Compositions and Methods for Epigenetic Editing

Abstract

Compositions and methods comprising an epigenetic editing system for epigenetic editing, or cells, nucleic acids and vectors comprising an epigenetic editing system, are disclosed herein. Also disclosed are epigenetically modified chromosomes.

Description

Technical Field

[0001] Cross-reference

[0001] This application claims the benefit of U.S. Provisional Application No. 63 / 354,969, filed Jun. 23, 2022; U.S. Provisional Application No. 63 / 488,639, filed Mar. 6, 2023; and U.S. Provisional Application No. 63 / 502,314, filed May 15, 2023, the entire contents of each application being incorporated herein by reference.

Background Art

[0002]

[0002] Genome editing has been regarded as a promising therapeutic approach for treating genetic diseases for decades. However, manipulation at the DNA level is still risky considering unwanted double-strand breaks, heterogenous repair including insertions and deletions of various sizes at the intended site, and the potential for toxicity.

Summary of the Invention

Means for Solving the Problems

[0003]

[0003] Provided herein are compositions for epigenetic modification related to an epigenetic editing system, and methods of using an epigenetic editing system in a target genome, including a host cell and a host organism, to generate epigenetic modifications without introducing changes to the genomic sequence.

[0004]

[0004] (a) A fusion protein comprising, from the N-terminus to the C-terminus, a transcriptional repressor domain, a first linker (linker RB), a DNA-binding domain, a second linker (linker BD), a first DNA methyltransferase (DNMT) domain, a third linker (linker DD), and a second DNMT domain; or (b) A fusion protein comprising, from the N-terminus to the C-terminus, a first DNMT domain, a first linker (linker DD), a second DNMT domain, a second linker (linker BD), a DNA-binding domain, a third linker (linker RB), and a transcriptional repressor domain; or (c) An epigenetic editing system comprising a nucleic acid molecule encoding the fusion protein of (a) or (b) is described herein.

[0005]

[0005] In some embodiments, linker RB, linker BD, and / or linker DD comprise a flexible or unstructured peptide linker. In some embodiments, linker RB, linker BD, and / or linker DD comprise a glycine-rich and / or serine-rich polypeptide sequence. In some embodiments, the glycine-rich and / or serine-rich polypeptide sequence comprises an amino acid sequence of at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 20, or at least 25 consecutive amino acid residues, and the amino acid sequence comprises at least 50% glycine and / or serine residues.

[0006]

[0006] In some embodiments, the glycine-rich and / or serine-rich polypeptide sequence has the sequence (G x S y ) z(wherein x is an integer from 1 to 10, y is an integer from 1 to 10, and z is an integer from 1 to 10). In some embodiments, x / y is at least 2, at least 3, at least 4, or at least 5. In some embodiments, x is 3. In some embodiments, x is 4. In some embodiments, x is 3 and y is 1. In some embodiments, x is 4 and y is 1. In some embodiments, z is 3, 4, or 5. In some embodiments, x is 4, y is 1, and z is 4).

[0007]

[0007] In some embodiments, linker RB comprises (GGGGS)4 (SEQ ID NO: 4) and / or linker DD comprises (GGGGS)4 (SEQ ID NO: 4). In some embodiments, linker DD comprises the amino acid sequence SSGNSNANSRGPSFSSGLVPLSLRGSH (SEQ ID NO: 5). In some embodiments, linker RB, linker BD, and / or linker DD comprise an XTEN linker. In some embodiments, linker RB, linker BD, and / or linker DD comprise an XTEN16 or XTEN80 linker. In some embodiments, linker RB and linker BD comprise an XTEN16 or XTEN80 linker. In some embodiments, linker RB comprises an XTEN16 linker and linker BD comprises an XTEN80 linker. In some embodiments, linker RB comprises an XTEN16 linker, linker BD comprises an XTEN80 linker, and linker DD comprises the amino acid sequence SSGNSNANSRGPSFSSGLVPLSLRGSH (SEQ ID NO: 5).

[0008]

[0008] In some embodiments, the transcriptional repressor domain is a KRAB domain. In some embodiments, the KRAB domain is a KRAB domain derived from KOX1. In some embodiments, the KRAB domain is a KRAB domain derived from ZNF10. In some embodiments, the KRAB domain is a KRAB domain derived from ZIM3. In some embodiments, the KRAB domain comprises the amino acid sequence of SEQ ID NO: 16 or an amino acid sequence that is at least 90% homologous thereto.

[0009]

[0009] In some embodiments, the first DNMT domain or the second DNMT domain is a DNMT3A domain. In some embodiments, the DNMT3A domain is a human DNMT3A domain. In some embodiments, the DNMT3A domain is a mouse DNMT3A domain. In some embodiments, the DNMT3A domain comprises an amino acid sequence that is at least 90% identical to SEQ ID NO: 13.

[0010]

[0010] In some embodiments, the first DNMT domain or the second DNMT domain is a DNMT3L domain. In some embodiments, the DNMT3L domain is a human DNMT3L (hD3L) domain. In some embodiments, the hD3L domain comprises an amino acid sequence that is at least 90% identical to SEQ ID NO: 14. In some embodiments, the DNMT3L domain is a mouse DNMT3L (mD3L) domain. In some embodiments, the mD3L domain comprises an amino acid sequence that is at least 90% identical to SEQ ID NO: 69.

[0011]

[0011] In some embodiments, the first DNMT domain is a DNMT3A domain and the second DNMT domain is a DNMT3L domain. In some embodiments, the first DNMT domain is a DNMT3L domain and the second DNMT domain is a DNMT3A domain.

[0012] In some embodiments, at least one of the DNA binding domains comprises a zinc finger motif or a zinc finger array. In some embodiments, at least one of the DNA binding domains comprises a nucleic acid-inducible DNA binding domain. In some embodiments, at least one of the DNA binding domains comprises an RNA-inducible DNA binding domain. In some embodiments, at least one of the DNA binding domains comprises the DNA binding domain of a CRISPR-Cas protein. In some embodiments, the CRISPR-Cas protein comprises nuclease-inactive Cas9 (dCas9), nuclease-inactive Cas12a (dCas12a), or nuclease-inactive CasX (dCasX).

[0013] Also described herein is an epigenetic editing system comprising: (a) a fusion protein comprising, from N-terminus to C-terminus, a DNMT3L domain, a first linker (linker BD), a DNA binding domain, a second linker (linker RB), and a transcriptional repressor domain; or (b) a fusion protein comprising, from N-terminus to C-terminus, a transcriptional repressor domain, a first linker (linker RB), a DNA binding domain, a second linker (linker BD), and a DNMT3L domain; or (c) a nucleic acid molecule encoding the fusion protein of (a) or (b).

[0014] In some embodiments, the fusion protein of (a) or the fusion protein of (b) does not contain a catalytically active DNA methyltransferase domain. In some embodiments, the fusion protein of (a) or the fusion protein of (b) does not contain a DNMT3A domain. In some embodiments, the fusion protein of (a) or the fusion protein of (b) does not contain a DNMT1 domain. In some embodiments, linker RB, linker BD, and / or linker DD comprise a flexible or unstructured peptide linker. In some embodiments, linker RB, linker BD, and / or linker DD comprise a glycine-rich and / or serine-rich polypeptide sequence. In some embodiments, the glycine-rich and / or serine-rich polypeptide sequence comprises an amino acid sequence of at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 20, or at least 25 consecutive amino acid residues, and the amino acid sequence comprises at least 50% glycine and / or serine residues. In some embodiments, the glycine-rich and / or serine-rich polypeptide sequence comprises the sequence (G x S y ) z (wherein x is an integer from 1 to 10, y is an integer from 1 to 10, and z is an integer from 1 to 10). In some embodiments, x / y is at least 2, at least 3, at least 4, or at least 5. In some embodiments, x is 3. In some embodiments, x is 4. In some embodiments, x is 3 and y is 1. In some embodiments, x is 4 and y is 1. In some embodiments, z is 3, 4, or 5. In some embodiments, x is 4, y is 1, and z is 4.

[0015] In some embodiments, linker RB comprises (GGGGS)4 (SEQ ID NO: 4), and / or linker DD comprises (GGGGS)4 (SEQ ID NO: 4). In some embodiments, linker DD comprises the amino acid sequence SSGNSNANSRGPSFSSGLVPLSLRGSH (SEQ ID NO: 5).

[0016] In some embodiments, linker RB, linker BD, and / or linker DD comprise an XTEN linker. In some embodiments, linker RB, linker BD, and / or linker DD comprise an XTEN16 or XTEN80 linker. In some embodiments, linker RB and linker BD comprise an XTEN16 or XTEN80 linker. In some embodiments, linker RB comprises an XTEN16 linker and linker BD comprises an XTEN80 linker. In some embodiments, linker RB comprises an XTEN16 linker, linker BD comprises an XTEN80 linker, and linker DD comprises the amino acid sequence SSGNSNANSRGPSFSSGLVPLSLRGSH (SEQ ID NO: 5).

[0017] In some embodiments, the transcriptional repressor domain is a KRAB domain. In some embodiments, the KRAB domain is a KRAB domain derived from KOX1. In some embodiments, the KRAB domain is a KRAB domain derived from ZIM3. In some embodiments, the KRAB domain comprises an amino acid sequence that is at least 90% identical to SEQ ID NO: 16 or thereto.

[0018] In some embodiments, the first DNMT domain or the second DNMT domain is a DNMT3A domain. In some embodiments, the DNMT3A domain is a human DNMT3A domain. In some embodiments, the DNMT3A domain is a mouse DNMT3A domain. In some embodiments, the DNMT3A domain comprises an amino acid sequence that is at least 90% identical to SEQ ID NO: 13 or thereto.

[0019]

[0019] In some embodiments, the first DNMT domain or the second DNMT domain is a DNMT3L domain. In some embodiments, the DNMT3L domain is a human DNMT3L (hD3L) domain. In some embodiments, the hD3L domain comprises an amino acid sequence that is at least 90% identical to SEQ ID NO: 14. In some embodiments, the DNMT3L domain is a mouse DNMT3L (mD3L) domain.

[0020]

[0020] In some embodiments, the mD3L domain comprises an amino acid sequence that is at least 90% identical to SEQ ID NO: 69. In some embodiments, the first DNMT domain is a DNMT3A domain and the second DNMT domain is a DNMT3L domain. In some embodiments, the first DNMT domain is a DNMT3L domain and the second DNMT domain is a DNMT3A domain.

[0021]

[0021] In some embodiments, at least one of the DNA binding domains comprises a zinc finger motif or a zinc finger array. In some embodiments, at least one of the DNA binding domains comprises a nucleic acid-inducible DNA binding domain. In some embodiments, at least one of the DNA binding domains comprises an RNA-inducible DNA binding domain. In some embodiments, at least one of the DNA binding domains comprises a DNA binding domain of a CRISPR-Cas protein. In some embodiments, the CRISPR-Cas protein comprises nuclease-inactive Cas9 (dCas9), nuclease-inactive Cas12a (dCas12a), or nuclease-inactive CasX (dCasX).

[0022]

[0022] (a) A first fusion protein comprising a first DNA binding domain, a first linker, and a first DNA methyltransferase (DNMT) domain, and a second fusion protein comprising a second DNA binding domain, a second linker, and a transcriptional repressor domain, wherein the first linker and / or the second linker comprises (GGGGS)4 (SEQ ID NO: 4); or (b) An epigenetic editing system comprising a nucleic acid molecule encoding the fusion protein of (a) is also described herein.

[0023]

[0023] (a) A first fusion protein comprising a first DNA binding domain, a first linker (linker BD), and a first DNMT domain, and a second fusion protein comprising a second DNA binding domain, a second linker (linker RB), and a transcriptional repressor domain, wherein the first fusion protein comprises, from the N-terminus to the C-terminus, the first DNMT domain, the first linker (linker BD), and the first DNA binding domain, and / or the second fusion protein comprises, from the N-terminus to the C-terminus, the transcriptional repressor domain, the second linker (linker RB), and the second DNA binding domain; or (b) An epigenetic editing system comprising a nucleic acid molecule encoding the fusion protein of (a) is also described herein.

[0024]

[0024] (a) A first fusion protein comprising a first DNA binding domain, a first linker (linker BD), and a first DNMT domain, wherein the first DNMT domain is a DNMT3L (hD3L) domain, and a second fusion protein comprising a second DNA binding domain, a second linker (linker RB), and a transcriptional repressor domain; or (b) An epigenetic editing system comprising a nucleic acid molecule encoding the fusion protein of (a) is also described herein.

[0025]

[0025] In some embodiments, the DNT3L domain is a human DNMT3L (hD3L) domain. In some embodiments, hD3L comprises SEQ ID NO: 13.

[0026] (a) A first fusion protein comprising a first DNA binding domain, a first linker (linker BD), and a first DNMT domain, and a second fusion protein comprising a second DNA binding domain, a second linker (linker RB), and a transcriptional repressor domain, wherein the transcriptional repressor domain optionally comprises an amino acid sequence that is at least 90% identical to SEQ ID NO: 16 and that comprises a KRAB domain derived from ZIM3, or a KRAB domain derived from KOX10, or a KRAB domain derived from ZNF10; or (b) An epigenetic editing system comprising a nucleic acid molecule encoding the fusion protein of (a) is also described herein.

[0026]

[0027] In some embodiments, the first fusion protein comprises, from N-terminus to C-terminus, a first DNMT domain, a first linker (linker BD), and a first DNA binding domain, and / or the second fusion protein comprises, from N-terminus to C-terminus, a transcriptional repressor domain, a second linker (linker RB), and a second DNA binding domain. In some embodiments, the epigenetic editing system further comprises a third fusion protein comprising a third DNA binding domain, a third linker (linker BD), and a second DNMT domain, and optionally, the third fusion protein comprises, from N-terminus to C-terminus, a second DNMT domain, a third linker (linker BD), and a third DNA binding domain.

[0027]

[0028] In some embodiments, linker RB, linker BD, and / or linker DD comprise a flexible or unstructured peptide linker. In some embodiments, linker RB, linker BD, and / or linker DD comprise a glycine-rich and / or serine-rich polypeptide sequence. In some embodiments, the glycine-rich and / or serine-rich sequence comprises an amino acid sequence of at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 20, or at least 25 consecutive amino acid residues, and the amino acid sequence comprises at least 50% glycine and / or serine residues.

[0028]

[0029] In some embodiments, the glycine-rich and / or serine-rich polypeptide sequence comprises the sequence (G x S y ) z (wherein x is an integer from 1 to 10, y is an integer from 1 to 10, and z is an integer from 1 to 10). In some embodiments, x / y is at least 2, at least 3, at least 4, or at least 5. In some embodiments, x is 3. In some embodiments, x is 4. In some embodiments, x is 3 and y is 1. In some embodiments, x is 4 and y is 1. In some embodiments, z is 3, 4, or 5. In some embodiments, x is 4, y is 1, and z is 4.

[0029]

[0030] In some embodiments, linker RB comprises (GGGGS)4 (SEQ ID NO: 4), and / or linker DD comprises (GGGGS)4 (SEQ ID NO: 4). In some embodiments, linker DD comprises the amino acid sequence SSGNSNANSRGPSFSSGLVPLSLRGSH (SEQ ID NO: 5).

[0030]

[0031] In some embodiments, linker RB, linker BD, and / or linker DD comprises an XTEN linker. In some embodiments, linker RB, linker BD, and / or linker DD comprises an XTEN16 or XTEN80 linker. In some embodiments, linker RB and linker BD comprise an XTEN16 or XTEN80 linker. In some embodiments, linker RB comprises an XTEN16 linker and linker BD comprises an XTEN80 linker. In some embodiments, linker RB comprises an XTEN16 linker, linker BD comprises an XTEN80 linker, and linker DD comprises the amino acid sequence SSGNSNANSRGPSFSSGLVPLSLRGSH (SEQ ID NO: 5).

[0031]

[0032] In some embodiments, the transcriptional repressor domain is a KRAB domain. In some embodiments, the KRAB domain is a KRAB domain derived from KOX1. In some embodiments, the KRAB domain is a KRAB domain derived from ZIM3. In some embodiments, the KRAB domain comprises an amino acid sequence that is at least 90% homologous to SEQ ID NO: 16 or thereto.

[0032]

[0033] In some embodiments, the first DNMT domain or the second DNMT domain is a DNMT3A domain. In some embodiments, the DNMT3A domain is a human DNMT3A domain. In some embodiments, the DNMT3A domain is a mouse DNMT3A domain. In some embodiments, the DNMT3A domain comprises an amino acid sequence that is at least 90% homologous to SEQ ID NO: 13 or thereto.

[0033]

[0034] In some embodiments, the first DNMT domain or the second DNMT domain is a DNMT3L domain. In some embodiments, the DNMT3L domain is a human DNMT3L (hD3L) domain. In some embodiments, the hD3L domain comprises an amino acid sequence that is at least 90% identical to SEQ ID NO: 14. In some embodiments, the DNMT3L domain is a mouse DNMT3L (mD3L) domain. In some embodiments, the mD3L domain comprises an amino acid sequence that is at least 90% identical to SEQ ID NO: 69.

[0034]

[0035] In some embodiments, the first DNMT domain is a DNMT3A domain and the second DNMT domain is a DNMT3L domain. In some embodiments, the first DNMT domain is a DNMT3L domain and the second DNMT domain is a DNMT3A domain.

[0035]

[0036] In some embodiments, at least one of the DNA binding domains comprises a zinc finger motif or a zinc finger array. In some embodiments, at least one of the DNA binding domains comprises a nucleic acid-inducible DNA binding domain. In some embodiments, at least one of the DNA binding domains comprises an RNA-inducible DNA binding domain.

[0036]

[0037] In some embodiments, at least one of the DNA binding domains comprises the DNA binding domain of a CRISPR-Cas protein. In some embodiments, the CRISPR-Cas protein comprises nuclease-inactive Cas9 (dCas9), nuclease-inactive Cas12a (dCas12a), or nuclease-inactive CasX (dCasX). In some embodiments, the first fusion protein comprises any one of the sequences of SEQ ID NOs: 18-68 or 70-84. In some embodiments, the second fusion protein comprises any one of the sequences of SEQ ID NOs: 18-68 or 70-84. In some embodiments, the third fusion protein comprises any one of the sequences of SEQ ID NOs: 18-68 or 70-84.

[0037]

[0038] Also described herein is an epigenetic editing system comprising a fusion protein comprising an array of any of SEQ ID NOs: 18 to 68 or 70 to 84.

[0039] Also provided herein is a method of modifying the epigenetic state of a target gene in a mammalian cell, the method comprising contacting the cell with an epigenetic editing system.

[0038]

[0040] Also provided herein is a method of regulating the expression of a target gene in a mammalian cell, the method comprising contacting the cell with an epigenetic editing system.

[0041] Also provided herein is a method of modifying the epigenetic state of a target gene in a cell in a subject, the method comprising administering an epigenetic editing system to the subject.

[0039]

[0042] In some embodiments, the modification or regulation occurs at one or more off-target sites, and the percentage of modification or regulation occurring at the one or more off-target sites is less than 10%. In some embodiments, the percentage is less than 5%. In some embodiments, the percentage is less than 1%. In some embodiments, the percentage is less than 0.5%. In some embodiments, the percentage is less than 0.1%. In some embodiments, the modification or regulation is stable after one active cell replication of mammalian cells. In some embodiments, the modification or regulation is stable after two active cell replications of mammalian cells. In some embodiments, the modification or regulation is stable after five active cell replications of mammalian cells. In some embodiments, the modification or regulation is stable one week after contact of mammalian cells or administration to a subject. In some embodiments, the modification or regulation is stable two weeks after contact of mammalian cells or administration to a subject. In some embodiments, the modification or regulation is stable one month after contact of mammalian cells or administration to a subject. In some embodiments, the modification or regulation is stable three months after contact of mammalian cells or administration to a subject.

[0040]

[0043] Further aspects and advantages of the present disclosure will be readily apparent to those skilled in the art from the following detailed description, which is described by showing only exemplary embodiments of the present disclosure. As will be recognized, the present disclosure is capable of other different embodiments, and some of its details are modifiable in various obvious respects, all without departing from the present disclosure. Accordingly, the drawings and description should be regarded as illustrative in nature and not restrictive.

[0041] Incorporation by reference

[0044] All publications, patents, and patent applications mentioned in this specification are hereby incorporated by reference to the same extent as if each individual publication, patent, or patent application was specifically and individually indicated to be incorporated by reference. To the extent that the incorporated publications and patents or patent applications conflict with the disclosure contained herein, this specification is intended to supersede and / or take precedence over any such conflicting material.

[0042] Brief Description of the Drawings

[0045] The novel features of the invention are set forth in the appended claims. The understanding of the features and advantages of the invention will be enhanced by reference to the following detailed description that describes exemplary embodiments that utilize the principles of the invention and the accompanying drawings (referred to herein as "FIGURES").

Brief Description of the Drawings

[0043]

Figure 1

[0046] Figure 1 shows a schematic diagram of a triple CRISPR-based engineered transcriptional repressor (ETR), a triple TALE- or ZFP-based ETR, and a two-element or three-element ETR.

Figure 2

[0047] Figure 2 is a schematic diagram of an exemplary construct containing two epigenesencing-based platforms: a triple ETR combination and CRISPRoff.

Figure 3

[0048] Figure 3A shows the CRISPRoff platform when cloned into the same mammalian expression vector used for the CRISPRoff platform and the ETR. Figure 3B shows that CRISPRoff in the ETR backbone improves the silencing efficiency by 2-fold when compared to CRIPSRoff.

Figure 4

[0049] Figures 4A-4B show a comparison of the episilencing efficiency between untreated (UT), or triplex ETR combination- or CRISPRoff-transfected K-562 B2MtdTomato cells (Figure 4A). A similar analysis is shown for Hepa 1-6 Pcsk9tdTomato cells (Figure 4B). In both the K-562 B2MtdTomato cell line and the Hepa 1-6 Pcsk9tdTomato cell line, the triplex ETR combination outperformed CRISPRoff by 1.4-fold and 1.8-fold, respectively.

Figure 5

[0050] Figure 5A is a schematic diagram of ETR combinations with different linker sequences. The G linker is the GSGGG spacer sequence. The X linker is the XTEN linker (such as XTEN16 or XTEN80). The GX4 linker is a 4-tandem repeat of the GGGGS linker. Figure 5B shows the episilencing efficiency of different linker types compared to the parental triplex ETR combination and CRISPRoff using an equal amount of the unsaturated plasmid.

Figure 6

[0051] Figure 6A shows a schematic diagram of effector domains with ZIM3 or truncated DNMT3L (both of mouse (mD3L) and human (hD3L) origin). Figure 6B shows the episilencing efficiency of various effector domain constructs compared to CRISPRoff.

Figure 7

[0052] Figure 7 shows the episilencing efficiency of various effector domain inversion constructs compared to CRISPRoff. In both the K-562 B2MtdTomato cell type and the Hepa 1-6 Pcsk9tdTomato cell type, the ETR combination construct outperformed CRISPRoff by 1.6-fold and 1.8-fold, respectively.

Figure 8

[0053] Figures 8A-8B show a comparison of the epigenetic silencing efficiency when it was confirmed that the XTEN linker was detrimental, but the GX4 linker had improved epigenetic silencing efficiency for the platform. The fold change in epigenetic silencing efficiency between CRISPRoff and the double ETR combination containing dCas9:GX4:K and dCas9:GX4:hD3L (hereinafter referred to as d.ETRv.1) was 1.8 for K-562 B2MtdTomato and 2.3 for Hepa 1-6 Pcsk9tdTomato.

Figure 9

[0054] Figure 9A shows a schematic diagram of alternative KRAB and DNMT3L effector domains. Figure 9B shows the epigenetic silencing efficiency of alternative effector domain constructs compared to CRISPRoff. In both the K-562 B2MtdTomato cell type and the Hepa 1-6 Pcsk9tdTomato cell type, the ETR combination constructs are 3-fold and 3.8-fold more performant than CRISPRoff, respectively.

Figure 10

[0055] Figure 10A shows a schematic diagram of the all-in-one fusion construct. Figure 10B shows a comparison of the relative efficiency of the all-in-one fusion construct evaluated by plasmid nucleofection compared to cells treated with CRISPRoff.

Figure 11

[0056] Figure 11A shows a schematic diagram of the ZIM3 construct. Figure 11B shows a comparison of the relative efficiency of the ZIM3 construct evaluated by plasmid nucleofection compared to cells treated with CRISPRoff.

Figure 12

[0057] Figure 12A shows a schematic diagram of the effector domain inversion construct. Figure 12B shows a comparison of the relative efficiency of the effector domain inversion construct evaluated by plasmid nucleofection compared to cells treated with CRISPRoff.

Figure 13

[0058] Figure 13A shows a schematic diagram of a two-element ETR construct having a KRAB ZNF10 domain. Figure 13B shows a comparison of the relative efficiencies of the indicated two-element ETR constructs evaluated by plasmid nucleofection compared to cells treated with CRISPRoff.

Figure 14

[0059] Figure 14A shows a schematic diagram of a two-element ETR construct having a KRAB ZIM3 domain. Figure 14B shows a comparison of the relative efficiencies of the indicated two-element ETR constructs evaluated by plasmid nucleofection compared to cells treated with CRISPRoff.

Figure 15

[0060] Figure 15A shows a schematic diagram of a domain-inverted two-element ETR construct. Figure 15B shows a comparison of the relative efficiencies of the domain-inverted two-element ETR constructs evaluated by plasmid nucleofection compared to cells treated with CRISPRoff.

Figure 16

[0061] Figures 16A - 16C show flow cytometry data of B2M epigenetic silencing. Figure 16A shows CRISPRoff as a control. Figure 16B shows that the three-element ETR hD3A:hD3L:X80:dCas9:X16:K functions 9.4-fold better than the CRISPRoff control in the silencing of B2M. Figure 16C shows that the two-element ETR Z:GX4:dCas9:G:hD3L functions 6.3-fold better than the CRISPRoff control in the silencing of B2M.

Figure 17

[0062] Figures 17A - 17B show a ZFP-based ETR used to target the mouse Pcsk9 gene. Figure 17A shows a schematic diagram of the ZFP-based ETR. Figure 17B shows the effectiveness of different pDNA doses of the ZFP-based ETR when targeting the mouse Pcsk9 gene.

Figure 18

[0063] Figure 18A shows schematic diagrams of two three-element ETRs and a two-element ETR containing a ZFP DNA-binding domain. Figure 18B shows the effectiveness of different pDNA doses of the three-element ETR and the two-element ETR.

Figure 19-1

[0064] Figures 19A-19C show that in vitro screening in Hepa 1-6 Pcsk9tdTomato cells identified ZFP-based ETR as the most effective platform for Pcsk9 epigenetic silencing. The top of Figure 19A shows a schematic of the Hepa 1-6 Pcsk9tdTomato cell line in which the 2A-tdTomato cassette was targeted in-frame into the last exon of Pcsk9. The bottom of Figure 19A shows schematics of different ETR platforms showing their relative binding to the CpG island (CGI) of Pcsk9. Figure 19B shows a diagram of the experimental procedure used to compare the efficiency of different ETR platforms in the Hepa 1-6 Pcsk9tdTomato cell line. ETR was delivered into the cells using mRNA nucleofection. As an editing control, the cells were co-transfected with mRNA encoding Cas9 and a gRNA targeting the first exon of Pcsk9. Figure 19C shows representative flow cytometry dot plots of untreated (UT) or Hepa 1-6 Pcsk9tdTomato cells transfected with the indicated ETR constructs. SSC-A: side scatter area.

Figure 19-2

[0064] Figures 19A-19C show that in vitro screening in Hepa 1-6 Pcsk9tdTomato cells identified ZFP-based ETR as the most effective platform for Pcsk9 epigenetic silencing.

