 4575), (iii) truncated gRNAs corresponding to pair number 10 for TET2 (TE19_15 and TE 20_15) or pair numbers 4 (TG7_15 and TG8_15) and 10 (TG19_15 and TG20_15) for TGFBR2. Cells were either transduced or not with AAV6 donor template to target the NY-ESO TCR into the TRAC locus, and treated T cells were analyzed by (i) flow cytometry to measure epigenetic silencing of B2M and gene editing of TRAC (i.e., disruption or targeting of the NY-ESO TCR by the absence or presence of the AAV6 donor) and (ii) ddPCR to quantify expression levels of TET2 and TGFBR2.

[0452] [Table 4]

[0453] Regarding the experimental conditions of multifunctional editing of B2M, TRAC, and TGFBR2 without AAV6 donor, the analysis showed that ZIM3:Cas9:DNMT3L could induce up to 11% and 95% of cells negative for B2M and endogenous TCR, respectively (Figure 31). ddPCR analysis of bulk treated cells showed that the expression level of TGFBR2 was significantly reduced in these samples, resulting in up to 50% episilencing (Figure 31). For samples treated with AAV6 donor, it was found that up to 16% of treated T cells were negative for B2M, while 59% and 26.7% were negative for endogenous TCR and positive for NY-ESO, respectively (Figure 32). In these cells, the episilencing efficiency of TGFBR2 was 54% (Figure 32). Importantly, molecular analysis of treated T cells, transduced or not with AAV6 donor, did not show any indication of reciprocal chromosomal translocations between the three target genes (Figure 33). Inconsistent with this latter data, experiments performed with ZIM3:Cas9:DNMT3L and full-length gRNAs against the examined genes showed clear evidence of reciprocal chromosomal translocations between B2M, TRAC, and TGFBR2 (Figure 33).

[0454] For the experimental conditions of multifunctional editing of B2M, TRAC, and TET2 without AAV6 donor, the analysis showed that ZIM3:Cas9:DNMT3L could induce up to 46% and 99% of cells negative for B2M and endogenous TCR, respectively (Figure 34). ddPCR analysis of bulk treated cells showed that the expression level of TET2 was significantly reduced in these samples, resulting in up to 63% episilencing (Figure 34). For the samples treated with AAV6 donor, it was found that up to 40% of treated T cells were negative for B2M, while 53% and 29% were negative for endogenous TCR and positive for NY-ESO, respectively (Figure 35). In these cells, the episilencing efficiency of TET2 was 60% (Figure 35). Importantly, molecular analysis of treated T cells, transduced or not with AAV6 donors, did not show any indication of reciprocal chromosomal translocations between the three target genes (Figure 36). Inconsistent with this latter data, experiments performed with ZIM3:Cas9:DNMT3L and full-length gRNAs against the examined genes showed clear evidence of numerous reciprocal chromosomal translocations between B2M, TRAC, and TET2 (Figure 36).

[0455] Finally, quadruple multifunctional editing of B2M, TRAC, TGFBR2, and TET2 was tested using ZIM3:Cas9:DNMT3L with or without AAV6 donor. In this experiment, truncated gRNAs for B2M, TGFBR2, and TET2, and full-length gRNA for TRAC were used. In the absence of AAV6 donor, up to 5.7% and 93% of treated cells proved to be negative for B2M and endogenous TCR, respectively (Figure 37). ddPCR analysis of these cells showed that TGFBR2 and TET2 transcripts were significantly reduced compared to mock-treated samples, resulting in up to 70% and 71% episilencing, respectively (Figure 37). For samples treated with AAV6 donor, up to 7% of treated T cells were found to be negative for B2M, while 54% and 26% were negative for endogenous TCR and positive for NY-ESO, respectively (Figure 38). In these cells, the episilencing efficiency of TGFBR2 and TET2 was 50% and 51%, respectively (Figure 38). Importantly, molecular analysis of treated T cells showed no indication of reciprocal chromosomal translocations between the three target genes, either transduced or not with AAV6 donor (Figure 39). Inconsistent with this latter data, experiments performed with ZIM3:Cas9:DNMT3L and full-length gRNAs against the examined genes showed clear evidence of numerous reciprocal chromosomal translocations between B2M, TRAC, and TET2 (Figure 39).

[0456] Overall, these data indicate that Cas9-based ETR (EMT) with truncated and full-length gRNAs can perform multiple orthogonal editing in T cells without inducing reciprocal chromosomal translocations.

[0457] Additional targets that can be silenced by epigenetic silencing include, for example, A2AR, CISH, PTPN11, PTPN6, PTPA, PTPN2, JUNB, TOX, TOX2, NR4A1, NR4A2, NR4A3, MAP4K1, REL, IRF4, DGKA, PIK3CD, HLA-A, USP16, DCK, and FAS.

[0458] Epigenetic silencing of these targets may be combined with gene editing of the TRAC, PD-1, and CTLA4 genes that do not have CpG islands (CGIs).