Figure 20-1

[0065] Figures 20A - 20G show the development of an effective epigenome editing platform for the persistent hit - end silencing of mouse Pcsk9. Figure 20A is a schematic showing, above, the Pcsk9 promoter region annotated with CpG islands (CGI), and, below, a zoom on the CGI showing the target sites of all the guide RNAs (gRNA; black arrows), ZFPs (black arrows) and TALEs (gray arrows) tested. The black arrow indicates the most active gRNA / DBD used for subsequent experiments. Figure 20B shows a schematic of the plasmid used for ETR expression, either after its direct transfection into cells or as a template for in vitro transcription (IVT) of the ETR mRNA. CMV: cytomegalovirus enhancer / promoter. T7: promoter for mRNA production. ATG: start codon; DBD: DNA - binding domain; SV40 NLS: nuclear localization signal from SV40; GSGGG: linker peptide; ED: effector domain of KRAB from either the ZNF10 protein, cdDNMT3A or DNMT3L; Wpre: woodchuck post - transcriptional regulatory element; 64A: stretch of 64 adenines; SpeI: restriction site used to linearize the plasmid for IVT; BGH polyA: polyadenylation signal from the bovine growth hormone gene. Figure 20C is a dot blot showing the percentage of Pcsk9tdTomato - negative cells over 22 days after delivery of a plasmid encoding the indicated dCas9 - based ETR and eight different gRNAs. gRNA#4 was the most active among the guides tested (black dots and connecting line) and was thus used for subsequent experiments. Data are reported as mean ± SD (n = 2). Figure 20D is a dot plot showing the percentage of Pcsk9tdTomato - negative cells over 22 days after delivery of a plasmid encoding 16 different ZFP DBDs fused to the KRAB domain of the ZNF10 protein. This experiment was meant to identify the most effective ZFP among those tested, using KRAB - mediated epigenetic silencing of Pcsk9 as a proxy for DBD efficiency.ZFP3, ZFP6, and ZFP8 were the most active among those tested (black dots and connecting lines) and were thus used for subsequent experiments. Data are reported as mean ± SD (n = 4). Figure 20E is similar to Figure 20D but shows experiments performed using DBDs based on 16 different TALEs. TALE2, TALE4, and TALE6 were the most active among those tested (black dots and connecting lines) and were thus used for subsequent experiments. Data are reported as mean ± SD (n = 4). The left panel of Figure 20F is a heatmap showing the percentage of Pcsk9tdTomato-negative cells on day 7 after delivery of combinations of plasmids encoding ETRs based on KRAB, DNMT3L, and cDNMT3A containing ZFPs. A matrix was constructed by transfecting all possible combinations of the three best-functioning ZFP DBDs (i.e., #3, #6, and #8) fused to either DNMT3L (Y-axis) or cdDNMT3A (X-axis) to any one of the ZFP-KRAB ETRs from Figure 20D. Considering their highest performance, triple ETR combinations containing ZFP8-KRAB, ZFP6-DNMT3L, and ZFP3-cDNMT3A were selected for further testing. Color intensity indicates mean silencing efficiency (n = 2). The right panel of Figure 20F is similar to the left panel of Figure 20F but shows experiments performed using the three best-functioning TALEs from Figure 20E. Considering their highest performance, triple ETR combinations containing TALE2-KRAB, TALE6-DNMT3L, and TALE4-cDNMT3A were selected for further testing. Color intensity indicates mean silencing efficiency (n = 3). Figure 20G shows time-course flow cytometry analysis of Pcsk9tdTomato-negative cells nucleofected with the selected ZFP-ETR, demonstrating the stability of episilencing. Data are reported as mean ± SD (n = 3).

Figure 20-2

[0065] Figures 20A - 20G show the development of an effective epigenome editing platform for sustained hit-end silencing of mouse Pcsk9.

Figure 20-3

[0065] Figures 20A - 20G show the development of an effective epigenome editing platform for persistent hit endoribosilencing of mouse Pcsk9.

Figure 21-1

[0066] Figures 21A - 21D show the editing of Pcsk9 in mice. Figure 21A is a bar graph showing the circulating levels of Pcsk9 (left) and the percentage of gene editing (right) on day 7 after injection of the indicated LNP formulation encapsulating mRNA encoding Cas9 and gRNA targeting the first exon of Pcsk9 (N = 4 for each group; treatment with LNP - A (NP ratio 9) and LNP - E (NP ratio 9) resulted in 2 and 1 deaths, respectively). Dots: data from individual mice normalized to pre - treatment levels. Bars: central levels for each group. Figure 21B is a bar graph showing the circulating levels of LDL - C in mice 30 days after the indicated treatment (N = 7 for ZFP - ETR, N = 3 for Cas9, N = 5 for mock, and N = 4 for vehicle - injected mice). Data are presented as mg / dL. Dots: individual mice. Bars: central levels for each group. Figure 21C shows the time course of transaminases (ALT: alanine transaminase; AST: aspartate aminotransferase) up to 30 days after treatment. Data are reported as the mean ± SD of plasma in ng / mL (N = 6 for any group). The gray area indicates physiological levels. Figure 21D shows the time course of circulating Pcsk9 up to 70 days after treatment. Data are reported as the mean ± SD of plasma in ng / mL (N = 22 for ETR, N = 7 for Cas9, N = 16 for mock, and N = 15 for vehicle - injected mice).

Figure 21-2

[0066] Figures 21A - 21D show the editing of Pcsk9 in mice.

Figure 22-1

[0067] Figures 22A - 22F show persistent epigenetic silencing of Pcsk9 in mouse hepatocytes and liver upon LNP - mediated delivery of ZFP - ETR. Figure 22A is a bar graph showing the levels of Pcsk9 in the supernatant of mouse hepatocytes upon transfection with three different doses of mRNA encoding either ZFP - ETR or eGFP (mock). Pcsk9 levels for each replicate and each group were normalized to the mean of the mock at the same dose. Data from individual replicates are reported as dots, while the bars represent the mean values (n = 3, mean ± SD). UD: undetectable. Figure 22B shows a schematic of the experimental procedure used to evaluate the efficacy and persistence of Pcsk9 editing in vivo. LNPs were loaded with mRNA encoding either ZFP - ETR, Cas9, or eGFP and injected separately into mice intravenously (IV). Blood samples were collected before and after LNP injection to measure the circulating levels of Pcsk9 by ELISA. LNP doses are shown as milligrams per kilogram (mg / Kg). PBS: vehicle. The left panel of Figure 22C is a bar graph showing the levels of Pcsk9 in the plasma of mice treated as indicated. Data (dots) for individual mice are reported as normalized to the mean of vehicle - treated mice. The bars represent the median value for any condition. The right panel of Figure 22C shows a time - course dot - plot of circulating Pcsk9 levels up to 330 days after LNP injection. Data are reported as mean ± SD (N = 7 for ZFP - ETR - injected mice, N = 3 for Cas9 - injected mice, N = 5 for mock - injected mice, and N = 4 for vehicle - injected mice; statistical analysis by two - way repeated - measures ANOVA and Dunnett's multiple - comparison test; *p - value ≤ 0.05, **p - value ≤ 0.01, ***p - value ≤ 0.001, ****p - value ≤ 0.0001). Figure 22D shows a schematic of the experimental procedure used to evaluate editing persistence during partial hepatectomy. BS - seq: bisulfite - sequencing. Figure 22E is a bar graph showing the circulating levels of Pcsk9 before and after partial hepatectomy for the indicated treatments.Data for individual mice are reported as normalized to the average of vehicle-treated mice (dots), while bars indicate the median (N = 4 for each experimental group; statistical analysis by two-way repeated measures ANOVA and Dunnett's multiple comparison test; *p value ≤ 0.05, **p value ≤ 0.01, ***p value ≤ 0.001, ****p value ≤ 0.0001). Figure 22F is a heatmap showing the average methylation levels at single CpG resolution within the Pcsk9 CGI region in treatment (ZFP-ETR) and control (vehicle) samples before and after partial hepatectomy (PH). The color intensity refers to the percentage of CpG methylation (average of N = 4 for each experimental group).

Figure 22-2

[0067] Figures 22A - 22F show persistent epigenetic silencing of Pcsk9 in mouse hepatocytes and liver upon LNP-mediated delivery of ZFP-ETR.

Figure 22-3

[0067] Figures 22A - 22F show persistent epigenetic silencing of Pcsk9 in mouse hepatocytes and liver upon LNP-mediated delivery of ZFP-ETR.

Figure 22-4

[0067] Figures 22A - 22F show persistent epigenetic silencing of Pcsk9 in mouse hepatocytes and liver upon LNP-mediated delivery of ZFP-ETR.

Figure 23

[0068] Figure 23A shows a triple ZFP ETR combination: the KRAB derived from ZNF10 operably linked to ZFP8 (referred to as ZFP8:G:K), the catalytic domain of DNMT3A operably linked to ZFP3 (referred to as ZFP3:G:D3A), and the human full-length DNMT3L operably linked to ZFP6 (referred to as ZFP6:G:hD3L), and the dCas9 domain of CRISPRoff, a ZFPoff construct replaced with ZFP8 used as a control. Figure 23B shows a comparison of the effectiveness of the triple ETR combination and ZFPoff in Hepa 1-6 Pcsk9tdTomato cells using plasmid-based transfection.

Figure 24A

[0069] Figure 24A shows the silencing efficiency of the optimized triple ETR combination, normalized to ZFPoff.

Figure 24B

Figure 25A

[0070] Figure 25A shows a two-element ETR structure tested for epigenetic silencing efficiency using ZFPoff as a control.

Figure 25B

Figure 26

[0071] Figure 26A shows data measuring the circulating levels of Pcsk9 from mice monitored over 30 days after injection of ETR packaged in lipid nanoparticles. Figure 26B shows the normalized data from Figure 26A.

Figure 27

[0072] Figures 27A - 27C show the specificity evaluation of ZFP-based ETR in volcano plots of differential gene expression. The vertical dotted line corresponds to the threshold of |log2FC| > 2. Upregulated genes are shown in purple, while downregulated genes are shown in yellow. Gray genes are not differentially expressed according to the applied threshold. Pcsk9 is shown. Figure 27A shows the differential gene expression in cells treated with the parental ZF-ETR. Figure 27B shows the differential gene expression in cells treated with h3Ls:X80:ZFP8:X16:K. Figure 27C shows the differential gene expression in cells treated with h3A:W:h3L:X80:ZFP8:X16:K.

Figure 28

[0073] Figures 28A - 28C show the specificity evaluation of dCas9 - based ETR in the volcano plot of differential gene expression. The vertical dotted line corresponds to the threshold of |log2FC|>2. Up - regulated genes are shown in purple, while down - regulated genes are shown in yellow. Gray genes are not differentially expressed according to the applied threshold. Pcsk9 is shown. Figure 28A shows the differential gene expression in cells treated with parental dCas9 - ETR. Figure 28B shows the differential gene expression in cells treated with m3Ls - XTEN80 - dCas9 - XTEN16 - ZIM3 (abbreviated as "m3Ls:X80:dCas9:X16:Z" in the drawing). Figure 28C shows the differential gene expression in cells treated with h3A - Wlink - h3L - XTEN80 - dCas9 - XTEN16 - KRAB (abbreviated as "h3A:W:h3L:X80:dCas9:X16:K" in the drawing).

Mode for Carrying Out the Invention

[0044] Detailed Description

[0074] Various embodiments of the present disclosure are shown and described herein, but it will be apparent to those skilled in the art that such embodiments are provided merely as examples. Numerous variations, modifications, and substitutions will occur to those skilled in the art without departing from the present disclosure. It is understood that various alternatives to the embodiments of the present disclosure described herein may be utilized.

[0045]

[0075] The practice of the present invention utilizes conventional techniques in chemistry, biochemistry, molecular biology, microbiology, and immunology, which are within the capabilities of those skilled in the art unless otherwise specified. Such techniques are explained in the literature. See, for example, Sambrook, J., Fritsch, E.F., and Maniatis, T. (1989) Molecular Cloning: A Laboratory Manual, 2nd ed., Cold Spring Harbor Laboratory Press; Ausubel, F.M. et al. (1995 and periodic supplements) Current Protocols in Molecular Biology, Chapters 9, 13, and 16, John Wiley & Sons; Roe, B., Crabtree, J., and Kahn, A. (1996) DNA Isolation and Sequencing: Essential Techniques, John Wiley & Sons; Polak, J.M., and McGee, J.O’D. (1990) In Situ Hybridization: Principles and Practice, Oxford University Press; Gait, M.J. (1984) Oligonucleotide Synthesis: A Practical Approach, IRL Press; and Lilley, D.M., and Dahlberg, J.E. (1992) Methods in Enzymology: DNA Structures Part A: Synthesis and Physical Analysis of DNA, Academic Press. Each of these general references is hereby incorporated by reference in its entirety into this specification.

[0046] Definitions

[0076] Whenever the terms "at least", "greater than", or "greater than or equal to" precede the first of a series of two or more numerical values, the terms "at least", "greater than", or "greater than or equal to" apply to each of the numerical values in that series. For example, 1, 2, or 3 or more is equivalent to 1 or more, 2 or more, or 3 or more.

[0047]

[0077] Whenever the terms "no more than", "less than", or "less than or equal to" precede the first of a series of two or more numerical values, the terms "no more than", "less than", or "less than or equal to" apply to each of the numerical values in that series. For example, 3, 2, or 1 or less is equivalent to 3 or less, 2 or less, or 1 or less.

[0048]

[0078] Absolute or sequential terms, such as "will", "will not", "shall", "shall not", "must", "must not", "first", "initially", "next", "subsequently", "before", "after", "lastly", and "finally", are not intended to limit the scope of the embodiments disclosed herein and are exemplary.

[0049]

[0079] As used herein, the singular forms "a", "an", and "the" are intended to include the plural forms as well, unless the context clearly dictates otherwise. Further, the terms "including", "includes", "having", "has", "with", or variations thereof are intended to be inclusive in the same manner as the term "comprising" as long as they are used in either the detailed description and / or claims.

[0050]

[0080] As used herein, the terms "hospital", "clinical site", "laboratory" or "laboratory site" refer to a hospital, clinic, pharmacy, research institution, pathology laboratory, or other commercial site where trained personnel are employed to process and / or analyze biological and / or environmental samples. These terms are contrasted with the clinical site, remote location, home, school, or other non-business, non-institutional site.

[0051]

[0081] The terms "determining", "measuring", "evaluating", "assessing", "assaying", and "analyzing" are frequently used interchangeably herein to refer to forms of measurement. These terms include determining whether an element is present or not (e.g., detection). These terms can include quantitative, qualitative, or both quantitative and qualitative determinations. Evaluations can be relative or absolute. "Detection of the presence of" can, depending on the context, include determining the amount of something present in addition to determining whether something is present or absent.

[0052]

[0082] The terms "subject", "patient" or "individual" are used interchangeably and frequently herein. A "subject" can be a biological entity containing expressed genetic material. The biological entity can be a plant, an animal, or a microorganism, including, for example, bacteria, viruses, fungi, and protozoa. A subject can be a tissue, cell, and their progeny of a biological entity obtained in vivo or cultured in vitro. A subject can be a mammal. The mammal can be a human. A subject can be diagnosed or suspected of having a high risk of a disease. In some cases, a subject is not necessarily diagnosed or suspected of having a high risk of a disease. A subject may or may not have been exposed to the pathogen of interest described herein, and may be symptomatic of a disease or condition associated with infection by or exposure to a pathogen described herein. In some embodiments, a subject is suspected of having been exposed to a pathogen, such as a virus. In some embodiments, a subject has been exposed to an antigen or protein representative of a particular pathogen, such as a virus, or cross-reacts with an antigen of a particular pathogen, such as a virus. In some embodiments, a subject has one or more symptoms indicative of a disease or condition associated with infection by or exposure to a pathogen described herein. In some embodiments, a subject is currently infected with a pathogen, such as a virus, described herein. In some embodiments, a subject has been previously infected with a pathogen described herein. In some embodiments, a subject is a carrier of a virus described herein. In some embodiments, a subject is a carrier of a fragment or remnant of a virus described herein. In some cases, a subject is a carrier of adaptive immunity resulting from a previous or current infection by a virus described herein. In some embodiments, a subject is a carrier of adaptive immunity resulting from a previous or current exposure to a different virus or pathogen other than the virus or pathogen of interest.

[0053]

[0083] The term "subject" includes mammals. Examples of mammals include any member of the class Mammalia, i.e., humans, non-human primates such as chimpanzees, other apes and monkey species; domestic animals such as cows, horses, sheep, goats, pigs; laboratory animals such as rabbits, dogs and cats; rodents such as rats, mice and guinea pigs, and the like, but are not limited thereto.

[0054]

[0084] The term "about" or "approximately" means within an acceptable error range for a particular value as determined by one of ordinary skill in the art, which is in part due to how that value is measured or determined, e.g., limitations of the measurement system. For example, "about" can mean within one or more than one standard deviation in the practice at a given value. When a particular value is recited in the application and claims, unless otherwise specified, the term "about" is assumed to mean an acceptable error range for the particular value.

[0055]

[0085] The expressions "at least one", "one or more" and "and / or" as used herein are open-ended expressions that are both conjunctive and disjunctive in practice. For example, each of the expressions "at least one of A, B and C", "at least one of A, B or C", "one or more of A, B and C", "one or more of A, B or C", "A, B and / or C" means A alone, B alone, C alone, A and B together, A and C together, B and C together, or A, B and C together.

[0056]

[0086] As used herein, the term "nucleic acid" refers to a polymer containing at least two nucleotides (i.e., deoxyribonucleotides or ribonucleotides) in either single-stranded or double-stranded form, including DNA and RNA. A "nucleotide" contains a sugar deoxyribose (DNA) or ribose (RNA), a base, and a phosphate group. Nucleotides are linked together via phosphate groups. A "base" includes purines and pyrimidines, which further include the natural compounds adenine, thymine, guanine, cytosine, uracil, inosine, as well as natural analogs, and synthetic derivatives of purines and pyrimidines, which include modifications that place new reactive groups, such as, but not limited to, amines, alcohols, thiols, carboxylates, and alkyl halides. Nucleic acids include nucleic acids containing known nucleotide analogs or modified backbone residues or backbone linkages, which are synthetic, natural, and non-natural and have binding properties similar to those of a reference nucleic acid. Examples of such analogs and / or modified residues include, but are not limited to, phosphorothioates, phosphoramidates, methylphosphonates, chiral methylphosphonates, 2'-O-methyl ribonucleotides, and peptide nucleic acids (PNAs).

[0057]

[0087] The term "nucleic acid" includes any oligonucleotide or polynucleotide having fragments containing up to 60 nucleotides, generally referred to as oligonucleotides, and longer fragments, referred to as polynucleotides. Deoxyribooligonucleotides consist of a pentose sugar called deoxyribose, which is covalently bound to phosphate esters at the 5' and 3' carbons of the sugar to form an unbranched alternating polymer. DNA can be, for example, in the form of antisense molecules, plasmid DNA, pre-condensed DNA, PCR products, vectors, expression cassettes, chimeric sequences, chromosomal DNA, or derivatives, and combinations of groups thereof. Ribooligonucleotides consist of a similar repeating structure, with the pentose sugar being ribose. Thus, the terms "polynucleotide" and "oligonucleotide" can refer to polymers or oligomers of nucleotides or nucleoside monomers consisting of native bases, sugars, and sugar-sugar (backbone) linkages. The terms "polynucleotide" and "oligonucleotide" can also include polymers or oligomers containing non-native monomers, or portions thereof that function similarly. Such modified or substituted oligonucleotides are generally preferred over their native counterparts, for example, due to properties such as enhanced cellular uptake, reduced immunogenicity, and increased stability in the presence of nucleases.

[0058]

[0088] The "nucleic acids" described in this specification may include one or more nucleotide variants that include non-standard nucleotides, unnatural nucleotides, nucleotide analogs, and / or modified nucleotides. Examples of modified nucleotides include, but are not limited to, diaminopurine, 5-fluorouracil, 5-bromouracil, 5-chlorouracil, 5-iodouracil, hypoxanthine, xanthine, 4-acetylcytosine, 5-(carboxyhydroxymethyl)uracil, 5-carboxymethylaminomethyl-2-thiouridine, 5-carboxymethylaminomethyluracil, dihydrouracil, beta-D-galactosylqueosine, inosine, N6-isopentenyladenine, 1-methylguanine, 1-methylinosine, 2,2-dimethylguanine, 2-methyladenine, 2-methylguanine, 3-methylcytosine, 5-methylcytosine, N6-adenine, 7-methylguanine, 5-methylaminomethyluracil, 5-methoxyaminomethyl-2-thiouracil, beta-D-mannosylqueosine, 5'-methoxycarboxymethyluracil, 5-methoxyuracil, 2-methylthio-N6-isopentenyladenine, uracil-5-oxyacetic acid (v), wybutoxosine, pseudouracil, queosine, 2-thiocytosine, 5-methyl-2-thiouracil, 2-thiouracil, 4-thiouracil, 5-methyluracil, methyl ester of uracil-5-oxyacetic acid, 5-methyl-2-thiouracil, 3-(3-amino-3-N-2-carboxypropyl)uracil, (acp3)w, 2,6-diaminopurine, etc. In some cases, the nucleotide includes a modification in its phosphate ester moiety, including a modification to a triphosphate ester moiety. Non-limiting examples of such modifications include fairly long phosphate ester chains (e.g., phosphate ester chains having 4, 5, 6, 7, 8, 9, 10 or more phosphate ester moieties), and modifications that include a thiol moiety (e.g., alpha thiotriphosphate ester and beta thiotriphosphate ester).

[0059]

[0089] As used herein, a "nucleic acid" may be modified in the base moiety (e.g., typically one or more atoms available for forming hydrogen bonds with complementary nucleotides and / or one or more atoms typically unable to form hydrogen bonds with complementary nucleotides), the sugar moiety, or the phosphate backbone. Backbone modifications may include, but are not limited to, phosphorothioate, phosphorodithioate, phosphorosenoate, phosphorodiselenoate, phosphoranilothioate, phosphoranilidate, phosphoramidate, and phosphorodiamidate linkages. A phosphorothioate linkage substitutes a non-bridging oxygen with a sulfur atom in the phosphate ester backbone, retarding nuclease degradation of the oligonucleotide. A phosphorodiamidate linkage (N3’→P5’) enables prevention of nuclease recognition and degradation. Backbone modifications may also include a peptide bond (e.g., N-(2-aminoethyl)-glycine units linked by peptide bonds in peptide nucleic acids), or a carbamate, amide, and linker groups containing linear and cyclic hydrocarbon groups, in place of phosphorus in the backbone structure. Oligonucleotides having modified backbones are reviewed in Micklefield, Backbone modification of nucleic acids: synthesis, structure and therapeutic applications, Curr. Med. Chem., 8(10):1157-1179, 2001 and Lyer et al., Modified oligonucleotides-synthesis, properties and applications, Curr. Opin. Mol. Ther., 1(3):344-358, 1999. The nucleic acid molecules described herein may contain a sugar moiety containing ribose or deoxyribose as present in native nucleotides, or a modified sugar moiety, or a sugar analog.Examples of modified sugar moieties include, but are not limited to, 2'-O-methyl, 2'-O-methoxyethyl, 2'-O-aminoethyl, 2'-fluoro, N3'→P5' phosphoramidate, 2'dimethylaminooxyethoxy, 2'2'dimethylaminoethoxyethoxy, 2'-guanidinium, 2'-O-guanidiniumethyl, carbamate-modified sugars, and bicyclic-modified sugars. 2'-O-methyl or 2'-O-methoxyethyl modifications promote an A-type or RNA-like conformation in oligonucleotides, increase binding affinity for RNA, and have enhanced nuclease resistance. Modified sugar moieties can also include additional bridge bonds (e.g., a methylene bridge connecting the 2'-O atom and the 4'-C atom of ribose in locked nucleic acids) or sugar analogs, such as a morpholine ring (e.g., as in phosphorodiamidate morpholino).

[0060]

[0090] Unless otherwise specified, particular nucleic acid sequences implicitly include their conservatively modified variants (e.g., degenerate codon substitutions), alleles, orthologs, SNPs, and complementary sequences, as well as the explicitly recited sequences. Specifically, degenerate codon substitutions can be achieved by generating sequences in which the third position of one or more selected (or all) codons is substituted with a mixture of bases and / or deoxyinosine residues (Batzer et al., Nucleic Acid Res., 19:5081 (1991); Ohtsuka et al., J. Biol. Chem., 260:2605-2608 (1985); Rossolini et al., Mol. Cell. Probes, 8:91-98 (1994)).

[0061]

[0091] The present disclosure includes isolated or substantially purified nucleic acid molecules and compositions containing such molecules. As used herein, an “isolated” or “purified” DNA or RNA molecule is a DNA or RNA molecule that exists separate from its natural environment. An isolated DNA or RNA molecule can exist in a purified form or in a non-natural environment, such as in a transgenic host cell. For example, an “isolated” or “purified” nucleic acid molecule or a biologically active portion thereof is substantially free of other biological materials or culture medium when produced by recombinant techniques, or substantially free of chemical precursors or other chemicals when chemically synthesized. In one embodiment, an “isolated” nucleic acid does not include sequences that are naturally adjacent to the nucleic acid in the genomic DNA of the organism from which the nucleic acid is derived (i.e., sequences located at the 5′ and 3′ ends of the nucleic acid). For example, in some embodiments, an isolated nucleic acid molecule may contain less than about 5 kb, 4 kb, 3 kb, 2 kb, 1 kb, 0.5 kb, or 0.1 kb of nucleotide sequences that are naturally adjacent to the nucleic acid molecule in the genomic DNA of the cell from which the nucleic acid is derived.

[0062]

[0092] As used herein, the terms "protein", "polypeptide" and "peptide" are used interchangeably and refer to a polymer of amino acid residues linked by peptide bonds and can consist of two or more polypeptide chains. The terms "polypeptide", "protein" and "peptide" refer to a polymer of at least two amino acid monomers linked to each other via amide bonds. Amino acids can be L - optical isomers or D - optical isomers. More specifically, the terms "polypeptide", "protein" and "peptide" refer to, for example, a molecule consisting of two or more amino acids in a specific order determined by the nucleotide base sequence in the gene or RNA encoding that protein. Proteins are essential for the structure, function and regulation of the body's cells, tissues and organs, and each protein has a unique function. Examples are hormones, enzymes, antibodies, and fragments of any of them. In some cases, a protein can be a portion of that protein, for example, a domain, sub - domain or motif of a protein. In some cases, a protein can be a variant (or mutation) of that protein, provided that one or more amino acid residues are inserted into, deleted from, and / or substituted within the native (or at least known) amino acid sequence of that protein. A polypeptide can be a single - straight - chain polymer of amino acids linked to each other by peptide bonds between the carboxyl group and amino group of adjacent amino acid residues. A polypeptide can be modified, for example, by carbohydrate addition, phosphorylation, etc. A protein can contain one or more polypeptides.

[0063]

[0093] The protein or its variant can be native or recombinant. Methods for detecting and / or measuring polypeptides in biological substances are well known in the art and include, but are not limited to, Western blotting, flow cytometry, ELISA, RIA, and various proteomics techniques. An exemplary method for measuring or detecting a polypeptide is an immunoassay, such as ELISA. This type of protein quantification can be based on an antibody that can capture a specific antigen and a secondary antibody that can detect the captured antigen. Exemplary assays for detecting and / or measuring polypeptides are described in Harlow, E. and Lane, D. Antibodies: A Laboratory Manual (1988), Cold Spring Harbor Laboratory Press.

[0064]

[0094] As used herein, the term "fragment" or equivalent may refer to a portion of a protein that is less than the full length of the protein and optionally maintains the function of that protein. Further, when a portion of that protein is BLASTed against the protein, a portion of the protein sequence can align with at least 80% identity to a portion of the protein sequence, for example.

[0065]

[0095] Any system, method, and platform described herein is modular and not limited to sequential steps. Thus, terms such as "first" and "second" do not necessarily imply precedence, importance, or order of actions.

[0066]

[0096] The term "modulate" refers to a change in amount, degree of function, or range. For example, the epigenetic modification composition disclosed herein can modulate the activity of a promoter sequence by binding to a motif within the promoter, and as a result, induce, enhance, or suppress the transcription of a gene operably linked to the promoter sequence. Alternatively, modulation can include inhibition of gene transcription, provided that the epigenetic editing system binds to the structural gene and blocks DNA-dependent RNA polymerase from reading across the gene, thereby inhibiting gene transcription. The structural gene can be, for example, a normal cell gene or a cancer gene. Alternatively, modulation can include inhibition of transcription of the transcript. Thus, "modulation" of gene expression includes both gene activation and gene repression.

[0067]

[0097] As used herein, the terms "administer" and its grammatical equivalents can refer to the step of providing one or more pharmaceutical compositions described herein to a subject, patient, or sample. By way of example and not limitation, "administer" can be effected by intravenous (i.v.) injection, subcutaneous (s.c.) injection, intradermal (i.d.) injection, intraperitoneal (i.p.) injection, intramuscular (i.m.) injection, intravascular injection, infusion (inf.), oral route (p.o.), topical (top.) administration, or rectal (p.r.) administration. One or more such routes can be utilized. Parenteral administration can be, for example, by bolus injection or by gradual perfusion over time.

[0068]

[0098] As used herein, the terms "treat", "treating", or "treatment" and grammatical equivalents include alleviation, reduction, or improvement of at least one symptom of a disease or condition, prevention of additional symptoms, inhibition of a disease or condition, e.g., preventing the onset of a disease or condition, reducing a disease or condition, regression of a disease or condition, alleviation of a condition caused by a disease or condition, or arrest, either prophylactically and / or therapeutically, of symptoms of a disease or condition. "Treating" can relate to administration of a vector, nucleic acid (e.g., mRNA), or LNP composition to a subject after the onset of, or suspected onset of, a disease or condition. "Treat" includes the concept of "alleviate", and "alleviate" relates to attenuation of the frequency of occurrence or recurrence, or of any symptom or other adverse effect associated with a disease or condition and / or the severity of side effects associated with a disease or condition. The term "treat" also includes the concept of "manage", and "manage" relates to reduction in the severity of a particular disease or condition in a patient, or delay in its recurrence, e.g., prolongation of a remission period in a patient suffering from a disease. The term "treat" further includes the concepts of "prevent", "preventing", and "prevention". It is recognized, although not excluded as a possibility, that treatment of a disorder or condition does not require complete elimination of the disorder, condition, or associated symptoms. As used herein, the term "treatment" encompasses treatment of any disease in mammals, particularly humans, and includes (a) prevention of the occurrence of a disease in a subject who is susceptible to the disease but has not yet been diagnosed as having the disease; (b) inhibition of a disease, i.e., preventing its expression; or (c) alleviation of a disease, i.e., sedation or improvement of the disease and / or its symptoms or conditions. As used herein, the term "prevention" relates to measures taken for the prevention or partial prevention of a disease or condition.

[0069]

[0099] "Treatment or prevention of a condition" means improvement of a condition, sign, or symptom associated with a disorder, either before or after the onset of the disorder. For example, alleviation of the symptoms of a disorder, as measured by any standard technique, may include a reduction or prevention of at least 3%, 5%, 10%, 20%, 40%, 50%, 60%, 80%, 90%, 95%, 98%, 99%, 99.5%, 99.9%, or 100% compared to an equivalent untreated control. In some embodiments, alleviation of the symptoms of a disorder may include a reduction or prevention of at least 2-fold, at least 3-fold, at least 4-fold, at least 5-fold, at least 10-fold, at least 20-fold, at least 25-fold, at least 30-fold, at least 40-fold, at least 50-fold, at least 60-fold, at least 70-fold, at least 80-fold, at least 90-fold, at least 100-fold, at least 200-fold, at least 300-fold, at least 400-fold, at least 500-fold, at least 600-fold, at least 700-fold, at least 800-fold, at least 900-fold, at least 1000-fold, at least 2000-fold, at least 3000-fold, at least 4000-fold, at least 5000-fold, at least 6000-fold, at least 7000-fold, at least 8000-fold, at least 9000-fold, or at least 10000-fold compared to an equivalent untreated control.

[0070]

[0100] As used herein, the term "pharmaceutical composition" and its grammatical equivalents may refer to a mixture or solution that should be administered to a subject in need of administration, such as a human, comprising a therapeutically effective amount of an active pharmaceutical ingredient together with one or more pharmaceutically acceptable excipients, carriers, and / or therapeutic agents.

[0071]

[0101] As used herein, the term "pharmaceutically acceptable" and its grammatical equivalents can generally refer to the attributes of materials that are safe, non-toxic, not biologically or otherwise harmful, and useful in the preparation of pharmaceutical compositions that are acceptable for veterinary as well as human pharmaceutical use. "Pharmaceutically acceptable" can refer to materials, such as carriers or diluents, that do not inactivate the biological activity or properties of a compound and are relatively non-toxic, i.e., the material can be administered to a subject without causing undesirable biological effects or interacting in a deleterious manner with any of the components of the pharmaceutical composition containing the material.

[0072]

[0102] "Pharmaceutically acceptable excipients, carriers or diluents" refer to excipients, carriers or diluents that can be administered to a subject together with a drug, do not destroy its pharmacological activity, and are non-toxic at the dosage sufficient to deliver a therapeutically effective amount of the drug.

[0073]

[0103] A "pharmaceutically acceptable salt" can be an acid salt or a base salt that is generally considered suitable for use in contact with human or animal tissue in the art without undue toxicity, irritation, allergic response, or other problems or complications. Such salts include salts of basic residues such as mineral and organic salts of amines, and salts of acidic residues such as alkalis or organic salts of carboxylic acids. Specific pharmaceutical salts include salts of acids such as hydrochloric acid, phosphoric acid, hydrobromic acid, malic acid, glycolic acid, fumaric acid, sulfuric acid, sulfamic acid, sulfanilic acid, formic acid, toluenesulfonic acid, methanesulfonic acid, benzenesulfonic acid, ethanedisulfonic acid, 2-hydroxyethylsulfonic acid, nitric acid, benzoic acid, 2-acetoxybenzoic acid, citric acid, tartaric acid, lactic acid, stearic acid, salicylic acid, glutamic acid, ascorbic acid, pamoic acid, succinic acid, fumaric acid, maleic acid, propionic acid, hydroxymaleic acid, hydroiodic acid, phenylacetic acid, alkanoic acids such as acetic acid, HOOC-(CH2)n-COOH (where n is from 0 to 4), etc., but are not limited thereto. Similarly, pharmaceutically acceptable cations include, but are not limited to, sodium, potassium, calcium, aluminum, lithium, and ammonium. One of ordinary skill in the art will recognize from the present disclosure and knowledge in the art that additional pharmaceutically acceptable salts include those listed by Remington’s Pharmaceutical Sciences, 17th Edition, Mack Publishing Company, Easton, PA, page 1418 (1985). Generally, pharmaceutically acceptable acid salts or base salts can be synthesized from the parent compound containing a basic or acidic moiety by any conventional chemical method. Briefly, such salts can be prepared by reacting the free acid or free base form of those compounds with a stoichiometric amount of the appropriate base or acid in a suitable solvent.

[0074]

[0104] As used herein, the term "therapeutically effective amount" means an amount of an agent (e.g., nucleic acid, drug, payload, composition, therapeutic agent, diagnostic agent, prophylactic agent, etc.) to be delivered that is sufficient to treat, ameliorate, diagnose the onset of, prevent, and / or delay the symptoms of an infection, disease, disorder and / or condition in a subject that has or is susceptible to that infection, disease, disorder and / or condition.

[0075]

[0105] The term "repressor domain" or "transcriptional repressor domain" relates to a transcriptional repressor protein or a part thereof, e.g., a transcription factor, that can form a complex with one or more DNA-binding domains in order to act as a negative regulatory domain. The repressor domain blocks the recruitment of RNA polymerase in order to suppress the transcription of a particular gene. The repressor domain enables precise control of gene expression by inhibiting the activation of transcription through interaction with other cellular components, such as, but not limited to, basal transcription factors, effector molecules, activator or co-activator proteins, repressors, and co-repressors.

[0076]

[0106] The term "KRAB" refers to the Kruppel-associated box, a transcriptional repressor protein domain. KRAB refers to homologs, orthologs and variants of the KRAB domain in which the basic function of inhibiting the transcription of structural genes is conserved or enhanced. The KRAB domain is one of a group of transcriptional repressor domains present in approximately 400 human zinc finger protein-based transcription factors. The KRAB domain typically contains from about 45 to about 75 amino acid residues. Descriptions including its function and use regarding the KRAB domain can be found, for example, in Ecco, G., Imbeault, M., Trono, D., KRAB zinc finger proteins, Development 144, 2017.

[0077]

[0107] The term "DNMT" refers to DNA methyltransferase. This term as used herein includes enzymes that catalyze the transfer of a methyl group to DNA, such as to standard cytosine-5, and DNMTs (e.g., DNMT1, DNMT3A, DNMT3B, and DNMT3C) that catalyze the addition of a methyl group to genomic DNA. This term also includes non-standard family members that do not themselves catalyze methylation but recruit (including activating) catalytically active DNMTs, and non-limiting examples of such DNA methyltransferases include DNMT3L. See, e.g., Lyko, Nat Review. (2018) 19:81-92. Unless otherwise specified, a DNMT domain can refer to a polypeptide domain derived from a catalytically active DNMT (e.g., DNMT1, DNMT3A, and DNMT3B) or from a catalytically inactive DNMT (e.g., DNMT3L).

[0078]

[0108] The term "DNA binding domain" refers to DNA binding domains from proteins selected from the family of CRISPR proteins, TAL proteins, zinc fingers, and other transcriptional regulators, and homologs, orthologs, and variants thereof that maintain or improve the basic function of those DNA binding proteins.

[0079]

[0109] The ranges provided in this specification are to be understood as a shorthand for all values within those ranges. For example, a range of 1 to 50 is understood to include any number, combination of numbers, or sub-range from the group consisting of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49 or 50, as well as all fractional values between said integers, for example, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8 and 1.9. With respect to sub-ranges, “nested sub-ranges” that extend from one endpoint of the range are specifically intended. For example, nested sub-ranges of an exemplary range of 1 to 50 can include, in one direction, 1 to 10, 1 to 20, 1 to 30 and 1 to 40, or in the other direction, 50 to 40, 50 to 30, 50 to 20 and 50 to 10.

[0080]

[0110] The term “therapeutic agent” can refer to any agent that, when administered to a subject, has a therapeutic effect, a diagnostic effect, and / or a prophylactic effect, and / or induces a desirable biological and / or pharmacological effect. A therapeutic agent can also be referred to as an “active” or “active agent”. Such agents include, but are not limited to, cytotoxins, radioactive ions, chemotherapeutic agents, small molecule drugs, proteins, and nucleic acids.

[0081]

[0111] As used herein, the term “ameliorate” can refer to a decrease, inhibition, attenuation, reduction, arrest, or stabilization of the expression or progression of a disease.

[0112] As used herein, "Delaying" the onset of a disease means extending, preventing, decelerating, retarding, stabilizing, and / or postponing the progression of the disease. This delay can vary in duration depending on the medical history and / or the individual being treated. A method for "delaying" or alleviating the onset of a disease, or delaying the development of a disease, is a method that reduces the likelihood of one or more symptoms of the disease occurring within any time frame and / or reduces the degree of symptoms within any time frame as compared to not using the method. Such comparisons are typically based on clinical trials involving a fairly large number of subjects sufficient to yield statistically significant results.

[0082]

[0113] "Expression" or "progression" of a disease means the initial symptoms of the disease and / or subsequent progression. The expression of a disease may be detectable and evaluable using standard clinical techniques well known in the art. However, expression also refers to progression that may not be detectable. For the purposes of the present disclosure, expression or progression refers to the biological progression of symptoms. "Expression" includes occurrence, recurrence, and onset.

[0083]

[0114] As used herein, "onset" or "occurrence" of a disease includes initial onset and / or recurrence. Conventional methods known to those of ordinary skill in the medical art can be used to administer the isolated polypeptide or pharmaceutical composition to a subject, depending on the type of disease or the site of the disease to be treated. The composition can also be administered via other conventional routes, for example, orally, parenterally, by inhalation spray, topically, rectally, nasally, buccally, vaginally, or via an implantable reservoir.

[0084]

[0115] As used herein, the term "parenteral" includes subcutaneous, intradermal, intravenous, intramuscular, intra-articular, intra-arterial, intra-synovial, intrasternal, intrathecal, intralesional, and intracranial injection, or infusion techniques. Further, for example, it may be administered to a subject via an injectable depot administration route using depot injection or biodegradable materials and methods for 1 month, 3 months, or 6 months.

[0085]

[0116] In addition to the specific proteins and nucleotides referred to herein, the present invention is understood to contemplate the use of their variants, derivatives, homologs and fragments. As used herein, a variant of any given sequence is a sequence in which the specific sequence of residues (whether amino acid residues or nucleic acid residues) in that sequence is modified in such a way that the polypeptide or polynucleotide substantially retains at least one of its native functions. Variant sequences can be obtained by addition, deletion, substitution, modification, replacement and / or variation of at least one residue present in the native protein. As used herein, a derivative of any given sequence of interest is, on the premise that the resulting protein or polypeptide substantially retains at least one of its native functions, any substitution, variation, modification, replacement, deletion and / or addition of one (or more) amino acid residues from or to that sequence. Amino acid substitutions can be made, for example, by substitutions from 1, 2 or 3 to 10 or 20, as long as the modified sequence substantially retains the required activity or ability. Amino acid substitutions can include the use of non-natural analogs. The proteins used in the present disclosure can also have deletions, insertions or substitutions of amino acid residues that result in a functionally equivalent protein without affecting the function of the protein. Intentional amino acid substitutions can be made based on the similarity of the polarity, charge, solubility, hydrophobicity, hydrophilicity and / or amphipathicity of the residues, as long as the native function is retained. For example, negatively charged amino acids include aspartic acid and glutamic acid; positively charged amino acids include lysine and arginine; amino acids with similar hydrophilicity values with uncharged polar head groups include asparagine, glutamine, serine, threonine and tyrosine.

[0086]

[0117] Homologs of any protein or nucleic acid sequence as used and intended herein include sequences having a certain degree of homology to the wild-type amino acid and nucleic acid sequences. Homologous sequences can include sequences that are at least 50%, 55%, 65%, 75%, 85% or 90% identical to the target sequence, for example, amino acid sequences. In certain embodiments, the homologous sequences can include amino acid sequences that are at least 95% or 97% or 99% identical to the target sequence.

[0087]

[0118] Sequence identity can be measured using sequence analysis software (e.g., the sequence analysis software package from Genetics Computer Group, University of Wisconsin Biotechnology Center, 1710 University Avenue, Madison, Wis. 53705, BLAST, BESTFIT, GAP, or the PILEUP / PRETTYBOX programs). Such software matches identical or similar sequences by assigning a degree of homology to various substitutions, deletions, and / or other modifications. Conservative substitutions typically include substitutions within the following groups: glycine, alanine; valine, isoleucine, leucine; aspartic acid, glutamic acid, asparagine, glutamine; serine, threonine; lysine, arginine; and phenylalanine, tyrosine. In one exemplary approach for determining the degree of identity, the BLAST program can be used with a probability score between e-3 and e-100 that indicates related sequences.

[0088]

[0119] It is understood that the numbering of a particular position or residue in each sequence depends on the particular protein and the numbering scheme used. The numbering may differ, for example, in the precursor of the mature protein and in the mature protein itself, and sequence differences between species may affect the numbering. One of ordinary skill in the art can identify each residue in any homologous protein and in each encoding nucleic acid by methods well known in the art, such as sequence alignment and determination of homologous residues.

[0089] Epigenetic editing system

[0120] An epigenetic editing system for epigenetic modification and expression regulation of a target gene is described herein. The epigenetic editing system used for gene silencing may include a fusion protein or a nucleic acid encoding the fusion protein. In some embodiments, the fusion protein includes a DNA binding domain. In some embodiments, the fusion protein includes a DNA methyltransferase (DNMT) domain. In some embodiments, the fusion protein includes a repressor domain.

[0090]

[0121] The epigenetic editing system used herein can be any agent that binds to a target polynucleotide and has epigenetic regulatory activity. In some embodiments, the epigenetic editing system uses a DNA binding domain to bind to a polynucleotide at a specific sequence. In some embodiments, the epigenetic editing system uses a nucleic acid-inducible DNA binding protein to bind to a polynucleotide at a specific sequence. In some embodiments, the epigenetic editing system includes an effector domain that can regulate the epigenetic state of a nucleic acid sequence in a target polynucleotide or a nucleic acid sequence adjacent to the target polynucleotide. In some embodiments, the epigenetic editing system can place an epigenetic editing mark on a chromatin region, nucleic acid sequence, or histone amino acid residue in or adjacent to the target polynucleotide. For example, the epigenetic editing system can methylate, demethylate, acetylate, deacetylate, ubiquitinate, or deubiquitinate a chromatin region, nucleic acid sequence, or histone amino acid residue in or adjacent to the target polynucleotide. In some embodiments, the epigenetic editing system can recruit one or more proteins or complexes involved in transcriptional regulation, such as transcription factors, transcriptional activators, transcriptional repressors, or insulators, to a chromatin region, nucleic acid sequence, or histone amino acid residue in or adjacent to the target polynucleotide.

[0091]

[0122] The epigenetic editing system provided herein can include one or more of the effector domains described. In some embodiments, the epigenetic editing system includes a plurality of effector domains. In some embodiments, the epigenetic editing system includes one effector domain. In some embodiments, the epigenetic editing system includes at least two, three, four, five, six, seven, eight, nine, ten, or more effector domains.

[0092]

[0123] In some embodiments, the epigenetic editing system includes a DNA methylation domain, a repression domain, and a nucleic acid binding domain. In some embodiments, the epigenetic editing system includes a DNA methylation domain and a nucleic acid binding domain. In some embodiments, the nucleic acid binding domain is at the C-terminus of the fusion protein. In some embodiments, the nucleic acid binding domain is at the N-terminus of the fusion protein. In some embodiments, the nucleic acid binding domain is in the middle of the fusion protein. In some embodiments, the DNA methylation domain is at the C-terminus of the fusion protein. In some embodiments, the DNA methylation domain is at the N-terminus of the fusion protein. In some embodiments, the DNA methylation domain is in the middle of the fusion protein. In some embodiments, the repression domain is at the C-terminus of the fusion protein. In some embodiments, the repression domain is at the N-terminus of the fusion protein. In some embodiments, the repression domain is in the middle of the fusion protein. In some embodiments, the epigenetic editing system includes a DNA demethylation domain and a histone acetylation domain. In some embodiments, the epigenetic editing system includes a DNA demethylation domain and an activation domain that recruits additional DNA demethylation proteins or histone acetylation proteins. In some embodiments, the epigenetic editing system includes a DNA demethylation domain, a histone acetylation domain, and a scaffold protein that recruits additional DNA demethylation proteins or histone acetylation proteins. In some embodiments, the epigenetic editing system includes two or more DNA demethylation domains, two or more histone acetylation domains, and / or two or more scaffold proteins that recruit additional DNA demethylation proteins or histone deacetylation proteins.

[0093]

[0124] In some embodiments, the epigenetic editing system includes a KRAB domain and a DNMT3 domain, both of which can act synergistically on the enhanced downregulation or silencing of the target gene expression as compared to an epigenetic effector having only one of the two repressor domains. In some embodiments, the epigenetic editing system includes a KRAB domain, a DNMT3A domain, and a DNMT3L domain. In some embodiments, the epigenetic editing system includes a configuration of a DNA binding domain sandwiched between a KRAB domain and a DNMT3A-DNMT3L fusion protein domain. In some embodiments, the epigenetic editing system includes the following configuration: N-[KRAB]-[DNA binding domain]-[Dnmt3A-Dnmt3L]-C (wherein, "]-[ " is any nuclear localization signal, any tag sequence, or any linker provided herein).

[0094]

[0125] The effector domain of the epigenetic editing system can be linked to another effector domain via direct fusion or via any linker described herein. The effector domain and the DNA binding domain of the epigenetic editing system can also be linked via direct fusion or via any linker described herein. The epigenetic editing system provided herein may be a two-element fusion protein. The epigenetic editing system provided herein may be a three-element fusion protein.

[0095]

[0126] In some embodiments, two or more effector domains are the same. In some embodiments, two or more effector domains belong to the same protein family. In some embodiments, two or more effector domains are different proteins included in the same transcriptional or regulatory mechanism.

[0096]

[0127] To achieve the activation or suppression of one or multiple target genes, multiple fusion proteins or constructs can be used. For example, an epigenetic editing system includes a fusion protein containing a DNA binding domain (e.g., dCas9 domain) and a methylation domain, as well as other fusion proteins containing a DNA binding domain and a repressor domain, and can be co-delivered by two or more guide RNAs, each targeting a different target DNA sequence. The two or more target DNA sequences may be within the same target gene or within different target genes.

[0097]

[0128] The epigenetic editing system may include two or more fusion proteins that result in the activation or suppression of a target gene or multiple target genes. The epigenetic editing system may include a first fusion protein containing a first DNA binding domain and a DNMT domain and a second fusion protein containing a second DNA binding domain and a transcriptional repressor domain. The epigenetic editing system may include a linker (e.g., (GGGGS)4) between the first DNA binding domain and the DNMT domain. The epigenetic editing system may include a linker (e.g., (GGGGS)4) between the second DNA binding domain and the transcriptional repressor domain. In some embodiments, the epigenetic editing system may include a third fusion protein containing a third DNA binding domain, a third linker, and a second DNMT domain.

[0098]

[0129] The epigenetic editing system may include a fusion protein that brings about the activation or suppression of a target gene or a plurality of target genes. The three-element fusion protein may include a transcriptional repressor domain, a DNA binding domain, a first DNMT domain, and a second DNMT domain. The three-element fusion protein construct may include a linker (e.g., (GGGGS)4) between the transcriptional repressor domain and the DNA binding domain. The three-element fusion protein construct may include a linker (e.g., (GGGGS)4) between the DNA binding domain and the first DNMT domain. The three-element fusion protein construct may include a linker (e.g., (GGGGS)4) between the first DNMT domain and the second DNMT domain.

[0099] Effector domain

[0130] The epigenetic system provided herein may include one or more effector protein domains that regulate the expression of a target gene. The effector domain can be used to contact a target polynucleotide sequence in the target gene to achieve an epigenetic modification, such as a change in the methylation state of DNA nucleotides in the target gene. Thus, an epigenetic editing system that includes one or more effector domains can provide the effect of regulating the expression of a target gene without changing the DNA sequence of the target gene. For example, in some embodiments, the effector domain results in the suppression or silencing of the expression of the target gene. In some embodiments, the effector domain results in the activation or upregulation of the expression of the target gene.

[0100]

[0131] Epigenetic effectors can chemically modify chromatin at the location of target genes. Non-limiting examples of chemical modifications include methylation, demethylation, acetylation, deacetylation, phosphorylation, SUMOylation, and / or ubiquitination of DNA or histone residues of chromatin. In some embodiments, the epigenetic effector can effect histone tail modification. In some embodiments, the epigenetic effector can add or remove active marks on histone tails. In some embodiments, active marks can include H3K4 methylation, H3K9 acetylation, H3K27 acetylation, H3K36 methylation, H3K79 methylation, H4K5 acetylation, H4K8 acetylation, H4K12 acetylation, H4K16 acetylation, and / or H4K20 methylation. In some embodiments, the epigenetic effector can add or remove repressive marks on histone tails. In some embodiments, those repressive marks can include H3K9 methylation and / or H3K27 methylation.

[0101]

[0132] In some embodiments, the effector domain in an epigenetic editing system changes the chemical modification state of a target gene containing a target sequence. For example, the effector domain can change the chemical modification state of nucleotides in the target gene. In some embodiments, the effector domain of the epigenetic editing system chemically modifies nucleotides in the target gene. In some embodiments, the effector domain of the epigenetic editing system chemically modifies histones associated with the target gene. In some embodiments, the effector domain of the epigenetic editing system removes chemical modifications at nucleotides in the target gene. In some embodiments, the effector domain of the epigenetic editing system removes chemical modifications of histones associated with the target gene. In some embodiments, the chemical modification increases the expression of the target gene. For example, the epigenetic editing system can include an effector domain having histone acetyltransferase activity. In some embodiments, the chemical modification decreases the expression of the target gene. For example, the epigenetic editing system can include an effector domain having DNA methyltransferase activity.

[0102]

[0133] Epigenetic modifications mediated by an epigenetic editing system can be in the vicinity of the target gene, can be distant from the target gene, or can spread from an initial epigenetic modification initiated by the epigenetic editing system at one or more nucleotides in the target sequence of the target gene.

[0103]

[0134] In some embodiments, the change in the chemical modification state is the DNA methylation state. For example, methylation can be introduced by an effector domain having DNA methyltransferase activity or removed by an effector domain having DNA demethylase activity. Alternatively, methylation can be introduced into an effector domain having recruitment activity (e.g., DNMT3L) to recruit an additional effector domain having DNA methyltransferase activity (e.g., DNMT3A / B). In some embodiments, the change in chemical modification, e.g., methylation, is in a hypomethylated nucleic acid sequence. For example, the chemically modified sequence at the target gene or chromosomal region can lack a methyl group on a 5-methylcytosine nucleotide (e.g., in CpG). Hypomethylation can occur, for example, in senescent cells or cancer (e.g., early stages of neoplasia) as opposed to young cells or non-cancerous cells, respectively. In some embodiments, the target polynucleotide sequence is within a CpG island. In some embodiments, the target gene is known to be associated with a disease or condition. In some embodiments, the target gene contains a specific copy of a disease-related sequence. In some embodiments, the target gene contains a target sequence associated with a disease.

[0104]

[0135] In some embodiments, the change in chemical modification, e.g., methylation, is in a hypermethylated nucleic acid sequence. In some embodiments, the chemical modification is within a CpG island.

[0105]

[0136] In some embodiments, the protein fusion construct can have one effector domain, two effector domains, four effector domains, five effector domains, six effector domains, seven effector domains, eight effector domains, nine effector domains, or ten effector domains.

[0106]

[0137] Methyltransferase domain

[0138] In some embodiments, the effector domain comprises a histone methyltransferase domain. Alternatively, the effector domain may comprise a recruiter domain that recruits a subsequent domain having enzymatic activity (e.g., a histone methyltransferase domain). For example, repression (or silencing) can result from a repressive chromatin marker, DNA methylation, methylation of histone residues (e.g., H3K9, H3K27), or deacetylation of histone residues on chromatin containing the target nucleic acid sequence. Without intending to be bound by theory, the method can be used to alter the epigenetic state, for example, by closing chromatin via methylation or by introducing a repressive chromatin marker on chromatin containing the target nucleic acid sequence (e.g., a gene).

[0107]

[0139] Specific epigenetic imprints dictate gene transcription or gene silencing. For example, DNA methylation, histone modification, repressor protein binding to silencer regions, and other transcriptional activities can alter gene expression without changing the underlying DNA sequence. Thus, transcriptional regulation enables the expression of specific genes in a particular way while suppressing other genes. In some cases, cell fate or function can be controlled during early differentiation (e.g., during the development of an organism) or to reprogram cells or cell types (e.g., during diseases such as cancer, chronic inflammation, autoimmune diseases, diseases related to various microbiomes of an organism, etc.). Histone modification plays a structural and biochemical role in gene transcription by forming or disrupting nucleosome structures that bind to histones and prevent gene transcription. Histones are generally found in the nuclei of eukaryotic cells, ranging from multicellular organisms including humans to single-celled organisms represented by fungi (filamentous fungi and yeasts), and are basic proteins that bind ionically to genomic DNA. Histones usually consist of five components (H1, H2A, H2B, H3, and H4) and are very similar across species. For example, in the case of histone H4, the budding yeast histone H4 (full-length 102 amino acid sequence) and human histone H4 (full-length 102 amino acid sequence) are identical in 92% of the amino acid sequence and differ only at 8 residues. Among the natural proteins assumed to exist in tens of thousands of organisms, histones are known to be the most highly conserved proteins among eukaryotic species. Genomic DNA is folded with histones by ordered binding, and both complexes form a basic structural unit called a nucleosome. Furthermore, the aggregation of nucleosomes forms the chromosomal chromatin structure. Histones can undergo modifications, such as acetylation, methylation, phosphorylation, ubiquitination, SUMOylation, etc., at their N-terminus, called the histone tail, to maintain or specifically transform the chromatin structure, thereby controlling responses that occur on chromosomal DNA, such as gene expression, DNA replication, DNA repair, etc.Post-translational modification of histones is an epigenetic regulatory mechanism and is considered essential for genetic regulation in eukaryotic cells. Recent studies have revealed that chromatin remodeling factors that promote DNA access to transcription factors through modification of the nucleosome structure, such as SWI / SNF, RSC, NURF, NRD, etc., histone acetyltransferases (HATs) that regulate the acetylation state of histones, and histone deacetylases (HDACs) act as important regulatory factors. DNA methylation mainly occurs at CpG sites (a simplified notation of "C-phosphate ester-G-" or "cytosine-phosphate ester-guanine"). Highly methylated DNA regions tend to have lower transcriptional activity than less methylated sites. Many mammalian genes are located near CpG islands (regions with a high frequency of CpG sites) or have promoter regions containing CpG islands.