[0459] Cell culture conditions Peripheral blood mononuclear cells (PBMCs) were freshly isolated from healthy donors using centrifugation on a Ficoll gradient (Lymphoprep™). CD3 positive lymphocytes were then purified by magnetic separation using a Pan T cell isolation kit (Miltenyi Biotech) according to the manufacturer's instructions. T lymphocyte purity was assessed by flow cytometry (FACSCanto™ II-BD Bioscience, Cytoflex-Beckman Coulter) using anti-CD3 (BD, 349201), CD4 (Biolegend, 317429), and CD8 (Biolegend, 344708) antibodies. T lymphocytes were stimulated at a 1:1 ratio with anti-CD3 / CD28 magnetic beads (Dynabeads human T-activator CD3 / CD28, Thermo Fisher) and maintained in culture in RPMI (Corning) supplemented with penicillin (100 IU / ml), streptomycin (100 μg / ml), 2% glutamine, 10% FBS (Euroclone), and 5 ng / ml each of IL-7 and IL-15 (PeproTech). dTomato The reporter cell line has been described previously (Amabile et al., supra) and was maintained in culture in RPMI supplemented with penicillin (100 IU / ml), streptomycin (100 μg / ml), 2% glutamine, and 10% FBS. All cells were incubated at 37 °C for 2 h at 5% CO 2 The cells were cultured at 37°C in a humidified atmosphere.

[0460] mRNA, gRNA, and donor templates The gRNAs used in these studies were designed using CHOPCHOP (Labun et al., Nucleic Acids Res. (2019) 47(W1):W171-4). For T cell experiments, previously described highly chemically modified gRNAs containing 2'-O-methyl residues and phosphorothioate modifications (Finn et al., Cell Rep (2018) 22(9):2227-35) were purchased from IDT. mRNAs encoding ETRs, Cas9-based ETRs, and Cas9 were either purchased from TriLink or produced in-house using the MEGAscript™ T7 Transcription Kit (Invitrogen) according to the manufacturer's instructions. In both cases, mRNAs were 5'-capped using CleanCap® Reagent (TriLink) and UTP was fully replaced by N1-methylpseudouridine-5'-triphosphate (TriLink). In-house produced mRNA was also concentrated using an Amicon® Ultra-15 Centrifugal Filter Unit (Sigma-Aldrich). A construct carrying homology arms for the TRAC locus, IG4 NY-ESO TCR alpha / beta, was obtained from Addgene (plasmid #112021) and cloned into an AAV transfer construct containing AAV2 inverted terminal repeats. AAV6 was produced by TIGEM Vector Core by triple transfection method and purified by ultracentrifugation. K-562 dTomato For experiments, full-length or truncated gRNAs were cloned downstream of the human U6 promoter as fusion transcripts with tracrRNA (Amabile et al., supra). ETR, Cas9 system ETR, and Cas9 sequences were cloned into expression plasmids under the control of the CMV promoter (Amabile et al., supra).

[0461] Gene editing procedures T cells were edited 2 days after purification. Dynabeads were removed prior to electroporation. 5×10 5 Cells were electroporated with 1.5 μg (unless otherwise specified) stabilized mRNA for each ETR / Cas9-ETR / Cas9 and 3 μg of each highly modified gRNA. Immediately after nucleofection, 80 μl of RMPI was added directly to the cuvette and cells were incubated at 37° C. for 15 min. Cells were then transferred to 96-U bottom wells and 100 μl of complete 2× medium (RPMI with 20% FBS, 4 mM L-glutamine, 2% P / S, and 10 ng / ml each of IL-7 and IL-15) was added. For gene targeting experiments, AAV6 NY-ESO TCR was also electroporated with 10 μl of RMPI. 5 The K-562 was added to the 2x medium at a dose of vg / cell. The percentage of B2M negative cells was assessed by flow cytometry using an anti-B2M antibody (Biolegend, 316312), and NY-ESO / TCR positive events were assessed by using an anti-Vβ13.1 antibody (Beckman Coulter) or an anti-human TCR alpha / beta antibody (Biolegend). Complete fresh medium was added to the cultures every 3 days. dTomato For the experiment, 5 × 10 cells were transfected using the Lonza 4D-Nucleofector™ (SF Cell Line solution, FF-120 program). 5 Each cell was electroporated with 600 ng of ETR / Cas9-ETR / Cas9 plasmid and 200 ng of gRNA plasmid. Immediately after nucleofection, cells were seeded into 96-U bottom wells in complete RPMI. dTomato negative cells were analyzed by flow cytometry. Flow cytometry analysis was performed using Flow Jo software (FLOWJO, LLC).