[0108]

[0140] In particular, the unstructured N-terminus of histones can be modified by at least one of acetylation, methylation, ubiquitination, phosphorylation, sumoylation, ribosylation, citrullination, O-GlcNAcylation, or crotonylation. For example, acetylation of lysines K14 and K9 of histone H3 by histone acetyltransferase enzymes can be linked to transcriptional capacity in humans. Lysine acetylation can directly or indirectly create binding sites for chromatin-modifying enzymes that regulate transcriptional activation. For example, histone acetyltransferase (HAT) utilizes acetyl-CoA as a cofactor to catalyze the transfer of an acetyl group to the epsilon amino group of the lysine side chain. Since it neutralizes the positive charge of lysine and weakens the interaction between histones and DNA, it opens the chromosome, allowing transcription factors to bind and initiate transcription. Similarly, methylation of lysine 9 of histone H3 can be associated with heterochromatin, or transcriptionally silent chromatin. Specific DNA methylation patterns can be established and modified by at least one or more, two or more, three or more, four or more, or five or more independent DNA methyltransferases, including DNMT1, DNMT3A, and DNMT3B.

[0109]

[0141] In some embodiments, the effector domain comprises a histone methyltransferase domain. In some embodiments, the effector domain comprises a DOT1L domain, a SET domain, a SUV39H1 domain, a G9a / EHMT2 protein domain, an EZH1 domain, an EZH2 domain, a SETDB1 domain, or any combination thereof. In some embodiments, the effector domain comprises a histone-lysine-N-methyltransferase SETDB1 domain.

[0110]

[0142] In some embodiments, the effector domain comprises a DNA methyltransferase domain or a histone methyltransferase domain. The DNA methyltransferase domain can mediate methylation at DNA nucleotides, for example, at any of A, T, G, or C nucleotides. In some embodiments, the methylated nucleotide is N6-methyladenosine (m6A). In some embodiments, the methylated nucleotide is 5-methylcytosine (5mC). In some embodiments, the methylation occurs in a CG (or CpG) dinucleotide sequence. In some embodiments, the methylation occurs in a CHG or CHH sequence, where H is any one of A, T, or C.

[0111]

[0143] In some embodiments, the effector domain comprises a DNA methyltransferase DNMT domain that catalyzes the transfer of a methyl group to cytosine, thereby suppressing the expression of the target gene through the recruitment of a repressive regulatory protein. In some embodiments, the effector domain comprises a DNA methyltransferase (DNMT) family protein domain. In some embodiments, the effector domain comprises a DNMT1 domain. In some embodiments, the effector domain comprises a TRDMT1 domain. In some embodiments, the effector domain comprises a DNMT3 domain. In some embodiments, the effector domain comprises a DNMT3A domain. In some embodiments, the effector domain comprises a DNMT3B domain. In some embodiments, the effector domain comprises a DNMT3C domain. In some embodiments, the effector domain comprises a DNMT3L domain. In some embodiments, the effector domain comprises a fusion of the DNMT3A-DNMT3L domains.

[0112]

[0144] Exemplary methyltransferases that can be part of the epigenetic effector domain are provided in Table 1 below.

[0113]

Table 1

[0114]

[0145] Repressor domain

[0146] In some embodiments, the effector domain suppresses the expression of the target gene. Alternatively, or additionally, in some embodiments, the effector domain recruits one or more protein domains that suppress the expression of the target gene. In some embodiments, the effector domain interacts with a scaffold protein domain that recruits one or more protein domains that suppress the expression of the target gene. For example, the effector domain may recruit or interact with a scaffold protein domain that recruits a PRMT protein, an HDAC protein, a SETDB1 protein, or a NuRD protein domain. In some embodiments, the effector domain comprises a Kruppel-associated box (KRAB) repression domain; a repressor element silencing transcription factor (REST) repression domain, a KRAB-associated protein 1 (KAP1) domain, a MAD domain, a FKHR (forkhead in rhabdomyosarcoma gene) repressor domain, an EGR-1 (early growth response gene product 1) repressor domain, an ets2 repressor factor repressor domain (ERD), a MAD smSIN3 interaction domain (SID), the WRPW motif of a hairy-related basic helix-loop-helix (bHLH) repressor protein; an HP1 alpha chromo shadow repression domain, or any combination thereof. In some embodiments, the effector domain comprises a KRAB domain. In some embodiments, the effector domain comprises tripartite motif-containing protein 28 (TRIM28, TIF1-beta or KAP1).

[0115]

[0147] In some embodiments, the effector domain comprises a protein domain that suppresses the expression of a target gene. For example, the effector domain may comprise a functional domain derived from a zinc finger repressor protein. In some embodiments, the effector domain comprises a functional inhibitory domain derived from the KOX1 / ZNF10 domain, KOX8 / ZNF708 domain, ZNF43 domain, ZNF184 domain, ZNF91 KRAB domain, HPE4 domain, HTF10 domain, HTF34 domain, or any combination thereof. In some embodiments, the effector domain comprises a functional inhibitory domain derived from the ZIM3 protein domain, ZNF436 domain, ZNF257 domain, ZNF675 domain, ZNF490 domain, ZNF320 domain, ZNF331 domain, ZNF816 domain, ZNF680 domain, ZNF41 domain, ZNF189 domain, ZNF528 domain, ZNF543 domain, ZNF554 domain, ZNF140 domain, ZNF610 domain, ZNF264 domain, ZNF350 domain, ZNF8 domain, ZNF582 domain, ZNF30 domain, ZNF324 domain, ZNF98 domain, ZNF669 domain, ZNF677 domain, ZNF596 domain, ZNF214 domain, ZNF37A domain, ZNF34 domain, ZNF250 domain, ZNF547 domain, ZNF273 domain, ZNF354A domain, ZFP82 domain, ZNF224 domain, ZNF33A domain, ZNF45 domain, ZNF175 domain, ZNF595 domain, ZNF184 domain, ZNF419 domain, ZFP28-1 domain, ZFP28-2 domain, ZNF18 domain, ZNF213 domain, ZNF394 domain, ZFP1 domain, ZFP14 domain, ZNF416 domain, ZNF557 domain, ZNF566 domain, ZNF729 domain, ZIM2 domain, ZNF254 domain, ZNF764 domain, ZNF785 domain, or any arbitrary combination thereof.In some embodiments, the domain is a ZIM3 domain, a ZNF554 domain, a ZNF264 domain, a ZNF324 domain, a ZNF354A domain, a ZNF189 domain, a ZNF543 domain, a ZFP82 domain, a ZNF669 domain, or a ZNF582 domain or any combination thereof. In some embodiments, the domain is a ZIM3 domain, a ZNF554 domain, a ZNF264 domain, a ZNF324 domain, or a ZNF354A domain or any combination thereof. In some embodiments, the domain is a ZIM3 domain.

[0116]

[0148] Exemplary functional domain sequences capable of reducing or silencing target gene expression are provided in Table 2 below. Further examples of repressors and repressor domains can be found in PCT / US2021 / 030643 and Tycko et al. (Tycko J, DelRosso N, Hess GT, Aradhana, Banerjee A, Mukund A, Van MV, Ego BK, Yao D, Spees K, Suzuki P, Marinov GK, Kundaje A, Bassik MC, Bintu L. High-Throughput Discovery and Characterization of Human Transcriptional Effectors. Cell. 2020 Dec 23;183(7):2020~2035.e16. doi: 10.1016 / j.cell.2020.11.024. Epub 2020 Dec 15. PMID: 33326746; PMCID: PMC8178797.), which are hereby incorporated by reference in their entirety.

[0117]

Table 2

[0118]

[0149] In some embodiments, the effector domain may include SEQ ID NO: 9. In some embodiments, the effector domain may be SEQ ID NO: 9. In some embodiments, the effector domain may be at least 99%, 98%, 97%, 96%, 95%, 90%, 85% or 80% similar to SEQ ID NO: 9.

[0119]

[0150] In some embodiments, the effector domain may include SEQ ID NO: 10. In some embodiments, the effector domain may be SEQ ID NO: 10. In some embodiments, the effector domain may be at least 99%, 98%, 97%, 96%, 95%, 90%, 85% or 80% similar to SEQ ID NO: 10.

[0120]

[0151] In some embodiments, the effector domain may include SEQ ID NO: 11. In some embodiments, the effector domain may be SEQ ID NO: 11. In some embodiments, the effector domain may be at least 99%, 98%, 97%, 96%, 95%, 90%, 85% or 80% similar to SEQ ID NO: 11.

[0121]

[0152] In some embodiments, the effector domain may include SEQ ID NO: 16. In some embodiments, the effector domain may be SEQ ID NO: 16. In some embodiments, the effector domain may be at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% similar to SEQ ID NO: 16.

[0122]

[0153] In some embodiments, the effector domain comprises a functional domain that suppresses or silences gene expression, and the functional domain is part of a larger protein, such as a zinc finger repressor protein. Functional domains that can regulate gene expression, such as suppressing or increasing gene expression, can be identified from larger proteins using known methods and the methods provided herein. For example, a functional effector domain that can reduce or silence target gene expression can be identified based on the sequence of a repressor protein or an activator protein. The amino acid sequence of a protein having a function of regulating gene expression can be obtained from an available genome browser, such as the UCSD genome browser or the Ensembl genome browser.

[0123]

[0154] Protein annotation databases such as UniProt or Pfam can be used to identify functional domains within the complete protein sequence. Using these means, a repression domain can be identified within the protein sequence. In some examples, various functional domains identified from larger proteins can be tested. The databases may differ in specific boundary domains. In further embodiments, the starting point region may be cleaved by 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50 or more amino acids at the N-terminus or C-terminus, and various cleavages can be tested to identify the minimal functional unit.

[0124]

[0155] In some embodiments, the effector domain comprises tripartite motif-containing protein 28 (TRIM28, TIF1-beta, or KAP1). In some embodiments, the effector domain comprises one or more KAP1 proteins. KAP1 proteins in the epigenetic editing system can form a complex with one or more other effector domains of the epigenetic editing system or one or more proteins involved in the regulation of gene expression in the cellular environment. For example, KAP1 can be recruited by the KRAB domain of a transcriptional repressor. In some embodiments, KAP1 interacts with or recruits histone deacetylase proteins, histone-lysine methyltransferase proteins (e.g., placing a methyl group on lysine 9 [K9] of histone H3 tail [H3K9]), chromatin remodeling proteins, and / or heterochromatin proteins. In some embodiments, the KAP1 protein interacts with or recruits one or more protein complexes that reduce or silence gene expression. In some embodiments, the KAP1 protein interacts with or recruits heterochromatin protein 1 (HP1) protein (e.g., via the chromodomain of the HP1 protein), SETDB1 protein, HDAC protein, and / or NuRD protein complex components. In some embodiments, the KAP1 protein recruits the CHD3 subunit of the nucleosome remodeling and deacetylation (NuRD) complex, thereby reducing or silencing the expression of the target gene. In some embodiments, the KAP1 protein recruits the SETDB1 protein (e.g., to the promoter region of the target gene), thereby reducing or silencing the expression of the target gene, for example, by H3K9 methylation associated with the promoter region of the target gene. In some embodiments, the recruitment of the SETDB1 protein results in heterochromatinization of the chromosomal region carrying the target gene, thereby reducing or silencing the expression of the target gene.In some embodiments, the KAP1 protein interacts with or recruits the HP1 protein, thereby reducing or silencing the expression of the target gene by reducing the acetylation of H3K9 or H3K14 on the histone tail associated with the target gene. The recruitment of SETDB1 induces heterochromatinization. In some embodiments, the KAP1 protein interacts with or recruits the ZFP90 protein (e.g., isoform 2 of ZFP90).

[0125] Linker

[0156] The epigenetic editing system provided herein may include one or more linkers that link one or more components of the epigenetic editing system. The linker can be a covalent bond or a polymeric linker that is many atoms in length. The linker can be a peptide linker or a non-peptide linker.

[0126]

[0157] In certain embodiments, a linker can be used to link either a peptide or a peptide domain of an epigenetic editing system. The linker can be as simple as a covalent bond or a polymeric linker that is many atoms in length. In certain embodiments, the linker is a polypeptide or is amino acid-based. In other embodiments, the linker is not peptidic. In certain embodiments, the linker is a covalent bond (e.g., carbon-carbon bond, disulfide bond, carbon-heteroatom bond, etc.). In certain embodiments, the linker is the carbon-nitrogen bond of an amide bond. In certain embodiments, the linker is a cyclic or acyclic, substituted or unsubstituted, branched or unbranched, aliphatic or heteroaliphatic linker. In certain embodiments, the linker is a polymer (e.g., polyethylene, polyethylene glycol, polyamide, polyester, etc.). In certain embodiments, the linker comprises monomers, dimers or polymers of aminoalkanoic acids. In certain embodiments, the linker comprises aminoalkanoic acids (e.g., glycine, ethanoic acid, alanine, beta-alanine, 3-aminopropanoic acid, 4-aminobutanoic acid, 5-pentanoic acid, etc.). In certain embodiments, the linker comprises monomers, dimers or polymers of aminohexanoic acid (Ahx). In certain embodiments, the linker is based on a carbocyclic moiety (e.g., cyclopentane, cyclohexane). In other embodiments, the linker comprises a polyethylene glycol moiety (PEG). In other embodiments, the linker comprises an amino acid. In certain embodiments, the linker comprises a peptide. In certain embodiments, the linker comprises an aryl or heteroaryl moiety. In certain embodiments, the linker is based on a phenyl ring. The linker can include a functionalized moiety to facilitate the binding of a nucleophile (e.g., thiol, amino) from a peptide to the linker. Any electrophile can be used as part of the linker. Exemplary electrophiles include, but are not limited to, activated esters, activated amides, Michael acceptors, alkyl halides, aryl halides, acyl halides and isothiocyanates.

[0127]

[0158] In some embodiments, the linker is a non-peptide linker. For example, the linker can be a carbon bond, a disulfide bond, or a carbon-heteroatom bond. In certain embodiments, the linker is the carbon-nitrogen bond of an amide bond. In certain embodiments, the linker is a cyclic or acyclic, substituted or unsubstituted, branched or unbranched, aliphatic or heteroaliphatic linker.

[0128]

[0159] In certain embodiments, the linker is a polymer (e.g., polyethylene, polyethylene glycol, polyamide, polyester, etc.). In certain embodiments, the linker comprises a monomer, dimer or polymer of aminoalkanoic acid. In certain embodiments, the linker comprises an aminoalkanoic acid (e.g., glycine, ethanoic acid, alanine, beta-alanine, 3-aminopropanoic acid, 4-aminobutanoic acid, 5-pentanoic acid, etc.). In certain embodiments, the linker comprises a monomer, dimer or polymer of aminohexanoic acid (Ahx). In certain embodiments, the linker is based on a carbocyclic moiety (e.g., cyclopentane, cyclohexane). In other embodiments, the linker comprises a polyethylene glycol moiety (PEG). In other embodiments, the linker comprises an amino acid. In certain embodiments, the linker comprises a peptide. In certain embodiments, the linker comprises an aryl or heteroaryl moiety. In certain embodiments, the linker is based on a phenyl ring. The linker may include a functionalized moiety to facilitate the binding of a nucleophile (e.g., thiol, amino) from the peptide to the linker. Any electrophile can be used as part of the linker. Exemplary electrophiles include, but are not limited to, activated esters, activated amides, alkyl halides, aryl halides, acyl halides and isothiocyanates.

[0129]

[0160] In some embodiments, one or more linkers of the epigenetic editing systems provided herein are peptide linkers. For example, a zinc finger array and a repressor domain can be linked by a peptide linker to form a zinc finger-repressor fusion protein. The peptide linker can be of any length applicable to the epigenetic editing system fusion proteins described herein. In some embodiments, the linker can comprise a peptide of amino acids between 1 and 200. In some embodiments, a DNA binding domain, such as a zinc finger array, and an effector domain are fused via a linker comprising an amino acid length of 1-5, 1-10, 1-20, 1-30, 1-40, 1-50, 1-60, 1-80, 1-100, 1-150, 1-200, 5-10, 5-20, 5-30, 5-40, 5-60, 5-80, 5-100, 5-150, 5-200, 10-20, 10-30, 10-40, 10-50, 10-60, 10-80, 10-100, 10-150, 10-200, 20-30, 20-40, 20-50, 20-60, 20-80, 20-100, 20-150, 20-200, 30-40, 30-50, 30-60, 30-80, 30-100, 30-150, 30-200, 40-50, 40-60, 40-80, 40-100, 40-150, 40-200, 50-60, 50-80, 50-100, 50-150, 50-200, 60-80, 60-100, 60-150, 60-200, 80-100, 80-150, 80-200, 100-150, 100-200, or 150-200. Longer or shorter linkers are also contemplated. In some embodiments, the peptide linker is 4, 16, 32, or 104 amino acids in length. In some embodiments, the peptide linker is a flexible linker. In some embodiments, the peptide linker is a rigid linker.

[0130]

[0161] The linker can be 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 25, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95 or 100 amino acids in length. In some embodiments, the linker is 5 amino acids in length. In some embodiments, the linker is 16 amino acids in length. In some embodiments, the linker is 20 amino acids in length. In some embodiments, the linker is 26 amino acids in length. In some embodiments, the linker is 80 amino acids in length.

[0131]

[0162] In some embodiments, the peptide linker is an XTEN linker. In some embodiments, the linker is an XTEN16 linker. In some embodiments, the linker comprises the amino acid sequence of SEQ ID NO: 2. In some embodiments, the linker comprises an amino acid sequence having at least 99%, 98%, 97%, 96%, 95%, 94%, 93%, 92%, 91% or 90% sequence similarity to SEQ ID NO: 2. In some embodiments, the peptide linker comprises an XTEN80 linker. In some embodiments, the peptide linker comprises the amino acid sequence of SEQ ID NO: 3. In some embodiments, the linker comprises an amino acid sequence having at least 99%, 98%, 97%, 96%, 95%, 94%, 93%, 92%, 91% or 90% sequence similarity to SEQ ID NO: 3.

[0132]

[0163] Between an effector domain (e.g., a repressor domain) and a DNA-binding protein (e.g., a Cas9 domain), between an effector domain and a second effector domain, or between any two components of an epigenetic editing system, various linker lengths and flexibilities can be used (e.g., ranging from very flexible linkers of the form (GGGGS)n, (GGGGS)n, and (G)n to more rigid linkers of the form (EAAAK)n, (SGGS)n, and (XP)n) to achieve an optimal length for effector domain activity for a particular application. In some embodiments, n is any integer from 3 to 30. In some embodiments, n is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15. In some embodiments, the linker comprises a (GGS)n motif, wherein n is 1, 3, or 7. In some embodiments, the linker comprises a (GGGGS)n motif, wherein n is 4 (SEQ ID NO: 4).

[0133]

[0164] In some embodiments, the linker in the epigenetic editing system comprises, for example, a nuclear localization signal of peptide sequence SEQ ID NO: 7. In some embodiments, the linker in the epigenetic editing system comprises a cleavable peptide, such as a T2A peptide, a p2A peptide, or a furin / p2A peptide. In some embodiments, the linker in the epigenetic editing system comprises an expression tag, such as a detectable tag like green fluorescent protein.

[0134]

[0165] In some embodiments, the linker comprises a nucleic acid. For example, one or more linkers of an epigenetic editing system may comprise a nucleic acid that can bind to, interact with, associate with, or form a complex with a polypeptide. In some embodiments, the nucleic acid linker can be an RNA linker that can bind to and / or interact with an RNA-binding protein domain, such as a phage-derived RNA-binding domain. In some embodiments, the nucleic acid linker can be fused to a guide polynucleotide that can bind to a Cas protein of an epigenetic editing system. In some embodiments, the nucleic acid linker comprises a K homology (KH) domain binding sequence, an MS2 coat protein binding sequence, a PP7 coat protein binding sequence, an SfMu COM coat protein binding sequence, a telomerase Ku binding motif binding sequence, an sm7 protein binding sequence, or other RNA recognition motif binding sequences thereof.

[0135]

[0166] In some embodiments, the linker comprises an affinity domain that specifically binds to a component of the epigenetic effector. For example, the epigenetic effector may comprise a programmable DNA binding domain and a linker that comprises an affinity domain having a specific binding affinity for the epigenetic effector domain. The affinity domain may include an antibody, single-chain antibody, nanobody, and antigen-binding sequences, antibodies, nanobodies, functional antibody fragments, single-chain variable fragments (scFv), Fab, single-domain antibodies (sdAb), VH domains, VL domains, VNAR domains, VHH domains, bispecific antibodies, diabodies, or functional fragments or combinations thereof. In some embodiments, the epigenetic effector domain comprises a programmable DNA binding domain and a KAP1 antibody that binds to the KAP1 protein. In some embodiments, the epigenetic effector domain comprises a programmable DNA binding domain and a KRAB antibody that binds to the KRAB protein. In some embodiments, the epigenetic effector domain comprises a programmable DNA binding domain and a DNMT1 antibody that binds to the DNMT1 protein. In some embodiments, the epigenetic effector domain comprises a programmable DNA binding domain and a DNMT3A antibody that binds to the DNMT3A protein. In some embodiments, the epigenetic effector domain comprises a programmable DNA binding domain and a DNMT3L antibody that binds to the DNMT3L protein. In some embodiments, the epigenetic effector domain comprises a programmable DNA binding domain and a ZIM3 antibody that binds to the ZIM3 protein. In some embodiments, the epigenetic effector domain comprises a programmable DNA binding domain and a TET1 antibody that binds to the TET1 protein. In some embodiments, the epigenetic effector domain comprises a programmable DNA binding domain and a VP16 or VP64 antibody that binds to the VP16 or VP64 protein.

[0136]

[0167] In some embodiments, the linker comprises a repeat peptide array. In some embodiments, the linker comprises an epitope tag, e.g., SunTag. In some embodiments, the epigenetic editing system comprises one or more peptide arrays comprising multiple copies of an epitope tag that can link multiple effector domains attached or fused to a peptide that recognizes the epitope tag. For example, the epitope tag array can link a DNA binding domain and multiple effector domains or multiple copies of an effector domain fused or attached to an antibody sequence that recognizes the epitope tag. In some embodiments, the epigenetic editing system comprises at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more epitope tag repeats that link at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more effector domains or copies of an effector domain. In some embodiments, the epigenetic editing system comprises multiple epitope tag repeats that link multiple effector domains and a detectable expression tag domain, e.g., GFP. In some embodiments, the repeat peptide array comprises a gene control non-depressible 4 (GCN4) peptide sequence. In some embodiments, the repeat peptide array is further linked by a binding peptide sequence of 15-50 amino acids. The repeat peptide arrays described in U.S. Patent Application Publication No. 20170219596 and U.S. Patent No. 10,612,044 are hereby incorporated by reference in their entireties.

[0137]

[0168] In some embodiments, the peptide linker is a W linker. In some embodiments, the linker comprises the amino acid sequence of SEQ ID NO: 5. In some embodiments, the linker comprises an amino acid sequence having at least 99%, 98%, 97%, 96%, 95%, 94%, 93%, 92%, 91%, or 90% sequence similarity to SEQ ID NO: 5.

[0138] Nuclear localization sequence

[0169] In some embodiments, the epigenetic editing system provided herein includes one or more nuclear targeting arrays. For example, the zinc finger - repressor fusion proteins described herein may further include one or more nuclear targeting arrays, such as a nuclear localization sequence (NLS). In some embodiments, the fusion protein includes multiple NLSs. In some embodiments, the fusion protein includes an NLS at its N - terminus or C - terminus. In some embodiments, the fusion protein includes NLSs at both its N - terminus and C - terminus. In some embodiments, the NLS is embedded in the middle of the fusion protein. In some embodiments, the NLS includes an amino acid sequence that promotes the import of the protein containing the NLS into the cell nucleus. In some embodiments, the NLS is fused to the N - terminus of the fusion protein. In some embodiments, the NLS is fused to the C - terminus of the fusion protein. In some embodiments, the NLS is fused to the N - terminus of a nucleic acid - binding protein, such as Cas9 or a zinc finger array. In some embodiments, the NLS is fused to the C - terminus of a nucleic acid - binding protein. In some embodiments, the NLS is fused to the N - terminus of an effector domain, such as a repressor domain. In some embodiments, the NLS is fused to the C - terminus of an effector domain, such as a repressor domain. In some embodiments, the NLS is fused to the fusion protein via one or more linkers. In some embodiments, the NLS is fused to the fusion protein without a linker. In some embodiments, the NLS includes the amino acid sequence of any one of the NLS sequences provided or cited herein. In some embodiments, the NLS includes the amino acid sequence of SEQ ID NO: 7. Further nuclear localization sequences are known in the art and will be apparent to those skilled in the art.

[0139] Tag

[0170] The epigenetic editing systems provided herein may include one or more additional sequences, domains, tags for tracking, detecting, and localizing the editor. In some embodiments, the epigenetic editing system includes one or more detectable tags. In some embodiments, the epigenetic editing system includes one, two, three, four, five, six, seven, eight, nine, ten or more detectable tags. Each of the detectable tags may be the same or different.

[0140]

[0171] For example, an epigenetic editing system fusion protein may include a cytoplasmic localization sequence, an export sequence, such as a nuclear export sequence, or other localization sequences, as well as sequence tags useful for solubilization, purification, or detection of the fusion protein. Suitable protein tags provided herein include, but are not limited to, biotin carboxylase carrier protein (BCCP) tag, myc tag, calmodulin tag, FLAG tag, hemagglutinin (HA) tag, histidine tag or polyhistidine tag also called His tag, maltose binding protein (MBP) tag, nus tag, glutathione-S-transferase (GST) tag, green fluorescent protein (GFP) tag, thioredoxin tag, S-tag, Softag (e.g., Softag1, Softag3), strep-tag, biotin ligase tag, FlAsH tag, V5 tag, and SBP tag. Additional suitable sequences will be apparent to those skilled in the art.

[0141]

[0172] In some embodiments, the epigenetic editing system includes 1-2 detectable tags. In aspects, the fusion protein includes one detectable tag. In aspects, the fusion protein includes two detectable tags. In aspects, the fusion protein includes three detectable tags. In aspects, the fusion protein includes four detectable tags. In aspects, the fusion protein includes five detectable tags.

[0142] Epigenetic Editing System Structure

[0173] The multiple components of the epigenetic editing system described in this specification can be in any order. In some embodiments, the epigenetic editing system includes the structure: N’]-[D1]-[D2]-[C’], where any one of D1 and D2 is a DNA binding domain, an effector domain, or a nucleic acid binding domain.

[0143]

[0174] In some embodiments, the epigenetic editing system includes the structure: N’]-[D1]-[D2]-[D3]-[C’], where any one of D1, D2, and D3 is a DNA binding domain, an effector domain, or a nucleic acid binding domain. In some embodiments, D1 is a DNA binding domain. In some embodiments, D2 is a DNA binding domain. In some embodiments, D3 is a DNA binding domain. In some embodiments, D1 is the only DNA binding domain. In some embodiments, D2 is the only DNA binding domain. In some embodiments, D3 is the only DNA binding domain.

[0144]

[0175] In some embodiments, the epigenetic editing system includes the structure: N’]-[D1]-[D2]-[D3]-[D4]-[C’], where any one of D1, D2, D3, and D4 is a DNA binding domain, an effector domain, or a nucleic acid binding domain. In some embodiments, D1 is a DNA binding domain. In some embodiments, D2 is a DNA binding domain. In some embodiments, D3 is a DNA binding domain. In some embodiments, D4 is a DNA binding domain. In some embodiments, D1 is the only DNA binding domain. In some embodiments, D2 is the only DNA binding domain. In some embodiments, D3 is the only DNA binding domain. In some embodiments, D4 is the only DNA binding domain.

[0145]

[0176] In some embodiments, the epigenetic editing system comprises the structure: N’]-[D1]-[D2]-[D3]-[D4]-[D5]-[C’, wherein any one of D1, D2, D3, D4, and D5 is a DNA binding domain, an effector domain, or a nucleic acid binding domain. In some embodiments, D1 is a DNA binding domain. In some embodiments, D2 is a DNA binding domain. In some embodiments, D3 is a DNA binding domain. In some embodiments, D4 is a DNA binding domain. In some embodiments, D5 is a DNA binding domain. In some embodiments, D1 is the only DNA binding domain. In some embodiments, D2 is the only DNA binding domain. In some embodiments, D3 is the only DNA binding domain. In some embodiments, D4 is the only DNA binding domain. In some embodiments, D5 is the only DNA binding domain.

[0146]

[0177] In some embodiments, the epigenetic editing system comprises at least one effector domain that is a DNMT domain. In some embodiments, the epigenetic editing system comprises at least one effector domain that is a KRAB domain. In some embodiments, the epigenetic editing system comprises at least one effector domain that is a ZIM domain. In some embodiments, the epigenetic effector comprises at least one effector domain that is a DNMT3A domain or a truncated version thereof. In some embodiments, the epigenetic effector comprises at least one effector domain that is a DNMT3L domain or a truncated version thereof.

[0147]

[0178] The components of an epigenetic editing system can be structured in different configurations. For example, a DNA binding domain can be at the C-terminus, N-terminus, or in the middle of two or more epigenetic effector domains or additional domains. In some embodiments, the DNA binding domain is at the C-terminus of the epigenetic editing system. In some embodiments, the DNA binding domain is at the N-terminus of the epigenetic editing system. In some embodiments, the DNA binding domain is linked to one or more nuclear localization signals. In some embodiments, the DNA binding domain is linked to two or more nuclear localization signals. In some embodiments, an epigenetic effector domain or additional domain is adjacent to the DNA binding domain on both ends. In some embodiments, the epigenetic editing system includes a configuration of N’]-[epigenetic effector domain 1]-[DNA binding domain]-[epigenetic effector domain 2]-[C’. In some embodiments, the epigenetic editing system includes a configuration of N’]-[epigenetic effector domain 1]-[DNA binding domain]-[epigenetic effector domain 2]-[epigenetic effector domain 3]-[C’. In some embodiments, the epigenetic editing system includes a configuration of N’]-[epigenetic effector domain 1]-[epigenetic effector domain 2]-[DNA binding domain]-[epigenetic effector domain 3]-[C’. In some embodiments, the epigenetic editing system includes a configuration of N’]-[epigenetic effector domain 1]-[epigenetic effector domain 2]-[DNA binding domain]-[epigenetic effector domain 3]-[epigenetic effector domain 4]-[C’. In some embodiments, the epigenetic editing system includes a configuration of N’]-[KRAB]-[DNA binding domain]-[DNMT3L]-[C’. In some embodiments, the epigenetic editing system includes a configuration of N’]-[DNMT3L]-[DNA binding domain]-[KRAB]-[C’.In some embodiments, the epigenetic editing system comprises the configuration of N’]-[KRAB]-[DNA binding domain]-[DNMT3L-[C’. In some embodiments, the epigenetic editing system comprises the configuration of N’]-[KRAB]-[DNA binding domain]-[Dnmt3A]-[Dnmt3L]-[C’. In some embodiments, the epigenetic editing system comprises the configuration of N’]-[Dnmt3A]-[Dnmt3L]-[DNA binding domain]-[KRAB]-[C’. In some embodiments, the epigenetic editing system comprises the configuration of N’]-[SETDB1]-[DNA binding domain]-[Dnmt3A]-[Dnmt3L]-[C’. In some embodiments, the epigenetic editing system comprises the configuration of N’]-[SETDB1]-[DNA binding domain]-[Dnmt3A]-[C’. In some embodiments, the epigenetic editing system comprises the configuration of N’]-[KRAB]-[DNA binding domain]-[Dnmt3A-Dnmt3L]-[C’, provided that Dnmt3A and Dnmt3L are directly fused by a peptide bond.

[0148]

[0179] In some embodiments, the linker structure “]-[” in any one of the epigenetic editing system structures is a linker, for example, a peptide linker. In some embodiments, the linker structure “]-[” in any one of the epigenetic editing system structures is a detectable tag. In some embodiments, the linker structure “]-[” in any one of the epigenetic editing system structures is a peptide bond. In some embodiments, the linker structure “]-[” in any one of the epigenetic editing system structures is a nuclear localization signal. In some embodiments, the linker structure “]-[” in any one of the epigenetic editing system structures is a promoter or regulatory sequence. In the epigenetic editing system structure, the plurality of linker structures “]-[” may be the same, and each may be a different linker, tag, NLS or peptide bond.

[0149]

[0180] The DNA binding domain (DBD) of the epigenetic editing system can include any one of the DNA binding domains described herein or known to those of skill in the art. In some embodiments, the DBD includes one or more zinc finger arrays. In some embodiments, the DBD includes a TALE DNA binding domain. In some embodiments, the DBD is an RNA-guided programmable DNA binding domain, such as a CRISPR-Cas protein domain. Suitable Cas proteins, including nuclease-inactive Cas proteins that do not cause target DNA strand cleavage for the purpose of epigenetic editing, are provided herein. Cas proteins in the epigenetic editing system can be nuclease-inactive Cas9 (dCas9), SaCas9d, SpCas9d, dCas9 with modified PAM specificity, high-fidelity dCas9, nuclease-inactive Cpf1 (dCpf1), dCpf1 with modified PAM specificity, high-fidelity dCpf1, dCas12e, dCasY, or any other Cas protein described herein.

[0150]

[0181] In some embodiments, the epigenetic editing system includes a DNA binding domain (DBD) and an effector domain that suppresses or silences the expression of a target gene. In some embodiments, the epigenetic editing system includes a configuration of N’]-[repressor domain]-[DBD]-[-C’, wherein the linker structure]-[is any one of the linkers described herein, a detectable tag, an affinity domain, a peptide bond, a nuclear localization signal, a promoter and / or a regulatory sequence. In some embodiments, the epigenetic editing system includes a configuration of N’]-[DBD]-[repressor domain]-[-C’, wherein the linker structure]-[is any one of the linkers described herein, a detectable tag, an affinity domain, a peptide bond, a nuclear localization signal, a promoter and / or a regulatory sequence.

[0151]

[0182] In some embodiments, the epigenetic editing system comprises a DNA binding domain (DBD) and a catalytically active DNMT that suppresses or silences the expression of a target gene by placing and / or recruiting one or more methylation marks on the target gene, and includes a DNA methyltransferase domain. In some embodiments, the epigenetic editing system comprises a configuration of N’-[DNA methyltransferase domain]-[DBD]-[-C’, wherein the linking structure]-[is any one of the linkers described herein, a detectable tag, an affinity domain, a peptide bond, a nuclear localization signal, a promoter, and / or a regulatory sequence. In some embodiments, the epigenetic editing system comprises a configuration of N’-[DBD]-[DNA methyltransferase domain]-[-C’, wherein the linking structure]-[is any one of the linkers described herein, a detectable tag, an affinity domain, a peptide bond, a nuclear localization signal, a promoter, and / or a regulatory sequence.

[0152]

[0183] In some embodiments, the epigenetic editing system comprises a DNA binding domain (DBD), a DNA methyltransferase domain, and an effector domain that suppresses or silences the expression of a target gene. In some embodiments, the epigenetic editing system comprises a configuration of N’-[DNA methyltransferase domain]-[DBD]-[suppressor domain]-[-C’, wherein the linking structure]-[is any one of the linkers described herein, a detectable tag, an affinity domain, a peptide bond, a nuclear localization signal, a promoter, and / or a regulatory sequence. In some embodiments, the epigenetic editing system comprises a configuration of N’-[suppressor domain]-[DBD]-[DNA methyltransferase domain]-[-C’, wherein the linking structure]-[is any one of the linkers described herein, a detectable tag, an affinity domain, a peptide bond, a nuclear localization signal, a promoter, and / or a regulatory sequence.

[0153]

[0184] In some embodiments, the epigenetic editing system comprises the configuration of N’-[DNA methyltransferase domain]-[repressor domain]-[DBD]-[-C’, wherein the linking structure]-[is any one of the linkers described herein, a detectable tag, an affinity domain, a peptide bond, a nuclear localization signal, a promoter and / or a regulatory sequence. In some embodiments, the epigenetic editing system comprises the configuration of N’-[repressor domain]-[DNA methyltransferase domain]-[DBD]-[-C’, wherein the linking structure]-[is any one of the linkers described herein, a detectable tag, an affinity domain, a peptide bond, a nuclear localization signal, a promoter and / or a regulatory sequence.

[0154]

[0185] The repressor domain in the epigenetic editing system can include any one of the expression repressing proteins known to those skilled in the art and described herein, or any homolog or combination thereof. In some embodiments, the repressor domain includes a histone deacetylase domain. In some embodiments, the repressor domain interacts with a scaffold protein domain that recruits one or more protein domains that suppress the expression of the target gene. For example, the repressor domain can recruit or interact with a scaffold protein domain that recruits a DNMT, or a PRMT protein, an HDAC protein, a SETDB1 protein or a NuRD protein domain. In some embodiments, the repressor domain interacts with an epigenetically marked DNA molecule in the target gene, resulting in suppressing or silencing the expression of the target gene. In some embodiments, the repressor domain includes an MECP2 domain. In some embodiments, the repressor domain includes a KAP1 domain or a DNMT3L domain. In some embodiments, the repressor domain includes any one of the domains in Table 2, or any combination or homolog thereof.

[0155]

[0186] The DNA methyltransferase domain in the epigenetic editing system may include any one of the DNA methyltransferase proteins described herein that are known to those skilled in the art, or any homolog or combination thereof. In some embodiments, the effector domain includes the DNMT3 domain. In some embodiments, the DNA methyltransferase domain includes the DNMT3A domain. In some embodiments, the DNA methyltransferase domain includes the DNMT3B domain. In some embodiments, the DNA methyltransferase domain includes the DNMT3C domain. In some embodiments, the DNA methyltransferase domain includes the DNMT3L domain. In some embodiments, the DNA methyltransferase domain includes a fusion of the DNMT3A-DNMT3L domains. The DNMT3A-DNMT3L fusion domain described herein can be, for example, in either the order of N-DNMT3A-DNMT3L-C or N-DNMT3L-DNMT3A-C. In some embodiments, the DNA methyltransferase domain includes any one of the domains in Table 1, or any combination or homolog thereof.

[0156]

[0187] In some embodiments, the epigenetic editing system includes a DNA binding domain (DBD) and an effector domain that upregulates the expression of a target gene. In some embodiments, the epigenetic editing system includes a configuration of N’]-[activation domain]-[DBD]-[-C’, wherein the linker structure]-[is any one of the linkers described herein, a detectable tag, an affinity domain, a peptide bond, a nuclear localization signal, a promoter, and / or a regulatory sequence. In some embodiments, the epigenetic editing system includes a configuration of N’]-[DBD]-[activation domain]-[-C’, wherein the linker structure]-[is any one of the linkers described herein, a detectable tag, an affinity domain, a peptide bond, a nuclear localization signal, a promoter, and / or a regulatory sequence.

[0157]

[0188] In some embodiments, the epigenetic editing system includes a DNA binding domain (DBD) and a DNA demethylation domain that increases the expression of a target gene by removing one or more methylation marks in the target gene. In some embodiments, the epigenetic editing system includes a configuration of N’]-[DNA demethylase domain]-[DBD]-[-C’, wherein the linking structure]-[is any one of the linkers described herein, a detectable tag, an affinity domain, a peptide bond, a nuclear localization signal, a promoter, and / or a regulatory sequence. In some embodiments, the epigenetic editing system includes a configuration of N’]-[DBD]-[DNA demethylase domain]-[-C’, wherein the linking structure]-[is any one of the linkers described herein, a detectable tag, an affinity domain, a peptide bond, a nuclear localization signal, a promoter, and / or a regulatory sequence.

[0158]

[0189] In some embodiments, the epigenetic editing system includes a DNA binding domain (DBD), a DNA demethylase domain, and an activation effector domain that increases the expression of the target gene. In some embodiments, the epigenetic editing system includes a configuration of N’]-[DNA demethylase domain]-[DBD]-[activation domain]-[-C’, wherein the linking structure]-[is any one of the linkers described herein, a detectable tag, an affinity domain, a peptide bond, a nuclear localization signal, a promoter, and / or a regulatory sequence. In some embodiments, the epigenetic editing system includes a configuration of N’]-[activation domain]-[DBD]-[DNA demethylase domain]-[-C’, wherein the linking structure]-[is any one of the linkers described herein, a detectable tag, an affinity domain, a peptide bond, a nuclear localization signal, a promoter, and / or a regulatory sequence.

[0159]

[0190] In some embodiments, the epigenetic editing system comprises the configuration of N']-[DNA demethylase domain]-[activation domain]-[DBD]-[-C', wherein the linking structure]-[is any one of the linkers described herein, a detectable tag, an affinity domain, a peptide bond, a nuclear localization signal, a promoter, and / or a regulatory sequence. In some embodiments, the epigenetic editing system comprises the configuration of N']-[activation domain]-[DNA demethylase domain]-[DBD]-[-C', wherein the linking structure]-[is any one of the linkers described herein, a detectable tag, an affinity domain, a peptide bond, a nuclear localization signal, a promoter, and / or a regulatory sequence.

[0160]

[0191] In some embodiments, an epigenetic editing system that reduces or silences the expression of a target gene comprises a DBD and an affinity domain that specifically binds to a repression domain. For example, the epigenetic editing system can comprise a DBD and a repression domain antibody. In some embodiments, the epigenetic editing system comprises a DBD and a KAP1 affinity domain. In some embodiments, the epigenetic editing system comprises a DBD and a KRAB affinity domain. In some embodiments, the epigenetic editing system comprises a DBD and a SETDB1 affinity domain. In some embodiments, the epigenetic editing system comprises a DBD and a MECP2 affinity domain. In some embodiments, the epigenetic editing system comprises a DNA methyltransferase and a repression domain-binding affinity domain.

[0161]

[0192] In some embodiments, an epigenetic editing system that reduces or silences the expression of a target gene includes a DBD and an affinity domain that specifically binds to a DNA methyltransferase domain. For example, the epigenetic editing system may include a DBD and an antibody to a DNA methyltransferase domain. In some embodiments, the epigenetic editing system includes a DBD and a Dnmt3A affinity domain. In some embodiments, the epigenetic editing system includes a DBD and a Dnmt3L affinity domain. In some embodiments, the epigenetic editing system includes a repression domain and a DNA methyltransferase binding affinity domain. In some embodiments, the epigenetic editing system includes a repression domain and a Dnmt3A binding affinity domain. In some embodiments, the epigenetic editing system includes a repression domain and a Dnmt3L affinity domain. In some embodiments, the epigenetic editing system includes one or more of the KAP1, KRAB, and MECP2 domains and a Dnmt3A binding affinity domain. In some embodiments, the epigenetic editing system includes one or more of the KAP1 domains and a Dnmt3A binding affinity domain. In some embodiments, the epigenetic editing system includes one or more of the KAP1, KRAB, and MECP2 domains and a Dnmt3L binding affinity domain. In some embodiments, the epigenetic editing system includes one or more of the KAP1 domains and a Dnmt3L binding affinity domain. The affinity domain can be an antibody, single-chain antibody, nanobody, and antigen-binding sequence, antibody, nanobody, functional antibody fragment, single-chain variable fragment (scFv), Fab, single-domain antibody (sdAb), VH domain, VL domain, VNAR domain, VHH domain, bispecific antibody, diabody, or a functional fragment or combination thereof.

[0162]

[0193] In some embodiments, an epigenetic editing system that reduces or silences the expression of a target gene includes a DBD, a first affinity domain that specifically binds to a DNA methyltransferase domain, and a second affinity domain that specifically binds to a repression domain. For example, the epigenetic editing system can include a DBD, an antibody to a DNA methyltransferase domain, and an antibody to a repression domain. In some embodiments, the epigenetic editing system includes a DBD, a KAP1 affinity domain, and a Dnmt3A affinity domain. In some embodiments, the epigenetic editing system includes a DBD, a KAP1 affinity domain, and a Dnmt3L affinity domain. In some embodiments, the epigenetic editing system includes a DBD, an MECP2 affinity domain, and a Dnmt3A affinity domain. In some embodiments, the epigenetic editing system includes a DBD, an MECP2 affinity domain, and a Dnmt3L affinity domain. In some embodiments, the epigenetic editing system includes a DBD, a KRAB affinity domain, and a Dnmt3A affinity domain. In some embodiments, the epigenetic editing system includes a DBD, a KRAB affinity domain, and a Dnmt3L affinity domain. The affinity domain can be an antibody, a single-chain antibody, a nanobody, and an antigen-binding sequence, an antibody, a nanobody, a functional antibody fragment, a single-chain variable fragment (scFv), a Fab, a single-domain antibody (sdAb), a VH domain, a VL domain, a VNAR domain, a VHH domain, a bispecific antibody, a diabody, or a functional fragment or combination thereof.

[0163] DNA binding domain

[0194] The epigenetic editing systems and epigenetic editing complexes described herein can include one or more nucleic acid binding protein domains, such as a DNA binding domain, that can direct the epigenetic editing system to a target gene associated with specific conditions.

[0164]

[0195] The target gene used in this specification may include the entire nucleotide sequence of the gene of interest. For example, the sequence or nucleotides of the target gene may include coding and non-coding sequences. The sequence of the target gene may include exons or introns. The sequence of the target gene may include regulatory regions, including promoters, enhancers, terminators, 5' untranslated regions or 3' untranslated regions. In some embodiments, the sequence of the target gene includes a distal enhancer sequence.

[0165]

[0196] The epigenetic editing system described in this specification may include any polynucleotide binding domain. In some embodiments, the nucleic acid binding domain includes one or more DNA binding domains, such as zinc finger proteins (ZFPs) or transcription activator-like effectors (TALEs). In some embodiments, the nucleic acid binding domain includes a polynucleotide-induced DNA binding domain, such as a nuclease-inactive CRISPR-Cas protein guided by a guide RNA.

[0166]

[0197] The nucleic acid binding domain of the epigenetic editing system described in this specification may be able to recognize and bind to any target gene of interest, such as a target gene associated with a disease or disorder. In some embodiments, the target gene associated with a disease or disorder contains a mutation compared to the wild-type gene. In some embodiments, the target gene associated with a disease or disorder contains a copy containing a mutation associated with the disease or disorder. In some embodiments, the target gene associated with a disease or disorder has one or both copies of the wild-type DNA sequence. In some embodiments, the target gene is not associated with a disease or disorder. In some embodiments, the target gene not associated with a disease or disorder contains a mutation compared to the wild-type gene. In some embodiments, the target gene not associated with a disease or disorder has one or both copies of the wild-type DNA sequence.

[0167]

[0198] The DNA-binding domain can be modular and / or programmable. In some embodiments, the DNA-binding domain comprises a zinc finger domain, a transcription activator-like effector (TALE) domain, a meganuclease DNA-binding domain, or a polynucleotide-inducible nucleic acid-binding domain. Examples of DNA-binding domains can be found in U.S. Patent No. 11,162,114, which is incorporated herein by reference in its entirety.

[0168]

[0199] Transcription activator-like effectors (TALEs) can be engineered to bind to virtually any desired DNA sequence. Methods for programming TALEs are known to those of skill in the art. Such methods are described, for example, in Carroll et al., Genetics Society of America, 188(4):773-782 (2011); Miller et al., Nature Biotechnology 25(7):778-785 (2007); Christian et al., Genetics 186(2):757-61 (2008); Li et al., Nucleic Acids Res. 39(1):359-372 (2010); and Moscou et al., Science 326(5959):1501 (2009), each of which is incorporated herein by reference.

[0169]

[0200] The DNA-binding domain can be guided by a nucleic acid sequence, such as an RNA sequence, for identifying a target gene. In some embodiments, the DNA-binding domain comprises a programmable nuclease. In some embodiments, the DNA-binding domain comprises a programmable nuclease having reduced or inhibited nuclease activity. For example, the programmable nuclease can contain one or two mutations within its catalytic domain that inactivate the nuclease but maintain the DNA-binding activity of the nuclease. In some embodiments, the DNA-binding domain comprises a CRISPR-Cas protein domain. In some embodiments, the CRISPR-Cas protein domain lacks nuclease activity or has reduced nuclease activity.

[0170]

[0201] In some embodiments, the epigenetic editing system provided herein includes a Cas protein, e.g., a Cas9 protein domain. The Cas9 domain can be either the Cas9 domain or the Cas9 protein provided herein (e.g., nuclease-inactive Cas9 or Cas9 nickase, or a Cas9 variant from any species). In some embodiments, either the Cas domain or the Cas protein provided herein can be fused to one or more of the effector protein domains described herein. In some embodiments, any of the Cas proteins provided herein can be fused to two or more of the effector protein domains described herein. Cas9 can refer to a polypeptide having at least about 50%, 60%, 70%, 80%, 90%, 100% sequence identity and / or sequence similarity to a wild-type exemplary Cas9 polypeptide (e.g., from S. pyogenes). Cas9 can refer to a modified form of a Cas9 protein that can include wild-type, or amino acid changes such as deletions, insertions, substitutions, variants, mutations, fusions, chimeras, or any combination thereof.

[0171]

[0202] Cas9 sequences and structures of variant Cas9 orthologs have been described in various species. Exemplary species from which the Cas9 protein or other components may be derived include Streptococcus pyogenes, Streptococcus thermophilus, Streptococcus spp., Staphylococcus aureus, Listeria innocua, Lactobacillus gasseri, Francisella novicida, Wolinella succinogenes, Sutterella wadsworthensis, Gamma proteobacterium, Neisseria meningitidis, Campylobacter jejuni, Pasteurella multocida, Fibrobacter succinogene, Rhodospirillum rubrum, Nocardiopsis dassonvillei, Streptomyces pristinaespiralis, Streptomyces viridochromogenes, Streptomyces viridochromogenes, Streptosporangium roseum, Alicyclobacillus acidocaldarius, Bacillus pseudomycoides, Bacillus selenitireducens, Exiguobacterium sibiricum, Lactobacillus delbrueckii, Lactobacillus salivarius, Lactobacillus buchneri, Treponema denticola, Microscilla marina, bacteria of the order Burkholderiales, Polaromonas naphthalenivorans, Polaromonas spp., Crocosphaera watsonii, Cyanothece spp., Microcystis aeruginosa, Synechococcus spp., Acetohalobium arabaticum, Ammonifex degensii, Caldicelulosiruptor becscii, CandidatusDesulforudis, Clostridium botulinum, Clostridium difficile, Finegoldia magna, Natranaerobius thermophilus, Pelotomaculum thermopropionium, Acidithiobacillus caldus, Acidithiobacillus ferrooxidans, Allochromatium vinosum, Marinobacter sp., Nitrosococcus halophilus, Nitrosococcus watsoni, Pseudoalteromonas haloplanktis, Ktedonobacter racemifer, Methanohalobium evestigatum, Anabaena variabilis, Nodularia spumigena, Nostoc sp., Arthrospira maxima, Arthrospira platensis, Arthrospira sp., Lyngbya sp., Microcoleus chthonoplastes, Oscillatoria sp., Petrotoga mobilis, Thermosipho africanus, Streptococcus pasteurianus, Neisseria cinerea, Campylobacter lari, Parvibaculum lavamentivorans, Coryne bacterium diphtheria, or Acaryochloris marina, including but not limited to these. In some embodiments, the Cas9 protein is from Streptococcus pyogenes. In some embodiments, the Cas9 protein can be from Streptococcus thermophilus. In some embodiments, the Cas9 protein is from Staphylococcus aureus.

[0172]

[0203] Additional suitable Cas9 proteins, orthologs, variants including nuclease-inactive variants, and sequences are apparent to those skilled in the art based on the present disclosure, and such Cas9 nucleases and sequences include Cas9 sequences from organisms and loci disclosed in Chylinski et al. (2013) RNA Biology 10:5, pages 726-737, which are incorporated herein by reference.

[0173]

[0204] The epigenetic editing system can include a nuclease-inactive Cas9 domain (dead Cas9 or dCas9). The dCas9 protein domain can have one, two, or more mutations that suppress its nuclease activity but retain DNA-binding activity compared to wild-type Cas9. For example, the DNA cleavage domain of Cas9 is known to include two subdomains, the HNH nuclease subdomain and the RuvC1 subdomain. The HNH subdomain cleaves the strand complementary to the gRNA, while the RuvC1 subdomain cleaves the non-complementary strand. Mutations within these subdomains can silence the nuclease activity of Cas9. For example, the mutations D10A and H840A completely inactivate the nuclease activity of Cas9 from S. pyogenes. In some embodiments, dCas9 includes at least one mutation in the HNH and RuvC subdomains that reduces or suppresses nuclease activity. In some embodiments, dCas9 includes only the RuvC subdomain. In some embodiments, dCas9 includes only the HNR subdomain. It should be understood that any mutation that inactivates the RuvC or HNH domain, such as an insertion, deletion, or single or multiple amino acid substitutions in the RuvC domain and / or the HNH domain, can be included in dCas9.

[0174]

[0205] Additional suitable mutations that inactivate Cas9 will be apparent to those skilled in the art based on the present disclosure and knowledge in the art, and are within the scope of the present disclosure. Such additional exemplary, suitable nuclease-inactive Cas9 domains include, but are not limited to, D839A, N863A, and / or K603R. Cas9, dCas9, or Cas9 variants further include Cas9, dCas9, or Cas9 variants from any organism. Furthermore, it is recognized that dCas9, Cas9 nickase, or other suitable Cas9 variants from any organism may be used in accordance with the present disclosure.

[0175]

[0206] In some embodiments, the epigenetic editing system includes a high-fidelity Cas9 domain. For example, a high-fidelity Cas9 domain that includes one or more mutations that reduce the electrostatic interaction between the Cas9 domain and the sugar-phosphate backbone of DNA can be incorporated into the epigenetic editing system to confer increased target binding specificity compared to the corresponding wild-type Cas9 domain. Without wishing to be bound by a particular theory, a high-fidelity Cas9 domain with reduced electrostatic interaction with the sugar-phosphate backbone of DNA may have a smaller off-target effect. In some embodiments, the Cas9 domain includes one or more mutations that reduce the association between the Cas9 domain and the sugar-phosphate backbone of DNA. In some embodiments, the Cas9 domain includes one or more mutations that reduce the association between the Cas9 domain and the sugar-phosphate backbone of DNA by at least 1%, at least 2%, at least 3%, at least 4%, at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70% or more. In some embodiments, the high-fidelity Cas9 domain includes one or more of the N497X, R661X, Q695X and / or Q926X mutations (wherein X is any amino acid) numbered in the wild-type Cas9 amino acid sequence Uniprot reference sequence: Q99ZW2, or the corresponding amino acids in other Cas9s. In some embodiments, the high-fidelity Cas9 domain includes one or more of the N497A, R661A, Q695A and / or Q926A mutations of the amino acid sequence provided in the wild-type Cas9 sequence, or the corresponding mutations numbered in the wild-type Cas9 amino acid sequence Uniprot reference sequence: Q99ZW2, or the corresponding amino acids in other Cas9s.It is recognized that any of the epigenetic editing systems provided herein, such as any of the epigenetic activators or repressors provided herein, can be converted into a high-fidelity epigenetic editing system by the described modifications of the Cas9 domain. In a preferred embodiment, the high-fidelity Cas9 domain is a nuclease-inactive Cas9 domain.

[0176]

[0207] In some embodiments, the DNA binding domain in the epigenetic editing system is a CRISPR protein that recognizes a protospacer adjacent motif (PAM) sequence in the target gene. The CRISPR protein may recognize a native or standard PAM sequence or may have altered PAM specificity. Cas9 domains that bind to non-standard PAM sequences are described in the art and will be apparent to those skilled in the art. For example, Cas9 domains that bind to non-standard PAM sequences are described in Kleinstiver, B.P. et al., "Engineered CRISPR-Cas9 nucleases with altered PAM specificities" Nature 523, pages 481-485 (2015) and Kleinstiver, B.P. et al., "Broadening the targeting range of Staphylococcus aureus CRISPR-Cas9 BY modifying PAM recognition" Nature Biotechnology 33, pages 1293-1298 (2015), the entire contents of each of which are incorporated herein by reference.

[0177]

[0208] In some embodiments, the Cas9 domain is the Cas9 domain from S. pyogenes (SpCas9). In some embodiments, SpCas9 recognizes the standard NGG PAM sequence, where "N" in "NGG" is adenine (A), thymine (T), guanine (G), or cytosine (C), and G is guanine. In some embodiments, the epigenetic editing system or fusion protein provided herein contains a SpCas9 domain that can bind to a nucleotide sequence that does not contain a standard (e.g., NGG) PAM sequence. In some embodiments, the SpCas9 domain, nuclease-inactive SpCas9 domain, or SpCas9 nickase domain can bind to a nucleic acid sequence having an NGG, NGA, or NGCG PAM sequence. In some embodiments, the Cas9 domain is a modified SpCas9 domain having specificity for the 5'-NGCG-3' PAM sequence, where N is any one of the nucleotides A, G, C, or T. In some embodiments, the Cas9 domain is a modified SpCas9 domain having specificity for the 5'-NGAN-3' or 5-NGNG-3' PAM sequence, where N is any one of the nucleotides A, G, C, or T. In some embodiments, the Cas9 domain is a modified SpCas9 domain having specificity for the 5'-NGN-3' PAM sequence, where N is any one of the nucleotides A, G, C, or T. In some embodiments, the Cas9 domain is a modified SpCas9 domain having specificity for the 5'-NRN-3' or 5'-NYN-3' PAM sequence, where N is any one of the nucleotides A, G, C, or T, R is the nucleotide A or G, and Y is the nucleotide C or T.