[0462] molecular analysis Genomic DNA from cell lines was 2 × 10 6Subcellular samples were extracted using the Maxwell 16 LEV Blood DNA kit (Promega). 5 Subcellular DNA was extracted using QuickExtract™ DNA Extraction Solution (Epicentre). Genetic indels were detected using the Surveyor Nuclease Assay (Surveyor Mutation Kit, IDT) according to the manufacturer's instructions. The following primers were used to measure mutations in the B2M locus:

[0463] [Table 5-1] [Table 5-2] To measure mutations in other loci of interest, the following primers were used:

[0464] [Table 6]

[0465] Translocation analysis was performed using GoTaq® DNA Polymerase (Promega) combining the forward and reverse primers listed above according to the gRNA employed in the experiment. Amplicons were run on a 1% agarose gel. The following primers were used to detect the genomic translocation of interest:

[0466] [Table 7]

[0467] For gene expression analysis, 10 6Total RNA was extracted from cells and reverse transcribed using random hexamers according to the manufacturer's instructions for the SuperScript III First-Strand Synthesis System (Invitrogen). Transcript levels were determined by digital droplet PCR using 0.2–10 ng of template cDNA. PCR reactions were performed by adding 1× TaqMan Gene Expression Assay (Applied Biosystems) according to the manufacturer's instructions (Biorad), read by a QX200 reader, and analyzed by QuantaSoft software (Biorad). Data were normalized by HPRT and mock-treated samples. Reagents used are listed below.

[0468] [Table 8A]

[0469] Sequence Listing The sequences disclosed in this disclosure are listed below.

[0470] [Table 8B-1] [Table 8B-2] [Table 8B-3] [Table 8B-4]

Claims

1. A combination for modifying the transcription, expression, and / or activity of one or more genes in a cell, comprising: (a) one or more fusion proteins, or polynucleotides encoding one or more fusion proteins, each comprising a catalytically active CRISPR / Cas endonuclease domain, collectively comprising a transcriptional repressor domain and a DNMT3L domain; (b) one or more guide RNAs (gRNAs) having a spacer sequence of a length that allows epigenetic editing of a first gene in a cell and does not allow gene editing, the first gene comprising a CpG island (CGI), or a polynucleotide encoding the one or more gRNAs; and (c) one or more gRNAs, or polynucleotides encoding one or more gRNAs, having a spacer sequence of a length that allows gene editing of a second gene in the cell. A combination including:

2. (i) one or more gRNAs in (b) have a spacer sequence of 11 to 17 nucleotides; and / or (ii) one or more gRNAs in (c) have a spacer sequence of 18 to 21 nucleotides; 2. The combination according to claim 1.

3. The combination of claim 1, comprising 1 to 3 fusion proteins.

4. The combination of claim 1, further comprising a donor DNA comprising 5' and 3' arms that are homologous to a sequence in a second gene.

5. 2. The combination of claim 1, wherein the endonuclease domain is derived from a Cas9 protein, optionally SpCas9.

6. The combination according to claim 1, wherein the cell is a mammalian cell, optionally a human cell.

7. The combination of claim 1 , wherein the one or more fusion proteins collectively comprise a DNMT1, DNMT3A, DNMT3B, or SETDB1 domain, optionally further comprising DNMT3A.

8. (a) 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 (b) a fusion protein optionally comprising, from N-terminus to C-terminus, a transcriptional repressor domain, a Cas endonuclease domain, and a DNMT3L domain.

2. The combination of claim 1 , comprising:

9. (a) 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 (b) A fusion protein comprising a transcriptional repressor domain, a Cas endonuclease domain, a DNMT3L domain, and a DNMT3A domain.

2. The combination of claim 1 , comprising:

10. The combination of claim 1, wherein the transcriptional repressor domain is a Krueppel-associated box (KRAB) domain, optionally derived from human Kox1 or ZIM3.

11. The combination of claim 10, optionally comprising a fusion protein comprising, from N-terminus to C-terminus, the KRAB domain, the dCas9 domain, and the DNMT3L domain from ZIM3.

12. The combination of claim 1, optionally comprising a fusion protein comprising, from N-terminus to C-terminus, a KRAB domain from ZIM3, a catalytically active Cas9 domain, and a DNMT3L domain.

13. 2. The combination of claim 1, wherein the gRNA is chemically modified, and optionally the chemically modified gRNA comprises phosphorothioate internucleoside linkages and / or 2'-O-methyl nucleotides at the 5' and / or 3' ends.

14. A pharmaceutical composition for treating a subject in need thereof, comprising a combination according to any one of claims 1 to 13, a cell comprising a combination according to any one of claims 1 to 13, or a progeny of said cell.

15. A cell comprising a combination according to any one of claims 1 to 13, or a progeny of said cell.

16. A method for modifying the transcription, expression and / or activity of one or more genes in a human cell, comprising introducing into the cell in vitro or ex vivo a combination according to any one of claims 1 to 13.

17. 17. A cell obtained by the method of claim 16, or a progeny of said cell.

18. A pharmaceutical composition for treating a subject in need thereof, comprising the cells of claim 17.

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