[0178]

[0209] In some embodiments, the Cas9 domain is Cas9 from Staphylococcus aureus (SaCas9). In some embodiments, the SaCas9 domain is nuclease-inactive SaCas9 (dSacas9). In some embodiments, the SaCas9 domain, the nuclease-inactive SaCas9 domain, or the SaCas9 nickase domain can bind to a nucleic acid sequence having a non-standard PAM sequence. In some embodiments, the SaCas9 domain, the SaCas9d domain, or the SaCas9n domain can bind to a nucleic acid sequence having the NNGRRT PAM sequence, provided that N = A, T, C, or G, and R = A or G. In some embodiments, the Cas9 domain is the Cas9 domain from Neisseria meningitidis (NmeCas9). In some embodiments, the NmeCas9 domain is nuclease-inactive NmeCas9 (dNmeCas9). NmeCas9 can have specificity for the 5'-NNNGATT-3' PAM, provided that N is any one of the nucleotides A, G, C, or T. In some embodiments, the Cas9 domain is the Cas9 domain from Campylobacter jejuni (CjCas9). In some embodiments, the CjCas9 domain is nuclease-inactive CjCas9 (dCjCas9). CjCas9 can have specificity for the 5'-NNNVRYM-3' PAM, provided that N is any one of the nucleotides A, G, C, or T, V is the nucleotide A, C, or G, R is the nucleotide A or G, Y is the nucleotide C or T, and M is the nucleotide A or C. In some embodiments, the Cas9 domain is the Cas9 domain from Streptococcus thermophilus (StCas9). In some embodiments, StCas9 is encoded by the St CRISPR1 locus (St1Cas9) of Streptococcus thermophilus. In some embodiments, the St1Cas9 domain is nuclease-inactive St1Cas9 (dSt1Cas9).St1Cas9 can have specificity for a 5'-NNAGAAW-3' PAM, provided that N is any one of the nucleotides A, G, C, or T, and W is the nucleotide A or T. In some embodiments, StCas9 is encoded by the St CRISPR3 locus of Streptococcus thermophilus (St3Cas9). In some embodiments, the St3Cas9 domain is nuclease-inactive St3Cas9 (dSt3Cas9). St3Cas9 can have specificity for a 5'-NGGNG-3' PAM, provided that N is any one of the nucleotides A, G, C, or T.

[0179]

[0210] In some embodiments, the Cas9 domain of any of the fusion proteins provided herein comprises an amino acid sequence that is at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 99.5% identical to any one of the Cas9 sequences provided herein.

[0180]

[0211] In some embodiments, the epigenetic editing system provided herein comprises a Cpf1 (or Cas12a) protein domain. For example, the epigenetic editing system can comprise a nuclease-inactive Cpf1 protein or a variant thereof. The Cpf1 protein has a RuvC-like endonuclease domain that is similar to the RuvC domain of Cas9 but does not have an HNH endonuclease domain, and the N-terminus of Cpf1 does not have an alpha-helix recognition lobe of Cas9.

[0181]

[0212] In some embodiments, the Cpf1 protein comprises an amino acid sequence that is at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 99.5% identical to the FnCpf1 sequences provided herein. It is recognized that Cpf1 from other bacterial species can also be used in accordance with the present disclosure.

[0182]

[0213] In some embodiments, Cpf1 is the Cpf1 protein from Lachnospiraceae bacterium (LbCpf1). LbCpf1 may have specificity for the 5'-TTTV-3' PAM sequence, where V is any one of the nucleotides A, G, or C. In some embodiments, the LbCpf1 protein has reduced nuclease activity. In some embodiments, the nuclease activity of the LbCpf1 protein is abolished (dLbCpf1). In some embodiments, Cpf1 is the Cpf1 protein from Acidaminococcus sp. (AsCpf1). AsCpf1 may have specificity for the 5'-TTTV-3' PAM sequence, where V is any one of the nucleotides A, G, or C. In some embodiments, the AsCpf1 protein has reduced nuclease activity. In some embodiments, the nuclease activity of the AsCpf1 protein is abolished (dAsCpf10). In some embodiments, the dAsCpf1 or AsCpf1 protein further comprises a mutation that improves the fidelity of target recognition of the protein. In some embodiments, the dAsCpf1 or AsCpf1 protein further comprises a mutation that results in a change in the PAM specificity of the protein.

[0183]

[0214] In some embodiments, the epigenetic editing system provided herein includes a Cas protein other than the Cas9 protein. In some embodiments, the Cas9 protein includes an inactivated nuclease domain. In some embodiments, the epigenetic editing system includes a Cas12a, Cas12b, Cas12c, Cas12d, Cas12e, Cas12h or Cas12i domain. In some embodiments, the Cas9 protein is an RNA nuclease or an inactivated RNA nuclease. In some embodiments, the epigenetic editing system includes a Cas12g, Cas13a, Cas13b, Cas13c or Cas13d domain. In some embodiments, the epigenetic editing system includes an Argonaute protein domain.

[0184]

[0215] The CRISPR / Cas system or Cas protein in the epigenetic editing system provided herein may include class 1 or class 2 Cas proteins. The class 1 or class 2 proteins used in the epigenetic editing system may be inactivated in terms of their nuclease activity. In some embodiments, the epigenetic editing system includes a Cas protein derived from a type II, IIA, IIB, IIC, V or VI Cas nuclease. In some embodiments, the epigenetic editing system includes a Cas protein derived from a class 2 Cas nuclease such as Cas1, Cas1B, Cas2, Cas3, Cas4, Cas5, Cas6, Cas7, Cas8, Cas10, Cas14a, Cas14b, Cas14c, CasX, CasY, CasPhi, C2c4, C2c8, C2c9, C2c10, Csy1, Csy2, Csy3, Cse1, Cse2, Csc1, Csc2, Csa5, Csn2, Csm2, Csm3, Csm4, Csm5, Csm6, Cmr1, Cmr3, Cmr4, Cmr5, Cmr6, Csb1, Csb2, Csb3, Csx17, Csx14, Csx10, Csx16, CsaX, Csx3, Csx1, Csx1S, Csf1, Csf2, CsO, Csf4, or a homolog or modified version thereof. In some embodiments, the Cas protein in the epigenetic editing system is a nuclease-inactivated Cas protein.

[0185]

[0216] In some embodiments, the epigenetic editing system comprises a CasX (Cas12e) protein. The CasX protein can have specificity for a 5'-TTCN-3' PAM sequence, where N is any one of the nucleotides A, G, T, or C. In some embodiments, the CasX protein has a reduced or abolished nuclease activity (dCasX). In some embodiments, the epigenetic editing system comprises a CasY (Cas12d) protein. The CasY protein can have specificity for a 5'-TA-3' PAM sequence. In some embodiments, the CasY protein has a reduced or abolished nuclease activity (dCasY). In some embodiments, the epigenetic editing system comprises a Casφ (CasPhi) protein. The Casφ protein can have specificity for a 5'-TTN-3' PAM sequence, where N is any one of the nucleotides A, T, G, or C. In some embodiments, the Casφ protein has a reduced or abolished nuclease activity (dCasφ).

[0186]

[0217] In some embodiments, the Cas protein is a circular permutant Cas protein. For example, the epigenetic editing system can include a circular permutant Cas9 as described in Oakes et al., Cell 176, pages 254 - 267 (2019), which is incorporated herein by reference in its entirety. As used herein, the term "circular permutant" refers to a variant polypeptide in which a portion of the amino acid primary sequence has been moved to a different position within the amino acid primary sequence of the polypeptide, but the local order of the amino acids has not changed and the three - dimensional structure of the protein is conserved (e.g., of the subject Cas protein). For example, a circular permutant of a wild - type 1000 - amino - acid polypeptide can have the N - terminal residue of residue number 500 (relative to the wild - type protein), and residues 1 - 499 of the wild - type protein are added to the C - terminus. Such a circular permutant can have, relative to the wild - type protein sequence, from the N - terminus to the C - terminus, 1 - 499 following amino acid numbers 500 - 1000, resulting in a circular permutant where amino acid 499 is the C - terminal residue. Thus, such an example of a circular permutant has the same total number of amino acids as the wild - type reference protein, and although the amino acids are in the same local order in a specific region of the circular permutant, the overall amino acid primary sequence has changed.

[0187]

[0218] In some embodiments, the epigenetic editing system includes a circularly permuted Cas protein, e.g., a circularly permuted Cas9 protein. In some embodiments, the epigenetic editing system includes a fusion of a circularly permuted Cas protein and an epigenetic effector domain, provided that the epigenetic effector domain is fused to an N - terminus or C - terminus of the circularly permuted Cas protein that is different from the termini of the wild - type Cas protein.

[0188]

[0219] In some embodiments, the circularly permuted Cas protein comprises the N-terminus of the N-terminal fragment of the wild-type Cas protein fused to the C-terminus of the C-terminal fragment of the wild-type Cas protein, thereby generating a new N-terminus and C-terminus. Without wishing to be bound by any theory, the N-terminus and C-terminus of the wild-type Cas protein may be confined to small regions that can cause steric hindrance and reduce access to the target DNA sequence when the Cas protein is fused to an effector domain. In some embodiments, an epigenetic editing system comprising a circularly permuted Cas protein has reduced steric incompatibility compared to an epigenetic editing system comprising a wild-type Cas protein counterpart. In some embodiments, an epigenetic editing system comprising a circularly permuted Cas protein has improved efficacy compared to an epigenetic editing system comprising a wild-type Cas protein counterpart. In some embodiments, an epigenetic editing system comprising a circularly permuted Cas protein has improved epigenetic editing accuracy compared to an epigenetic editing system comprising a wild-type Cas protein counterpart. In some embodiments, an epigenetic editing system comprising a circularly permuted Cas protein has reduced off-target editing effects compared to an epigenetic editing system comprising a wild-type Cas protein counterpart. Guide polynucleotide

[0220] In some embodiments, the epigenetic editing system comprises a guide polynucleotide (or guide nucleic acid). For example, an epigenetic editing system having a DNA-binding domain comprising a CRISPR-Cas protein may also comprise a guide nucleic acid that can form a complex with the CRISPR-Cas protein.

[0189]

[0221] Methods for using a guide nucleotide sequence-programmable DNA binding protein, such as Cas9, for site-specific DNA targeting (e.g., for modifying a genome) are known in the art. A guide RNA (gRNA) can direct a programmable DNA binding protein, such as a class 2 Cas protein like Cas9, to a target sequence on a target nucleic acid molecule, where the gRNA hybridizes and the programmable DNA binding protein creates a modification at or near the target sequence. In some embodiments, the gRNA and an epigenetic editing system fusion protein can form a ribonucleoprotein (RNP), such as a CRISPR / Cas complex.

[0190]

[0222] A guide nucleotide sequence, such as a guide RNA sequence, can include two parts: 1) a nucleotide sequence that shares homology to a target nucleic acid (and, e.g., directs binding of the guide nucleotide sequence-programmable DNA binding protein to the target); and 2) a nucleotide sequence that binds to a nucleic acid-guided programmable DNA binding protein, such as a CRISPR-Cas protein. The nucleotide sequence in 1) can include a spacer sequence that hybridizes to the target sequence. The nucleotide sequence in 2) can be referred to as a scaffold sequence, tracrRNA, or an activation region of the guide nucleic acid and can include a stem-loop structure. The scaffold sequences of guide nucleic acids described in Jinek et al., Science 337:816-821 (2012), US Patent Application Publication Nos. 20160208288 and 20160200779 are hereby incorporated by reference in their entireties.

[0191]

[0223] The guide polynucleotide may be a single molecule or may comprise two separate molecules. For example, the aforementioned portions 1) and 2) may be fused to form one single guide (e.g., single guide RNA or sgRNA), or may be two separate molecules. In some embodiments, the guide polynucleotide is a double polynucleotide linked by a linker. In some embodiments, the guide polynucleotide is a double polynucleotide linked by a non-nucleic acid linker, e.g., a peptide linker or a chemical linker.

[0192]

[0224] Methods for selecting, designing, and validating gRNAs and targeting sequences (or spacer sequences) are described herein and are known to those of skill in the art. Software tools can be used to optimize the gRNA corresponding to the target nucleic acid sequence, e.g., to minimize the overall off-target activity across the genome. For example, a DNA sequence search algorithm can be used to identify the target sequence in the crRNA of the gRNA used with Cas9. Exemplary gRNA design tools, including those described in Bae et al., Cas-OFFinder: A fast and versatile algorithm that searches for potential off-target sites of Cas9 RNA-guided endonucleases. Bioinformatics 30, pages 1473-1475 (2014), are incorporated herein by reference in their entirety.

[0193]

[0225] Guide polynucleotides can be of various lengths. In some embodiments, the length of the spacer or targeting array depends on the CRISPR / Cas components of the epigenetic editing system and the components used. For example, different Cas proteins from different bacterial species have various optimal targeting array lengths. Thus, the spacer sequence can include nucleotides of lengths 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, or more than 50. In some embodiments, the spacer includes 18 - 24 nucleotides in length. In some embodiments, the spacer includes 19 - 21 nucleotides in length. In some embodiments, the spacer includes 20 nucleotides in length. In some embodiments, the guide nucleic acid (e.g., guide RNA) is 15 - 100 nucleotides in length and includes a sequence of at least 10 consecutive nucleotides that is complementary to the target sequence. In some embodiments, the guide RNA is 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49 or 50 nucleotides in length. In some embodiments, the guide RNA includes a sequence of 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49 or 50 consecutive nucleotides that is complementary to the target sequence. In some embodiments, the target sequence is a DNA sequence. In some embodiments, the degree of complementarity between the targeting sequence of the gRNA and the target sequence on the target nucleic acid molecule is at least about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%, 99% or 100%. In some embodiments, the targeting sequence of the gRNA and the target sequence on the target nucleic acid molecule can be 100% complementary.In other embodiments, the targeting sequence of the gRNA and the target sequence on the target nucleic acid molecule may contain at least one mismatch. For example, the targeting sequence of the gRNA and the target sequence on the target nucleic acid molecule may contain 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 mismatches.

[0194]

[0226] In some embodiments, the target sequence is in the genome of a virus. In some embodiments, the target sequence is in the genome of a bacterium. In some embodiments, the target sequence is in the genome of a eukaryote. In some embodiments, the target sequence is a sequence in the genome of a mammal. In some embodiments, the target sequence is a sequence in the genome of a human. In some embodiments, the 3' end of the target sequence is immediately adjacent to a canonical PAM sequence (NGG). In some embodiments, the guide nucleic acid (e.g., guide RNA) is complementary to a sequence associated with a disease or disorder.

[0195]

[0227] In some embodiments, the guide RNA is a shortened form. The shortening may include any number of nucleotide deletions. For example, the shortening may include 1, 2, 3, 4, 5, 10, 15, 20, 25, 30, 40, 50, or more nucleotides. In some embodiments, the guide polynucleotide includes RNA. In some embodiments, the guide polynucleotide includes DNA. In some embodiments, the guide polynucleotide includes a mixture of DNA and RNA.

[0196]

[0228] The guide polynucleotide can be modified. The modifications can include chemical changes, synthetic modifications, nucleotide additions, and / or nucleotide removals. Modified nucleosides or nucleotides can be present in the gRNA. For example, the gRNA can include one or more non-natural and / or natural components or configurations that are used in place of or in addition to the standard A, G, C, and U residues. Modified RNAs can include one or both of the non-bridging phosphate oxygens and / or one or more changes or substitutions of the bridging phosphate oxygens in the phosphodiester backbone linkages, changes in the ribose sugar, such as changes in the 2'-hydroxyl on the ribose sugar (an exemplary sugar modification), changes in the phosphate ester moiety, modifications or substitutions of natural nucleobases, substitutions or modifications of the ribose phosphate backbone, modifications of the 3'- or 5'-ends of the oligonucleotide, or substitutions of the terminal phosphate groups, or conjugation of moieties, caps, or linkers, or one or more of any combination thereof.

[0197]

[0229] In some embodiments, the ribose group (or sugar) can be modified. In some embodiments, the modified ribose can control the oligonucleotide binding affinity to a complementary strand, duplex formation, or interaction with nucleases. Examples of chemical modifications to the ribose group include, but are not limited to, 2'-O-methyl (2'-OMe), 2'-fluoro (2'-F), 2'-deoxy, 2'-O-(2-methoxyethyl) (2'-MOE), 2'-NH2, 2'-O-allyl, 2'-O-ethylamine, 2'-O-cyanoethyl, 2'-O-acetal ester, or bicyclic nucleotides, such as locked nucleic acid (LNA), 2'-(5-forced ethyl (S-cEt)), forced MOE, or 2'-0,4'-C-aminomethylene bridged nucleic acid (2',4'-BNANC). In some embodiments, the 2'-O-methyl modification can increase the binding affinity of the oligonucleotide. In some embodiments, the 2'-O-methyl modification can improve the nuclease stability of the oligonucleotide. In some embodiments, the 2'-fluoro modification can increase the oligonucleotide binding affinity and nuclease stability.

[0198]

[0230] In some embodiments, the phosphate group can be chemically modified. Examples of chemical modifications to the phosphate group include, but are not limited to, phosphorothioate (PS), phosphonoacetate (PACE), thiophosphonoacetate (thioPACE), amide, triazole, phosphonate or phosphotriester modifications. In some embodiments, a PS bond can refer to a bond in which sulfur replaces one non-bridging phosphate oxygen, for example, in a phosphodiester bond between nucleotides. "s" can be used to indicate a PS modification in the gRNA sequence. In some embodiments, the gRNA or sgRNA can include phosphorothioate (PS) bonds at the 5' end or the 3' end. In some embodiments, the gRNA or sgRNA can include phosphorothioate (PS) bonds at the 5' end. In some embodiments, the gRNA or sgRNA can include phosphorothioate (PS) bonds at the 3' end. In some embodiments, the gRNA or sgRNA can include phosphorothioate (PS) bonds at both the 5' end and the 3' end. In some embodiments, the gRNA or sgRNA can include one, two, three or more than three phosphorothioate bonds at the 5' end or the 3' end. In some embodiments, the gRNA or sgRNA can include three phosphorothioate (PS) bonds at the 5' end or the 3' end. In some embodiments, the gRNA or sgRNA can include three phosphorothioate bonds at the 3' end. In some embodiments, the gRNA or sgRNA can include two or fewer (i.e., only two) consecutive phosphorothioate (PS) bonds at the 5' end or the 3' end. In some embodiments, the gRNA or sgRNA can include three consecutive phosphorothioate (PS) bonds at the 5' end or the 3' end. In some embodiments, the gRNA or sgRNA can include the sequence 5'-UsUsU-3' at the 3' end or the 5' end, where U represents uridine and s represents a phosphorothioate (PS) bond.

[0199]

[0231] In some embodiments, the nucleobases can be chemically modified. Examples of chemical modifications to nucleobases include, but are not limited to, 2-thiouridine, 4-thiouridine, N6-methyladenosine, pseudouridine, 2,6-diaminopurine, inosine, thymidine, 5-methylcytosine, 5-substituted pyrimidines, isoguanine, isocytosine, or halogenated aromatic groups.

[0200]

[0232] The chemical modification can be performed on a part or the whole of the guide polynucleotide. In some embodiments, a guide RNA of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 base pairs is chemically modified. In some embodiments, a guide RNA of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50 base pairs is chemically modified. In some embodiments, a guide RNA of 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, or 150 base pairs is chemically modified. The chemical modification can be performed in the protospacer region, tracrRNA, crRNA, stem-loop, or any combination thereof. Zinc finger protein

[0233] In some embodiments, the epigenetic editing systems described herein include a nucleic acid binding domain that includes a zinc finger domain.

[0201]

[0234] Zinc finger proteins are DNA-binding proteins that contain one or more zinc fingers. In some embodiments, a zinc finger (ZF) contains a relatively small polypeptide domain that includes approximately 30 amino acids. A zinc finger can further include an α-helix adjacent to an antiparallel β-sheet (known as the ββα fold) and can coordinate with zinc ions between four Cys and / or His residues, as described further below. In some embodiments, the ZF domain recognizes and binds to a nucleic acid triplet or overlapping quadruplet within a double-stranded DNA target sequence. In certain embodiments, ZF can also bind to RNA and proteins.

[0202]

[0235] As used herein, the term "zinc finger" (ZF) or "zinc finger motif" (ZF motif) refers to an individual "finger" that includes a beta-beta-alpha (ββα) protein fold stabilized by a zinc ion, as described elsewhere herein. In some embodiments, each finger includes approximately 30 amino acids. In some embodiments, a ZF protein or ZF protein domain is a protein motif that contains multiple fingers or finger-like protrusions that make tandem contacts with its target molecule. For example, a ZF finger can bind to a triplet or (overlapping) quadruplet nucleotide sequence. Thus, a tandem array of ZF fingers can be designed for a non-naturally occurring ZF protein to bind to a desired target.

[0203]

[0236] Zinc finger proteins are widespread in eukaryotic cells. An exemplary motif characterizing one class of these proteins (the C2H2 class) is Cys-(X)2-4-Cys-(X)12-His-(X)3-5His, where X is any amino acid. A single finger domain can be about 30 amino acids in length. In some embodiments, a single finger contains an alpha helix that coordinates, via zinc, two invariant histidine residues with two cysteines of a single beta turn.

[0204]

[0237] In some embodiments, the amino acid sequence of a zinc finger protein, such as the Zif268 protein, can be varied by creating amino acid substitutions at helix positions (e.g., positions 1, 2, 3, and 6 of Zif268) on the zinc finger recognition helix. For example, a modified zinc finger having a non-native DNA recognition specificity in which an appropriate DNA sub-site is replaced with an altered DNA triplet can be generated by phage display and a combinatorial library containing randomized side chains in either the first finger or the central finger of Zif268, and then isolated using the altered Zif268 binding site.

[0205]

[0238] In some embodiments, a zinc finger contains a C2H2 finger. In some embodiments, a zinc finger protein contains a ZF array that includes contiguous C2H2-ZFs, each ZF contacting three or more contiguous bases. In some embodiments, zinc finger protein structures, such as the zinc finger protein Zif268 and its variants bound to DNA, exhibit a semi-conserved interaction pattern in which typically three amino acids from the alpha helix of the zinc finger contact three adjacent base pairs in the DNA. Thus, in embodiments, the zinc finger DNA binding domain functions in a modular fashion by a one-to-one interaction between the zinc finger and a three-base pair nucleotide sequence in the DNA sequence.

[0206]

[0239] In some embodiments, the epigenetic editing system includes a zinc finger motif consisting of the sequence: N’--(Helix 1)- -(Helix 2)- -(Helix 3)- -(Helix 4)- -(Helix 5)- -(Helix 6)- -C’, where (Helix) is a six consecutive amino acid residue peptide that forms a short alpha helix. In some embodiments, the epigenetic editing system includes a zinc finger motif consisting of the sequence: N’--(Helix 1)- -(Helix 2)- -(Helix 3)- -(Helix 4)--(Helix 5)-- -C’, where (Helix) is a six consecutive amino acid residue peptide that forms a short alpha helix.

[0207]

[0240] In some embodiments, to achieve specific recognition and binding of a continuous DNA sequence, two or more zinc fingers are linked together in a tandem array. The zinc finger or zinc finger array in the epigenetic editing system may be natural or may be artificially engineered with respect to the desired DNA binding specificity. For example, the DNA binding characteristics of individual zinc fingers can be manipulated by randomization of the amino acids at the alpha helix positions of the zinc fingers involved in DNA binding and the use of selection methods such as phage display to identify desirable variants that can bind to the target DNA site of interest. Alternatively, two or more zinc fingers can be linked together in a tandem array to achieve specific recognition and binding of a non - continuous sequence.

[0208]

[0241] An engineered zinc finger binding domain can have a novel binding specificity compared to a native zinc finger protein. Zinc fingers with desirable DNA binding specificities can be designed and selected via various approaches. For example, a database containing triplet (or quadruplet) nucleotide sequences and individual zinc finger amino acid sequences, where each triplet or quadruplet nucleotide sequence in the database is associated with the amino acid sequence of one or more zinc fingers that bind to a specific triplet or quadruplet sequence, can be used to design a zinc finger array for a specific DNA sequence. See, for example, U.S. Patent Nos. 6,453,242, 6,534,261, and 8,772,453, which are incorporated herein by reference in their entireties. In some embodiments, a zinc finger array can be designed and selected from a library of zinc fingers, such as a randomized zinc finger library. In some embodiments, zinc fingers with novel DNA binding specificities are generated by a selection-based method using a combinatorial library. For example, zinc fingers can be selected by successive rounds of affinity selection using biotinylated target DNA to enrich phage expressing a protein that can bind to a specific target sequence, following phage display involving the presentation of zinc finger proteins on the surface of filamentous phage. The bacterial two-hybrid (B2H) system can also be used to select zinc fingers that bind to a specific target site from a randomized library. For example, the zinc finger binding site can be placed upstream of a weak promoter that drives the expression of two selectable markers in a host cell, such as an E. coli cell. A library of zinc fingers fused to a fragment of the reporter protein, yeast Gal11P protein, can be expressed in the cells, and binding of the zinc finger to the target site activates transcription to recruit the RNA polymerase-Gal4 fusion, enabling cell survival on a selective medium.The rational design and selection of zinc fingers as described in Maeder et al., 2008, Mol. Cell, 31:294-301; Joung et al., 2010, Nat. Methods, 7:91-92; Isalan et al., 2001, Nat. Biotechnol., 19:656-660, Rebar et al., Science 263, 671-673 (1994), and Joung et al., Proc Natl Acad Sci USA 97, 7382-7387 (2000), each of which is incorporated herein by reference in its entirety.

[0209]

[0242] In some embodiments, the zinc fingers can be evolved and selected using a phage-assisted continuous evolution (PACE) system that includes a host cell, e.g., an E. coli cell, a "helper phagemid" that is present in all host cells and encodes all phage proteins except for one phage protein (e.g., the g3p protein), an "accessory plasmid" that is present in all host cells and expresses the g3p protein in response to an active library member, and a "selection phagemid" that expresses a library of evolving proteins or nucleic acids, is replicated, and is packaged into secreted phage particles. The helper plasmid and the accessory plasmid can be combined into a single plasmid. The new host cells can only be infected by phage particles containing g3p. Adaptive selection phagemids encoding library members that induce g3p expression from the accessory plasmid can be packaged into phage particles containing g3p. Phage particles containing g3p can infect new cells and further replicate the adaptive selection phagemids, but phage particles lacking g3p are non-infectious, so low fitness selection phagemids cannot proliferate. A selection system combined with a continuous flow of host cells through a lagoon that permits replication of the phagemid but not of the host cell can be used to rapidly select zinc fingers. The PACE system described in U.S. Patent No. 9,023,594 is incorporated herein by reference in its entirety.

[0210]

[0243] The zinc finger DNA binding domain of an epigenetic editing system can include one or more zinc fingers. For example, the zinc finger DNA binding domain can include two, three, four, five, six, seven, eight, nine, ten, eleven, twelve or more zinc fingers. In some embodiments, the zinc finger DNA binding domain has at least three zinc fingers. In some embodiments, the zinc finger DNA binding domain has at least four, five or six zinc fingers. In some embodiments, the zinc finger DNA binding domain has three zinc fingers. In some embodiments, the zinc finger DNA binding domain has at least two zinc fingers. In some embodiments, the zinc finger DNA binding domain has an array of two-finger units.

[0211]

[0244] The zinc finger DNA binding domains of an epigenetic editing system can be designed towards optimized specificity. In some embodiments, a sequential selection strategy is used to design a multi-finger ZF domain. For example, in a multi-finger ZF domain, the first finger can be randomized and selected by phage display, and a small pool of the selected fingers can be led to the next stage where the second finger is randomized and selected. This process can be repeated multiple times depending on the number of fingers in the ZF domain. In some embodiments, parallel optimization is used to design a multi-finger ZF domain. For example, a master randomized library can be screened using a B2H system with low selection stringency to identify various individual fingers that can bind to each 3-base pair sub-site of the target site. Then, the three selected populations can be randomly shuffled to generate a library of multi-finger proteins, and subsequently, the library can be screened under high stringency selection conditions to identify 3-finger proteins that target a specific 9-base pair site. In further embodiments, a number of low stringency selections can be used to generate a master library of single fingers, from which multi-finger proteins, such as 3-finger ZF proteins, can be selected. For example, the master library or archive can contain a pre-selected zinc finger pool, each containing a mixture of fingers that target different 3-base pair sub-sites of the DNA sequence at a certain position within a 3-finger ZF protein. In certain embodiments, the zinc finger archive contains at least 192 finger pools (64 potential 3bp target sub-sites for each position in a 3-finger protein). In some embodiments, the zinc finger archive contains at least one zinc finger pool containing at least 10, 20, 30, 40, 50, 60, 70, 80, 90, 95, 100 or more different fingers. In some embodiments, smaller libraries are created from the archive for screening using a reporting system, such as a bacterial two-hybrid selection system.

[0212]

[0245] In some embodiments, a multi-finger ZF domain, e.g., a 3-finger ZF domain, can be designed and selected using two complementary libraries. For example, a 3-finger ZF domain can be designed using two pre-made zinc finger phage display libraries, where the first library contains randomized DNA-binding amino acid positions in fingers 1 and 2, and the second library contains randomized DNA-binding amino acid positions in fingers 2 and 3. The two libraries are complementary. Because the first library contains randomization at all base contact positions of finger 1 and certain base contact positions of finger 2, while the second library contains randomization at the remaining base contact positions of finger 2 and all base contact positions of finger 3. Selection of "one-and-a-half" fingers from each master library can be done in parallel using a DNA sequence with 5 nucleotides fixed for the target sequence. Subsequently, PCR can be used to amplify the zinc finger coding sequences from the recovered phage, and sets of "one-and-a-half" fingers can be paired to yield a recombinant 3-finger DNA binding domain.

[0213]

[0246] In some embodiments, a multi-finger ZF domain can be designed according to the context effects of adjacent fingers. In some embodiments, a multi-finger ZF domain is designed without selection. For example, a 3-finger ZF domain can be assembled using an N-terminal finger and a C-terminal finger identified in other arrays containing a common middle finger using a library containing an archive of 3-finger ZF arrays, including pre-selected and / or tested 3-finger arrays.

[0214]

[0247] Software for the design and selection of ZF arrays, such as ZiFit (http: / / bindr.gdcb.iastate.edu / ZiFiT / ; http: / / www.zincfingers.org / software-tools.htm), is available and known to those skilled in the art.

[0215]

[0248] Accordingly, the zinc finger DNA binding domain of an epigenetic editing system can include one or more zinc fingers. For example, the zinc finger DNA binding domain can include two, three, four, five, six, seven, eight, nine, ten, eleven, twelve or more zinc fingers. In some embodiments, the zinc finger DNA binding domain has at least three zinc fingers. In some embodiments, the zinc finger DNA binding domain has at least four, five or six zinc fingers. In some embodiments, the zinc finger DNA binding domain has three zinc fingers. In some embodiments, a zinc finger DNA binding domain comprising at least three zinc fingers recognizes a target DNA sequence of 9 or 10 nucleotides. In some embodiments, a zinc finger DNA binding domain comprising at least four zinc fingers recognizes a target DNA sequence of 12-14 nucleotides. In some embodiments, a zinc finger DNA binding domain comprising at least six zinc fingers recognizes a target DNA sequence of 18-21 nucleotides.

[0216]

[0249] In some embodiments, the epigenetic editing systems disclosed herein are non-natural and suitably include three or more zinc fingers. In some embodiments, the epigenetic editing system includes four, five, six, eight, nine, ten, eleven, twelve, thirteen, fourteen, fifteen, sixteen, seventeen, eighteen, or more (e.g., up to about thirty or thirty-two at most) zinc finger motifs arranged tandemly adjacent to each other, forming an array of ZF motifs. In some embodiments, the epigenetic editing system includes at least three ZF motifs, at least four ZF motifs, at least five ZF motifs, or at least six ZF motifs, at least seven ZF motifs, at least eight ZF motifs, at least nine ZF motifs, at least ten ZF motifs, at least eleven or at least twelve ZF motifs in the nucleic acid binding domain. In some embodiments, the epigenetic editing system includes up to six, seven, eight, ten, eleven, twelve, sixteen, seventeen, eighteen, twenty-two, twenty-three, twenty-four, twenty-eight, twenty-nine, thirty, thirty-four, thirty-five, thirty-six, forty, forty-one, forty-two, forty-six, forty-seven, forty-eight, fifty-four, fifty-five, fifty-six, fifty-eight, fifty-nine, or sixty ZF motifs in the nucleic acid binding domain. Alternatively, the zinc finger motifs can be arranged with spacers to skip by at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, or 50 nucleotides.

[0217]

[0250] In some embodiments, zinc fingers or zinc finger arrays that target specific DNA sequences are designed using a modular assembly approach. For example, two or more preselected zinc fingers can be fused in tandem.

[0218]

[0251] In some embodiments, the zinc finger array comprises a plurality of zinc fingers fused via peptide bonds. In some embodiments, the zinc finger array comprises a plurality of zinc fingers, one or more of which are linked by a peptide linker. For example, the zinc fingers in a multi-finger array can be linked by a peptide linker of 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or more amino acids in length. In some embodiments, the zinc fingers in a multi-finger array are linked by a 5-amino acid-long peptide linker. In some embodiments, the zinc fingers in a multi-finger array are linked by a 6-amino acid-long peptide linker.

[0219]

[0252] In some embodiments, the ZF-containing protein may contain a ZF array of two or more ZF motifs, and those arrays may be directly adjacent to each other (i.e., separated by a short (standard) linker sequence), or may be separated by a longer, flexible or structured polypeptide sequence. In some embodiments, directly adjacent fingers bind to a continuous nucleic acid sequence, i.e., adjacent trinucleotides / triplets. In some embodiments, adjacent fingers cross-link between their respective target triplets, which can help enhance or improve the recognition of the target sequence and result in the binding of overlapping quadruplet sequences. In some embodiments, distant ZF domains within the same protein may recognize (or bind) non-continuous nucleic acid sequences and may also bind to different molecules (e.g., proteins rather than nucleic acids).

[0220]

[0253] In some embodiments, the epigenetic editing system comprises zinc fingers comprising more than three fingers. In some embodiments, the epigenetic editing system comprises at least six zinc fingers in the DNA binding domain. In some embodiments, the epigenetic editing system comprises six zinc fingers that bind to an 18 bp target sequence in the DNA binding domain. In some embodiments, the 18 bp target sequence is unique in the human genome. In some embodiments, the 18 bp target sequence is unique in the viral genome. In some embodiments, the 18 bp target sequence is unique in the bacterial genome. In some embodiments, the 18 bp target sequence is unique in the eukaryotic genome. In some embodiments, the epigenetic editing system comprises zinc fingers comprising at least seven, eight, nine, ten, eleven, twelve, thirteen, fourteen, fifteen or more zinc fingers. In some embodiments, the strong affinity of the three-finger protein will reduce specificity because some longer arrays will be able to bind to DNA. Without wishing to be bound by any theory, zinc finger proteins comprising multiple two-finger units or three-finger units linked by an extended linker may confer higher DNA binding specificity compared to arrays with fewer fingers or the same number of fingers simply linked via peptide bonds. In some embodiments, the epigenetic editing system comprises at least three two-finger units linked by a peptide linker, each of the two-finger units binding to one sub-site in the target DNA sequence. In some embodiments, the epigenetic editing system comprises at least four two-finger units linked by a peptide linker, each of the two-finger units binding to one sub-site in the target DNA sequence. In some embodiments, the epigenetic editing system comprises at least five two-finger units linked by a peptide linker, each of the two-finger units binding to one sub-site in the target DNA sequence.In some embodiments, the epigenetic editing system includes at least 6, 7, 8, 9, 10 or more two-finger units linked by a peptide linker, and each of these two-finger units binds to one sub-site in the target DNA sequence. In some embodiments, the epigenetic editing system includes at least two three-finger units linked by a peptide linker, and each of these three-finger units binds to one sub-site in the target DNA sequence. In some embodiments, the epigenetic editing system includes at least three three-finger units linked by a peptide linker, and each of these three-finger units binds to one sub-site in the target DNA sequence. In some embodiments, the epigenetic editing system includes at least four three-finger units linked by a peptide linker, and each of these three-finger units binds to one sub-site in the target DNA sequence. In some embodiments, the epigenetic editing system includes at least five three-finger units linked by a peptide linker, and each of these three-finger units binds to one sub-site in the target DNA sequence. In some embodiments, the epigenetic editing system includes at least 6, 7, 8, 9, 10 or more three-finger units linked by a peptide linker, and each of these three-finger units binds to one sub-site in the target DNA sequence.

[0221]

[0254] In some embodiments, multiple zinc fingers, each recognizing at least two specific DNA nucleotides or two-nucleotide "subsites", are assembled to target a specific DNA sequence in a target gene. In some embodiments, such DNA subsites are contiguous sequences in the target gene. In some embodiments, one or more of the DNA subsites are separated by gaps in the target gene; for example, a multi-finger ZF can recognize DNA subsites spanning a subsite gap of 1, 2, 3, or more base pairs between adjacent subsites. In some embodiments, the zinc fingers in a multi-finger ZF are linked by a peptide linker. The peptide linker can be 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or more amino acids in length. In some embodiments, the linker comprises 5 or more amino acids. In some embodiments, the linker comprises 7-17 amino acids. In some embodiments, the linker is a flexible linker. In some embodiments, the linker is a rigid linker, e.g., a linker comprising one or more prolines.

[0222]

[0255] A zinc finger array having sequence-specific DNA binding activity can be fused to a functional effector domain, such as an epigenetic effector domain described herein, to impart an epigenetic modification to a DNA sequence or associated histones in a target gene. In some embodiments, the epigenetic editing systems described herein include a zinc finger array having specificity for a target DNA sequence. In some embodiments, two linkers of the zinc finger array are the same. In some embodiments, two linkers of the zinc finger array are different.

[0223]

[0256] In some embodiments, the programmable DNA-binding protein comprises an Argonaute protein. An example of such a nucleic acid programmable DNA-binding protein is the Argonaute protein from Natronobacterium gregoryi (NgAgo). NgAgo is an ssDNA-guided endonuclease. NgAgo binds to a 5'-phosphorylated ssDNA (gDNA) of -24 nucleotides, guides it to its target site, and makes a DNA double-strand break at the gDNA site. In contrast to Cas9, the NgAgo-gDNA system does not require a protospacer adjacent motif (PAM). The use of nuclease-inactive NgAgo (dNgAgo) can greatly expand the bases that can be targeted. The characterization and use of NgAgo are described in Gao et al., Nat Biotechnol., July 2016; 34(7):768-73. PubMed PMID: 27136078; Swarts et al., Nature. 507(7491) (2014):258-61; and Swarts et al., Nucleic Acids Res. 43(10) (2015):5120-9, each of which is incorporated herein by reference.

[0224]

[0257] In some embodiments, the nucleic acid binding domain comprises a virus-derived RNA binding domain that is guided by an RNA sequence to bind to a target gene. In some embodiments, the nucleic acid binding domain comprises a K homology (KH) domain, an MS2 coat protein domain, a PP7 coat protein domain, an SfMu Com coat protein domain, a sterile alpha motif, a telomerase Ku-binding motif and Ku protein, a telomerase Sm7-binding motif and Sm7 protein, or any other RNA recognition motif.

[0225]

[0258] In some embodiments, the nucleic acid binding domain comprises an inactivated nuclease, for example, an inactivated meganuclease. Further non-limiting examples of DNA binding domains include the tetracycline repressor (tetR) DNA binding domain, leucine zipper, helix-loop-helix (HLH) domain, helix-turn-helix domain, zinc finger, β-sheet motif, steroid receptor motif, bZIP domain, homeodomain, and AT hook.

[0226] Target sequence

[0259] As used herein, a "target polynucleotide sequence" can be a nucleic acid sequence present in a gene of interest. The target sequence can be within the genome of a cell or can be expressed within the cell. In one aspect, the epigenetic editing systems provided herein are used to bind to a target polynucleotide sequence to effect epigenetic modification of a target gene and / or transcriptional regulation of a target gene. For example, the target sequence may be recognized by a zinc finger array of an epigenetic editing system or may hybridize with a guide RNA sequence complexed with a nuclease-inactivated CRISPR protein of the epigenetic editing system. In embodiments where the epigenetic editing system comprises a gRNA-dCas-effector domain complex, the gRNA is designed to have complementarity to the target sequence (or identity to the opposing strand of the target sequence, e.g., the protospacer sequence). In some embodiments, the gRNA comprises a spacer sequence that is 100% identical to the protospacer sequence in the target sequence. In some embodiments, the gRNA sequence comprises a spacer sequence that is about 95%, 90%, 85%, or 80% identical to the protospacer sequence in the target sequence.

[0227]

[0260] In some embodiments, the target sequence is the endogenous sequence of an endogenous gene of the host cell. In some embodiments, the target sequence is a foreign sequence.

[0261] The target array can be any region of a polynucleotide (e.g., a DNA sequence) suitable for epigenetic editing. For example, the target polynucleotide sequence can be any part of a target gene. In some embodiments, the target polynucleotide sequence is part of a transcriptional regulatory sequence. In some embodiments, the target polynucleotide sequence is part of a promoter, enhancer or silencer. In some embodiments, the target polynucleotide sequence is part of a promoter. In some embodiments, the target polynucleotide sequence is part of an enhancer. In some embodiments, the target polynucleotide sequence is part of a silencer. In some embodiments, the target polynucleotide sequence is within about 3000, 2900, 2800, 2700, 2600, 2500, 2400, 2300, 2200, 2100, 2000, 1900, 1800, 1700, 1600, 1500, 1400, 1300, 1200, 1100, 1000, 900, 800, 700, 600, 500, 400, 300, 200 or 100 base pairs (bp) adjacent to the transcription start site. In some embodiments, the target polynucleotide sequence is within about 1000, 900, 800, 700, 600, 500, 400, 300, 200 or 100 base pairs (bp) adjacent to the transcription start site. In some embodiments, the target polynucleotide sequence is within about 500, 400, 300, 200 or 100 base pairs (bp) adjacent to the transcription start site.

[0228]

[0262] In some embodiments, the target polynucleotide sequence is within about 100 base pairs (bp) adjacent to the transcription start site.

[0263] In some embodiments, the target polynucleotide sequence is a hypomethylated nucleic acid sequence. In some embodiments, the target polynucleotide sequence is a hypermethylated nucleic acid sequence. In some embodiments, the target polynucleotide sequence is in, near, or within a promoter sequence. In some embodiments, the target polynucleotide sequence is in, near, or within a promoter sequence. In some embodiments, the target polynucleotide sequence is adjacent to a CpG island. In some embodiments, the target polynucleotide sequence is known to be associated with a disease or condition.

[0229] Regulation of target gene expression

[0264] In some embodiments, the present disclosure provides epigenetic editing systems, compositions, and methods for epigenetic modification of target polynucleotides in a target gene encoding a protein or a target non-coding gene. In some embodiments, the epigenetic editing system effects epigenetic modification, such as DNA methylation, in the coding region of the target gene, thereby reducing or silencing the expression of the target gene. In some embodiments, the epigenetic editing system effects epigenetic modification, such as DNA methylation, in a regulatory sequence of the target gene, such as a promoter or enhancer, thereby reducing or silencing the expression of the target gene. In some embodiments, the epigenetic editing system effects transcriptional repression of the target gene or recruits a transcriptional repressor to the coding region of the target gene, thereby reducing or silencing the expression of the target gene. In some embodiments, the epigenetic editing system recruits a transcriptional repressor to a regulatory sequence of the target gene, such as a promoter or enhancer, thereby reducing or silencing the expression of the target gene. In some embodiments, the epigenetic editing system effects epigenetic modification, such as DNA demethylation, in the coding region of the target gene, thereby increasing the expression of the target gene. In some embodiments, the epigenetic editing system effects epigenetic modification, such as DNA demethylation, in a regulatory sequence of the target gene, such as a promoter or enhancer, thereby increasing the expression of the target gene. In some embodiments, the epigenetic editing system effects transcriptional activation of the target gene or recruits a transcriptional activator to the coding region of the target gene, thereby increasing the expression of the target gene. In some embodiments, the epigenetic editing system recruits a transcriptional activator to a regulatory sequence of the target gene, such as a promoter or enhancer, thereby increasing the expression of the target gene.

[0230]

[0265] In some embodiments, the target gene and / or the encoded protein is associated with a disease, disorder or pathogenic condition. Alternatively, or in addition, the target gene may not have a direct association with the disease, but instead may be able to complement a disease-related gene. In some embodiments, the target gene may be a transcription factor or other gene used to manipulate cells.

[0231]

[0266] The epigenetic modifications achieved by the epigenetic editing system described herein are sequence-specific. In some embodiments, the modification is at a specific site of the target polynucleotide. In some embodiments, the modification is at a specific allele of the target gene. Thus, the epigenetic modification can result in the regulation of the expression of one copy of the target gene containing the specific allele, e.g., a decrease or increase in expression, while the other copy of the target gene does not. In some embodiments, the specific allele is associated with a disease, condition or disorder.

[0232]

[0267] Epigenetic modifications can be performed on a target gene in a genome of interest, such as a prokaryotic genome, a plant genome, a viral genome, a mammalian genome, or a human genome. The target gene can be any organism and its genome or can be derived therefrom. For example, the target gene can be a prokaryotic gene, a eukaryotic gene, a viral gene, an animal gene, a plant gene, a mouse gene, a rat gene, a rabbit gene, a fish gene, a bird gene, a monkey gene, or a human gene. In some embodiments, the target gene is a reporter gene whose expression can be easily traced and monitored. Reporter genes and reporter systems include, for example, sequences encoding green fluorescent protein, red fluorescent protein, enhanced yellow protein, enhanced cyan protein, or luciferase protein. In some embodiments, the target gene encodes a selectable marker, such as beta-galactosidase, chloramphenicol acetyltransferase, or an antibiotic resistance marker. In some embodiments, the target gene is associated with a disease, condition, or disorder or contains one or more mutations associated with a disease, condition, or disorder. Non-limiting exemplary target genes include HBB, HBA, hMSH2, HMLH1, growth factor GM-SCF, VEGF, EPO, Erb-B2, and hGH.

[0233]

[0268] Target genes also include plant genes whose suppression or activation results in an improvement in plant characteristics, such as improved crop production, disease resistance, or herbicide resistance. For example, suppression of the expression of the FAD2-1 gene results in a favorable increase in oleic acid and a decrease in linoleic acid and linolenic acid.

[0234]

[0269] In some embodiments, the epigenetic editing system provided herein effects epigenetic modification in a gene containing a target sequence. In some embodiments, the epigenetic editing system modulates the expression of the protein encoded by that gene. In some embodiments, the epigenetic editing system reduces the level of the protein encoded by that gene. In some embodiments, the epigenetic editing system increases the level of the protein encoded by that gene.

[0235]

[0270] To generate epigenetic editing at a target gene, a target gene polynucleotide can be contacted with an epigenetic editing composition disclosed herein that includes a target DNA binding domain, an epigenetic effector domain, such as an epigenetic repressor domain, provided that the DNA binding domain directs the epigenetic effector domain to a target polynucleotide sequence in the target gene, resulting in an epigenetic modification, such as a modification of the methylation state. In some embodiments, the epigenetic editing system results in a change in the methylation state of a target DNA sequence in the target gene. In some embodiments, the epigenetic editing system results in a change in the methylation state of a specific allele in the target gene. In some embodiments, the epigenetic editing system results in a change in the methylation state of a histone protein associated with the target gene.

[0236]

[0271] In some embodiments, the epigenetic modification reduces the transcription of a target gene containing the target sequence. In some embodiments, the epigenetic modification abolishes the transcription of a target gene containing the target sequence. In some embodiments, the epigenetic modification reduces the transcription of a copy of the target gene containing a specific allele recognized by the epigenetic editing system. In some embodiments, the epigenetic modification abolishes the transcription of a copy of the target gene containing a specific allele recognized by the epigenetic editing system. In some embodiments, the epigenetic editing system reduces the level of the protein encoded by the target gene. In some embodiments, the epigenetic editing system eliminates the expression of the protein encoded by the target gene. In some embodiments, the epigenetic editing system reduces the level of the protein encoded by a copy of the target gene containing a specific allele recognized by the epigenetic editing system. In some embodiments, the epigenetic editing system eliminates the expression of the protein encoded by a copy of the target gene containing a specific allele recognized by the epigenetic editing system.

[0237]

[0272] In some embodiments, the epigenetic modification increases the transcription of a target gene containing the target sequence. In some embodiments, the epigenetic modification increases the transcription of a copy of the target gene containing a specific allele recognized by the epigenetic editing system. In some embodiments, the epigenetic editing system increases the level of the protein encoded by the target gene. In some embodiments, the epigenetic editing system increases the level of the protein encoded by a copy of the target gene containing a specific allele recognized by the epigenetic editing system.

[0238]

[0273] The target gene can be epigenetically modified in vitro, ex vivo, or in vivo. Thus, epigenetic modification of the target gene can regulate the expression of the target gene or its allele in ex vivo cells or in in vivo subjects. In some embodiments, the target polynucleotide sequence is a locus in the genomic DNA of a cell. In some embodiments, the cell is a cultured cell. In some embodiments, the cell is in vitro. In some embodiments, the cell is ex vivo. In some embodiments, the cell is in vivo. For example, an epigenetic editing system, such as a zinc finger array and a fusion protein comprising an effector domain, or an sgRNA complexed with a Cas protein-effector domain fusion, can be expressed in a cell in which regulation of the expression of the target gene is desired, such that the target gene is capable of contacting the epigenetic editing system described herein. In some embodiments, the cell is from a mammal. In some embodiments, the mammal is a human. In some embodiments, the mammal is a rodent. In some embodiments, the rodent is a mouse. In some embodiments, the rodent is a rat.

[0239]

[0274] In some embodiments, the epigenetic editing system described herein measures by transcription of a target gene in a cell, tissue, or subject, and reduces the expression of the target gene by at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 95%, at least about 99% or more compared to a control cell, control tissue, or control subject. In some embodiments, the epigenetic editing system described herein measures by transcription of a copy of a target gene in a cell, tissue, or subject, and reduces the expression of the copy of the target gene by at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 95%, at least about 99% or more compared to a control cell, control tissue, or control subject. In some embodiments, the copy of the target gene contains a specific sequence or specific allele recognized by the epigenetic editing system. In some embodiments, the copy to be epigenetically modified encodes a functional protein. Thus, in some embodiments, the epigenetic editing system compositions disclosed herein reduce or abolish the expression and / or function of the protein encoded by the target gene by reducing or abolishing the expression of the functional protein encoded by the target gene. For example, the methods and compositions disclosed herein can reduce the expression and / or function of the protein encoded by the target gene in a cell, tissue, or subject by at least 3-fold, at least 4-fold, at least 5-fold, at least 6-fold, at least 7-fold, at least 8-fold, at least 9-fold, at least 10-fold, at least 11-fold, at least 12-fold, at least 13-fold, at least 14-fold, at least 15-fold, at least 20-fold, at least 25-fold, at least 30-fold, at least 35-fold, at least 40-fold, at least 45-fold, at least 50-fold, at least 60-fold, at least 70-fold, at least 80-fold, at least 90-fold, or at least 100-fold compared to a control cell, control tissue, or control subject.

[0240]

[0275] In some embodiments, the epigenetic editing system described herein measures the transcription of a target gene in a cell, tissue, or subject and increases the expression of the target gene by at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 95%, at least about 99%, at least 100%, at least 110%, at least 120%, at least 130%, at least 140%, at least 150%, at least 160%, at least 170%, at least 180%, at least 190%, at least 200%, at least 250%, at least 300%, at least 350%, at least 400%, at least 450%, at least 500% or more compared to a control cell, control tissue, or control subject. In some embodiments, the epigenetic editing system described herein measures the transcription of a copy of a target gene in a cell, tissue, or subject and increases the expression of the copy of the target gene by at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 95%, at least about 99%, at least 100%, at least 110%, at least 120%, at least 130%, at least 140%, at least 150%, at least 160%, at least 170%, at least 180%, at least 190%, at least 200%, at least 250%, at least 300%, at least 350%, at least 400%, at least 450%, at least 500% or more compared to a control cell, control tissue, or control subject. In some embodiments, the copy of the target gene contains a specific sequence or specific allele recognized by the epigenetic editing system. In some embodiments, the copy to be epigenetically modified encodes a functional protein. Thus, in some embodiments, the epigenetic editing system composition disclosed herein increases the expression and / or function of the protein encoded by the target gene by increasing the expression of the functional protein encoded by the target gene.For example, the methods and compositions disclosed herein can increase the expression and / or function of the protein encoded by the target gene in a cell, tissue, or subject by at least 3-fold, at least 4-fold, at least 5-fold, at least 6-fold, at least 7-fold, at least 8-fold, at least 9-fold, at least 10-fold, at least 11-fold, at least 12-fold, at least 13-fold, at least 14-fold, at least 15-fold, at least 20-fold, at least 25-fold, at least 30-fold, at least 35-fold, at least 40-fold, at least 45-fold, at least 50-fold, at least 60-fold, at least 70-fold, at least 80-fold, at least 90-fold, or at least 100-fold compared to a control cell, control tissue, or control subject.

[0241]

[0276] Methods for determining the expression level of a gene, such as a target of an epigenetic editing system, are known in the art. For example, the transcript level of a gene can be determined by reverse transcription PCR, quantitative RT-PCR, droplet digital PCR (ddPCR), Northern blot, RNA sequencing, DNA sequencing (e.g., sequencing of complementary deoxyribonucleic acid (cDNA) obtained from RNA); Next-Gen sequencing, nanopore sequencing, pyrosequencing, or nanostring sequencing. The protein level expressed from a gene can be determined by Western blotting, enzyme linked immuno-absorbance assay, mass spectrometry, immunohistochemistry, or flow cytometry analysis. The gene expression product level can be normalized to an internal standard, such as total messenger ribonucleic acid (mRNA), or the expression level of a specific gene, such as a housekeeping gene.

[0242]

[0277] In some embodiments, the effect of an epigenetic editing system in regulating target gene expression can be examined using a reporter system. For example, an epigenetic editing system can be designed to target a reporter gene that encodes a reporter protein, such as a fluorescent protein. In such a model system, the expression of the reporter gene can be monitored, for example, by flow cytometry, fluorescence-activated cell sorting (FACS), or fluorescence microscopy. In some embodiments, a population of cells can be transfected with a vector containing the reporter gene. The vector can be constructed such that the reporter gene is expressed when the cell is transfected with the vector. Suitable reporter genes include genes that encode fluorescent proteins, such as green, yellow, cherry, cyan, or orange fluorescent proteins. A population of cells having a reporter system can be transfected with DNA, mRNA, or a vector encoding an epigenetic editing system that targets the reporter gene. The level of expression of the reporter gene can be quantified using appropriate techniques, such as FACS.

[0243]

[0278] The epigenetic editing system described in this specification may be transiently expressed in a host cell or may be integrated into the genome of the host cell. Both the transiently expressed epigenetic editing system and the integrated epigenetic editing system can achieve stable epigenetic modification. For example, after introducing an epigenetic editing system containing a DNA binding domain specific to a target gene and an epigenetic repression domain into a host cell, the target gene in the host cell can be stably or permanently repressed. In some embodiments, the expression of the target gene is reduced compared to the level of expression in the absence of the epigenetic editing system for at least 1 week, at least 2 weeks, at least 3 weeks, at least 4 weeks, at least 5 weeks, at least 6 weeks, at least 7 weeks, at least 2 months, at least 3 months, at least 5 months, at least 6 months, at least 1 year, at least 2 years, or over the entire lifetime of the cell or the subject having the cell. In some embodiments, the expression of the target gene is silenced compared to the level of expression in the absence of the epigenetic editing system for at least 1 week, at least 2 weeks, at least 3 weeks, at least 4 weeks, at least 5 weeks, at least 6 weeks, at least 7 weeks, at least 2 months, at least 3 months, at least 5 months, at least 6 months, at least 1 year, at least 2 years, or over the entire lifetime of the cell or the subject having the cell. In some embodiments, after introducing an epigenetic editing system containing a DNA binding domain specific to a target gene and an epigenetic activation domain into a host cell, the target gene in the host cell is stably or permanently activated. In some embodiments, the expression of the target gene is increased compared to the level of expression in the absence of the epigenetic editing system for at least 1 week, at least 2 weeks, at least 3 weeks, at least 4 weeks, at least 5 weeks, at least 6 weeks, at least 7 weeks, at least 2 months, at least 3 months, at least 5 months, at least 6 months, at least 1 year, at least 2 years, or over the entire lifetime of the cell or the subject having the cell.

[0244]

[0279] The epigenetic modifications described herein can be inherited by the progeny of a host cell that has been contacted with or introduced to an epigenetic editing system. For example, in some embodiments, after introducing an epigenetic editing system that includes a DNA binding domain specific to a target gene and an epigenetic repression domain into a stem cell, e.g., a hematopoietic stem cell, the expression of the target gene is repressed in the cells differentiated from that stem cell as compared to the cells differentiated from control stem cells in the absence of the epigenetic editing system. In some embodiments, the expression of the target gene is silenced in the cells differentiated from the stem cell. In some embodiments, after introducing an epigenetic editing system that includes a DNA binding domain specific to a target gene and an epigenetic activation domain into a stem cell, e.g., a hematopoietic stem cell, the expression of the target gene is increased in the cells differentiated from that stem cell as compared to the cells differentiated from control stem cells in the absence of the epigenetic editing system.

[0245]

[0280] The regulation of target gene expression can be evaluated by determining any parameter that is indirectly or directly affected by the expression of the target gene. Such parameters include, for example, changes in RNA or protein levels; changes in protein activity; changes in product levels; changes in downstream gene expression; changes in the transcription or activity of reporter genes such as luciferase, CAT, beta-galactosidase or GFP; changes in signal transduction; changes in phosphorylation and dephosphorylation; changes in receptor-ligand interactions; changes in the concentration of second messengers such as cGMP, cAMP, IP3 and Ca2+; changes in cell proliferation, angiogenesis, and / or any functional effect of gene expression. The measurement can be performed in vitro, in vivo, and / or ex vivo. Such functional effects can be measured by conventional methods such as measurement of RNA or protein levels, measurement of RNA stability, and / or identification of downstream or reporter gene expression. Readouts can be, for example, chemiluminescence, fluorescence, colorimetric reactions, antibody binding, inducible markers, ligand binding assays; changes in intracellular second messengers such as cGMP and inositol 3 phosphate (IP3); changes in intracellular calcium levels; cytokine release, etc.

[0246]

[0281] To determine the level of gene expression regulation by ZFP, the degree of inhibition or activation is examined in cells contacted with ZFP compared to, for example, control cells without a zinc finger protein or with a non-specific ZFP. A relative gene expression activity value of 100% is assigned to the control sample. Regulation / inhibition of gene expression is achieved when the gene activity value relative to the control is about 80%, preferably 50% (i.e., 0.5× the activity of the control), more preferably 25%, more preferably 5 - 0%. Regulation / activation of gene expression is achieved when the gene activity value relative to the control is 110%, more preferably 150% (i.e., 1.5× the activity of the control), more preferably 200 - 500%, more preferably 1000 - 2000% or more.

[0247]

[0282] In some embodiments, modification or regulation of the epigenetic state of the target gene may occur at one or more off-target sites. In some embodiments, the percentage of modification or regulation occurring at the one or more off-target sites is lower than about 20%, lower than about 19%, lower than about 18%, lower than about 17%, lower than about 16%, lower than about 15%, lower than about 14%, lower than about 13%, lower than about 12%, lower than about 11%, lower than about 10%, lower than about 9%, lower than about 8%, lower than about 7%, lower than about 6%, lower than about 5%, lower than about 4%, lower than about 3%, lower than about 2%, lower than about 1%, lower than about 0.9%, lower than about 0.8%, lower than about 0.7%, lower than about 0.6%, lower than about 0.5%, lower than about 0.4%, lower than about 0.3%, lower than about 0.2%, lower than about 0.1%, lower than about 0.09%, lower than about 0.08%, lower than about 0.07%, lower than about 0.06%, lower than about 0.05%, lower than about 0.04%, lower than about 0.03%, lower than about 0.02%, or lower than about 0.01%.

[0248]

[0283] In some embodiments, modification or regulation of the epigenetic state of the target gene may be stable after one round of active cell replication. In some embodiments, the modification or regulation may be stable after at least about 1, at least about 2, at least about 3, at least about 4, at least about 5, at least about 6, at least about 7, at least about 8, at least about 9, at least about 10, at least about 20, at least about 30, at least about 40, at least about 50 or more rounds of active cell replication.

[0249]

[0284] In some embodiments, modification or regulation of the epigenetic state of a target gene can be stable at least about 1 day, at least about 2 days, at least about 3 days, at least about 4 days, at least about 5 days, at least about 6 days, at least about 1 week, at least about 2 weeks, at least about 3 weeks, at least about 1 month, at least about 2 months, at least about 3 months, at least about 4 months, at least about 5 months, at least about 6 months, at least about 7 months, at least about 8 months, at least about 9 months, at least about 10 months, at least about 11 months, at least about 1 year, at least about 2 years, at least about 3 years, at least about 4 years, at least about 5 years or more later.

[0250] Delivery

[0285] In one aspect, provided herein is a composition for regulating gene expression comprising an epigenetic editing system provided herein that generates an epigenetic modification in a target gene. The epigenetic editing system, or a nucleic acid encoding the epigenetic editing system or a component thereof (e.g., a nucleic acid encoding an epigenetic editing system fusion protein comprising a zinc finger-repressor fusion, a Cas9-repressor fusion, and / or a nucleic acid encoding one or more guide RNAs) can be introduced into cells by a variety of methods known in the art. For example, in some embodiments, the epigenetic editing system is for delivery into a host cell, integration into the genome of a host cell, or transient expression within a host cell.

[0251]

[0286] In some embodiments, the nucleic acid encoding the epigenetic editing system or a component thereof is operably linked to a promoter and / or regulatory sequence. As used herein, the term "operably linked" means that the nucleotide sequence of interest is linked to the regulatory sequence in a manner that enables expression of that nucleotide sequence. As used herein, the term "regulatory sequence" includes, but is not limited to, promoters, enhancers, and other expression control elements. Such control sequences are well known in the art and are described, for example, in Goeddel; Gene Expression Technology: Methods in Enzymology 185, Academic Press, San Diego, CA (1990).

[0252]

[0287] In some embodiments, the composition further comprises a vector comprising a nucleic acid sequence encoding an epigenetic editing system protein. In some embodiments, the vector can be an expression vector. In some embodiments, the vector is a plasmid or viral vector. As used herein, the term "vector" refers to a nucleic acid molecule capable of transporting another nucleic acid to which it is linked. In some examples, the vector is an expression vector and can direct the expression of a nucleic acid operably linked to itself. Examples of expression vectors include, but are not limited to, plasmid vectors, viral vectors based on vaccinia virus, poliovirus, adenovirus, adeno-associated virus, SV40, herpes simplex virus, human immunodeficiency virus, retroviruses (e.g., murine leukemia virus, spleen necrosis virus, and retrovirus-derived vectors such as Rous sarcoma virus, Harvey sarcoma virus, avian leukosis virus, lentivirus, human immunodeficiency virus, myeloproliferative sarcoma virus, and mammary tumor virus), and other recombinant vectors. In some embodiments, the vector is a virus-like particle (VLP).

[0253]

[0288] Non-viral delivery systems include, but are not limited to, DNA delivery methods and RNA delivery methods, such as transfection. As used herein, transfection includes the process of using a non-viral vector to deliver a gene, DNA fragment, gene transcript, RNA, RNA fragment, circularized DNA, or circularized RNA into a target cell. Exemplary transfection methods include, but are not limited to, electroporation, DNA gene gun, lipid-mediated transfection, condensed DNA-mediated transfection, liposomes, immunoliposomes, exosomes, lipofection, cationic agent-mediated transfection, or cationic facial amphiphile (CFA).

[0254]

[0289] In some embodiments, the epigenetic editing system is delivered into a host cell for transient expression, e.g., via a transient expression vector. Transient expression of the epigenetic editing system can result in long-term or permanent epigenetic modification of the target gene. For example, the epigenetic modification can be stable for at least 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 7 weeks, 8 weeks, 9 weeks, 11 weeks, 12 weeks, 3 months, 4 months, 5 months, 6 months, 7 months, 8 months, 9 months, 10 months, 11 months, 12 months or longer after introduction of the epigenetic editing system into the host cell. The epigenetic modification can be maintained after one or more mitotic events of the host cell. The epigenetic modification can be maintained after one or more meiotic events of the host cell. In some embodiments, the epigenetic modification is maintained across generations of progeny produced from or derived from the host cell.

[0255]

[0290] In some embodiments, the nucleic acid sequence encoding the epigenetic editing system or its components is DNA, RNA or mRNA, or a modified nucleic acid sequence. For example, the mRNA sequence encoding the epigenetic editing system fusion protein can be chemically modified or can include a 5' cap, or one or more 3' modifications.

[0256]

[0291] The nucleic acid encoding the epigenetic editing system can be delivered directly to cells as naked DNA or RNA, for example, by transfection or electroporation, or can be conjugated to a molecule (e.g., N-acetylgalactosamine) that facilitates uptake by target cells. Nucleic acid vectors, such as vectors, can also be used. In certain embodiments, a polynucleotide encoding the epigenetic editing system or its functional components, such as mRNA, can be co-electroporated with a combination of multiple guide RNAs described herein.

[0257]

[0292] The nucleic acid vector can include one or more sequences encoding a fusion protein or a domain of the epigenetic editing system as described herein. The vector can also include a sequence encoding a signal peptide (e.g., nuclear localization, nucleolar localization, or mitochondrial localization) associated with (e.g., inserted or fused to) the protein-encoding sequence. As an example, the nucleic acid vector can include one or more nuclear localization sequences (e.g., nuclear localization sequences from SV40), and one or more effector domains, e.g., a Cas9 coding sequence including a repression domain.

[0258]

[0293] In certain embodiments, all or part of the fusion protein, protein domain, or epigenetic editing system component is encoded by a polynucleotide present in a viral vector (e.g., adeno-associated virus (AAV), AAV3, AAV3b, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAVrh8, AAV10, and variants thereof), or is the appropriate capsid protein of any viral vector. Thus, in some aspects, the present disclosure relates to viral delivery of the fusion protein. Examples of viral vectors include retroviral vectors (e.g., Moloney murine leukemia virus, MML-V), adenoviral vectors (e.g., AD100), lentiviral vectors (HIV and FIV-based vectors), and herpes viral vectors (e.g., HSV-2).

[0259]

[0294] In some embodiments, the epigenetic editing system protein is encoded by a polynucleotide present in an adeno-associated virus (AAV) vector. In some embodiments, the epigenetic editing system protein includes a zinc finger array in the DNA binding domain. Without wishing to be bound by any theory, an epigenetic editing system that uses a zinc finger array instead of a larger DNA binding domain, such as a Cas protein domain, may be conveniently packaged into a viral vector, such as an AAV vector, considering the small size of the zinc fingers. In some embodiments, the polynucleotide encoding the epigenetic editing system is about 1000 bp, 1.1 kilobases (kb), 1.2 kb, 1.3 kb, 1.4 kb, 1.5 kb, 1.6 kb, 1.7 kb, 1.8 kb, 1.9 kb, 2.0 kb, 2.1 kb, 2.2 kb, 2.3 kb, 2.4 kb, 2.5 kb, 2.6 kb, 2.7 kb, 2.8 kb, 2.9 kb, 3.0 kb, 3.1 kb, 3.2 kb, 3.3 kb, 3.4 kb, 3.5 kb, 3.6 kb, 3.7 kb, 3.8 kb, 3.9 kb, 4.0 kb or less in length. In some embodiments, the polynucleotide encoding the epigenetic editing system is about 2.0 kb, 2.1 kb, 2.2 kb, 2.3 kb, 2.4 kb, 2.5 kb, 2.6 kb, 2.7 kb, 2.8 kb, 2.9 kb, 3.0 kb, 3.1 kb, 3.2 kb, 3.3 kb, 3.4 kb, 3.5 kb, 3.6 kb, 3.7 kb, 3.8 kb, 3.9 kb, 4.0 kb, 4.1 kb, 4.2 kb, 4.3 kb, 4.4 kb, 4.5 kb, 4.6 kb, 4.7 kb, 4,8 kb, 4.9 kb, 5 kb or less in length.

[0260]

[0295] Any AAV serotype, such as human AAV serotypes, can be used, including but not limited to AAV serotype 1 (AAV1), AAV serotype 2 (AAV2), AAV serotype 3 (AAV3), AAV serotype 4 (AAV4), AAV serotype 5 (AAV5), AAV serotype 6 (AAV6), AAV serotype 7 (AAV7), AAV serotype 8 (AAV8), AAV serotype 9 (AAV9), AAV serotype 10 (AAV10), AAV serotype 11 (AAV11), their variants, or their shuffled variants (e.g., their chimeric variants). In some embodiments, the AAV variant has at least 90%, such as 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more amino acid sequence identity to wild-type AAV. The AAV1 variant can have at least 90%, such as 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more amino acid sequence identity to wild-type AAV1. The AAV2 variant can have at least 90%, such as 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more amino acid sequence identity to wild-type AAV2. The AAV3 variant can have at least 90%, such as 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more amino acid sequence identity to wild-type AAV3. The AAV4 variant can have at least 90%, such as 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more amino acid sequence identity to wild-type AAV4. The AAV5 variant can have at least 90%, such as 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more amino acid sequence identity to wild-type AAV5. The AAV6 variant can have at least 90%, such as 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more amino acid sequence identity to wild-type AAV6.The AAV7 variant can have at least 90%, for example, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more amino acid sequence identity to wild-type AAV7. The AAV8 variant can have at least 90%, for example, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more amino acid sequence identity to wild-type AAV8. The AAV9 variant can have at least 90%, for example, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more amino acid sequence identity to wild-type AAV9. The AAV10 variant can have at least 90%, for example, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more amino acid sequence identity to wild-type AAV10. The AAV11 variant can have at least 90%, for example, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more amino acid sequence identity to wild-type AAV11. The AAV12 variant can have at least 90%, for example, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more amino acid sequence identity to wild-type AAV12.

[0261]

[0296] In some cases, to generate an AAV chimeric virus, one or more regions of at least two different AAV serotype viruses are shuffled and reassembled. For example, the chimeric AAV can contain inverted terminal repeats (ITRs) that are of a heterologous serotype compared to the serotype of the capsid. The resulting chimeric AAV virus can have different antigen reactivity or antigen recognition compared to its parental serotype. In some embodiments, the chimeric variant of AAV contains amino acid sequences from 2, 3, 4, 5 or more different AAV serotypes.

[0262]

[0297] Descriptions of AAV variants and methods for their generation can be found, for example, in Weitzman and Linden, Chapter 1 - Adeno - Associated Virus Biology in Adeno - Associated Virus: Methods and Protocols, Methods in Molecular Biology, Volume 807, edited by Snyder and Moullier, Springer, 2011; Potter et al., Molecular Therapy - Methods & Clinical Development, 2014, 1, 14034; Bartel et al., Gene Therapy, 2012, 19, pages 694 - 700; Ward and Walsh, Virology, 2009, 386(2):237 - 248; and Li et al., Mol Ther, 2008, 16(7):1252 - 1260, each of which is incorporated herein by reference in its entirety. The AAV virions (e.g., viral vectors or viral particles) described herein can be transduced into cells to introduce an epigenetic editing system or any of its components into the cells. The epigenetic editing system can be packaged into an AAV viral vector according to any method known to those skilled in the art. An example of a useful method is described in McClure et al., J Vis Exp, 2001, 57:3378.

[0263]

[0298] The nucleic acid vectors described herein can also include any suitable number of regulatory / control elements, such as promoters, enhancers, introns, polyadenylation signals, Kozak consensus sequences, or internal ribosome entry sites (IRES). These elements are well - known in the art.

[0264]

[0299] The nucleic acid vectors according to the present disclosure include recombinant viral vectors. Exemplary viral vectors are described hereinabove. Other viral vectors known in the art can also be used. Further, viral particles can be used to deliver genome editing system components in the form of nucleic acids and / or peptides. For example, "empty" viral particles can be assembled to contain any suitable cargo. Viral vectors and viral particles can also be engineered to incorporate targeting ligands to alter target tissue specificity.

[0265]

[0300] In addition to viral vectors, non-viral vectors can be used to deliver nucleic acids encoding the genome editing systems according to the present disclosure. One category of non-viral nucleic acid vectors are nanoparticles, which can be organic or inorganic. Nanoparticles are well-known in the art. Any suitable nanoparticle design can be used to deliver genome editing system components, or nucleic acids encoding such components. For example, organic (e.g., lipid and / or polymer) nanoparticles may be suitable for use as delivery vehicles in certain embodiments of the present disclosure.

[0266]

[0301] In other aspects, lipid nanoparticles (LNPs) comprising the compositions provided herein are provided herein. As used herein, "lipid nanoparticle (LNP) composition" or "nanoparticle composition" refers to a composition comprising one or more of the described lipids. LNP compositions are typically on the order of micrometers or less in size and may include a lipid bilayer. Nanoparticle compositions include lipid nanoparticles (LNPs), liposomes (e.g., lipid vesicles), and lipoplexes. In some embodiments, LNP refers to any particle having a diameter of less than 1000 nm, 500 nm, 250 nm, 200 nm, 150 nm, 100 nm, 75 nm, 50 nm, or 25 nm. In some embodiments, the size of the nanoparticles can range from 1 to 1000 nm, 1 to 500 nm, 1 to 250 nm, 25 to 200 nm, 25 to 100 nm, 35 to 75 nm, or 25 to 60 nm.

[0267]

[0302] In some embodiments, the LNP can be made from cationic lipids, anionic lipids, or neutral lipids. In some embodiments, the LNP can include a neutral lipid, such as the fusogenic phospholipid 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE) or the membrane component cholesterol, as a helper lipid to improve transfection activity and nanoparticle stability. In some embodiments, the LNP can include hydrophobic lipids, hydrophilic lipids, or both hydrophobic and hydrophilic lipids. Any lipid or combination of lipids known in the art can be used to produce the LNP. Examples of lipids used to produce the LNP include, but are not limited to, DOTMA (N[1-(2,3-dioleyloxy)propyl]-N,N,N-trimethylammonium chloride), DOSPA (N,N-dimethyl-N-([2-sperminecarboxamido]ethyl)-2,3-bis(dioleyloxy)-1-propaniminium pentachloride), DOTAP (1,2-dioleoyl-3-trimethylammonium propane), DMRIE (N-(2-hydroxyethyl)-N,N-dimethyl-2,3-bis(tetradecyloxy)-1-propanaminium bromide), DC-cholesterol (3β-[N-(N’,N’-dimethylaminoethane)-carbamoyl]cholesterol), DOTAP-cholesterol, GAP-DMORIE-DPyPE, and GL67A-DOPE-DMPE(,2-bis(dimethylphosphino)ethane)-polyethylene glycol (PEG). Examples of cationic lipids include, but are not limited to, 98N12-5, C12-200, DLin-KC2-DMA (KC2), DLin-MC3-DMA (MC3), XTC, MD1, and 7C1. Examples of neutral lipids include, but are not limited to, DPSC, DPPC (dipalmitoylphosphatidylcholine), POPC (1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine), DOPE, and SM (sphingomyelin). Examples of PEGylated lipids include, but are not limited to, PEG-DMG (dimyristoyl glycerol), PEG-CerC14, and PEG-CerC20.In some embodiments, lipids can be combined in any number of molar ratios to produce LNPs. In some embodiments, polynucleotides can be combined with lipids in a wide range of molar ratios to produce LNPs.

[0268] Method of treatment

[0303] Further provided herein is a method of treating or preventing a condition in a subject in need thereof, the method comprising administering to the subject an epigenetic editing system composition described herein, provided that the epigenetic editing system complex or protein achieves epigenetic modification of a target polynucleotide in a target gene associated with a disease, condition or disorder in the subject and regulates the expression of the target, resulting in treatment or prevention of the disease, condition or disorder.

[0269]

[0304] The epigenetic modifications achieved by the epigenetic editing systems described herein are sequence-specific. In some embodiments, the modification is at a specific site of the target polynucleotide. In some embodiments, the modification is in a specific allele of the target gene. Thus, the epigenetic modification can result in the regulation of the expression of one copy of the target gene containing the specific allele, e.g., a decrease or increase in expression, while the other copy of the target gene does not. In some embodiments, the specific allele is associated with a disease, condition or disorder.

[0270]

[0305] In some embodiments, the epigenetic editing system decreases the expression of a target gene associated with a disease, condition or disorder. Alternatively, or additionally, the target gene may not have a direct association with the disease, but instead can complement a disease-related gene. In some embodiments, the target gene may be a transcription factor or other gene used to manipulate cells.

[0271]

[0306] The epigenetic editing systems described herein can be administered in a therapeutically effective amount to a subject in need thereof for treating a disease, condition, or disorder. Alternatively, or in addition, ex vivo manipulated cells conditioned with the epigenetic editing systems described herein can be administered in a therapeutically effective amount to a subject in need thereof for treating a disease, condition, or disorder.

[0272]

[0307] In another aspect, provided herein is a method of treating or preventing a condition in a subject in need thereof, the method comprising administering to the subject an epigenetic editing complex, vector, nucleic acid, protein, engineered cell, or composition provided herein, wherein the nucleic acid binding domain of the epigenetic editing system directs an effector domain to create an epigenetic modification in a target polynucleotide sequence in the cells of the subject, thereby regulating the expression of a target gene and treating or preventing the condition.

[0273]

[0308] In some embodiments, the modification reduces the expression of a functional protein encoded by the target gene in the subject.

[0309] A patient being treated for a condition, disease, or disorder is one diagnosed by a physician as having such a condition. The diagnosis is obtained by any suitable means. Diagnosis and monitoring can include, for example, detecting the presence of diseased cells, dying cells, or dead cells in a biological sample (e.g., a tissue biopsy, blood test, or urine test), detecting the presence of plaques, detecting the levels of alternative markers in a biological sample, or detecting symptoms associated with the condition. A patient being prophylactically treated for the manifestation of a condition may or may not have such a diagnosis. One of ordinary skill in the art will understand that those patients may have undergone the same standard tests as described above or may have been identified as being at high risk without testing due to the presence of one or more risk factors (e.g., family history or genetic predisposition).

[0274]

[0310] For the purpose of curing, healing, alleviating, reducing, changing, correcting, ameliorating, improving, or affecting a disease, disease symptom, or disease tendency, a subject may have a disease, disease symptom, or disease tendency. In some embodiments, the subject has hypercholesterolemia. In some embodiments, the subject has atherosclerotic vascular disease. In some embodiments, the subject has hypertriglyceridemia. In some embodiments, the subject has diabetes. In some embodiments, the subject is a mammal. In some embodiments, the subject is a non-human primate. In some embodiments, the subject is a human. Alleviation of a disease includes delaying the onset or progression of the disease or reducing the severity of the disease. Alleviation of a disease does not necessarily require a curative result.

[0275]

[0311] As used herein, "delay" of disease onset means to extend, impede, decelerate, retard, stabilize, and / or postpone the progression of the disease. This delay can vary in duration depending on the medical history and / or the individual being treated. A method for "delaying" or alleviating the onset of a disease, or delaying the development of a disease, is a method that reduces the likelihood that one or more symptoms of the disease will appear within any time frame and / or reduces the degree of symptoms within any time frame, compared to not using the method. Such comparisons are typically based on clinical trials that handle a sufficient number of subjects to yield statistically significant results.

[0276]

[0312] "Onset" or "progression" of a disease means the initial symptoms of the disease and / or subsequent progression. The onset of a disease may be detectable and evaluable using standard clinical techniques well known in the art. However, onset also refers to progression that may not be detectable. For the purposes of the present disclosure, onset or progression refers to the biological progression of symptoms. "Onset" includes occurrence, recurrence, and development.

[0277]

[0313] As used herein, "onset" or "occurrence" of a disease includes initial onset and / or recurrence. Conventional methods known to those of ordinary skill in the art can be used to administer the isolated polypeptide or pharmaceutical composition to a subject, depending on the type of disease or the site of the disease to be treated. The composition can also be administered via other conventional routes, for example, orally, parenterally, by inhalation spray, topically, rectally, nasally, buccally, vaginally, or via an implanted reservoir.

[0278]

[0314] The methods of treatment of the present disclosure can be performed on a subject showing a pathology resulting from a disease or condition, a subject suspected of showing a pathology resulting from a disease or condition, and a subject at risk of showing a pathology resulting from a disease or condition. For example, a subject having a genetic predisposition to a disease or condition can be prophylactically treated. A subject showing symptoms associated with a condition, disease, or disorder can be treated to reduce the symptoms or to slow or prevent further progression of the symptoms. Physical changes associated with an increase in the severity of a disease or condition are shown herein to be progressive. Thus, in embodiments of the present disclosure, a subject showing mild signs of a pathology associated with a condition or disease can be treated to improve the symptoms and / or to prevent further progression of the symptoms.

[0279]

[0315] The dosage and frequency (single dose or multiple doses) administered to a mammal can vary depending on a variety of factors, such as whether the mammal is suffering from other diseases and the route of administration; the size, age, sex, health, weight, body mass index, and diet of the recipient; the nature and extent of the symptoms of the disease being treated, the type of co-treatment, complications from the disease being treated, or other health-related problems. Adjustment and manipulation of established dosages (e.g., frequency and duration) are well within the ability of those of ordinary skill in the art. Treatments as disclosed herein can be administered to a subject daily, twice a day, bi-weekly, monthly, or on any applicable basis, and are therapeutically effective. In embodiments, the treatment is only as needed, for example, in response to the appearance of signs or symptoms of a condition or disease.

[0280]

[0316] The toxicity and therapeutic efficacy of the compositions of the present disclosure can be determined by standard pharmaceutical procedures for the determination of, for example, LD50 (the dose lethal to 50% of a population) and ED50 (the therapeutically effective dose in 50% of a population) in cell culture or animal models. The dose ratio between the toxic effect and the therapeutic effect (the ratio LD50 / ED50) is the therapeutic index. Agents that exhibit a high therapeutic index are preferred. The dose of the agent is preferably within the range of circulating concentrations that include the ED50 with little or no toxicity. Although agents that exhibit toxic side effects can be used, care should be taken to design a delivery system that targets such agents to the site of the diseased tissue in order to minimize the potential for damage to non-infected cells and thereby reduce side effects.

[0281]

[0317] One of ordinary skill in the art will understand that certain factors, including the degree of the disease or disorder, previous treatments, the health, sex, weight and / or age of the subject, as well as other current diseases, among others, can affect the dosage and frequency of administration required to effectively treat the subject. Further, treatment of a subject with a therapeutically effective amount of the composition may include a single treatment or, preferably, a series of treatments. It is also recognized that the effective dosage of the compositions of the present disclosure used in the treatment may increase or decrease during the course of a particular treatment. Changes in dosage may result from and be apparent from the results of the diagnostic assays described herein. The therapeutically effective dosage will generally be a function of the condition of the patient at the time of administration. The exact amount can be determined by routine experimentation but will ultimately be within the discretion of the clinician, for example, by monitoring the patient for signs of disease and adjusting the treatment accordingly.

[0282]

[0318] The frequency of administration can be determined and adjusted during the course of treatment and, although not necessarily so, will generally be based on the treatment and / or suppression and / or amelioration and / or retardation of the disease. Alternatively, a sustained continuous release formulation of the polypeptide or polynucleotide may be appropriate. A variety of formulations and devices for achieving sustained release are known in the art. In some embodiments, the dosage is daily, every other day, every three days, every four days, every five days, or every six days. In some embodiments, the dosing frequency is once a week, once every two weeks, once every four weeks, once every five weeks, once every six weeks, once every seven weeks, once every eight weeks, once every nine weeks, or once every ten weeks, or once a month, once every two months, or once every three months, or more than once. The progress of this treatment is readily monitored by conventional techniques and assays.

[0283]

[0319] The dosing schedule (including the compositions disclosed herein) can vary over time. In some embodiments, for a standard weight adult subject, dosages ranging from about 0.01 to 1000 mg / kg can be administered. In some embodiments, the dosage is between 1 and 200 mg. The specific dosing schedule, i.e., dosage, timing and repetition, will depend on the particular subject, and the subject's medical history, as well as the characteristics of the polypeptide or polynucleotide (e.g., the half-life of the polypeptide or polynucleotide, and other considerations well known in the art).

[0284]

[0320] For the purposes of the present disclosure, the appropriate therapeutically effective dosage of the compositions described herein will depend on the particular agent (or composition thereof) utilized, the formulation and route of administration, the type and severity of the disease, whether the polypeptide or polynucleotide is administered for prophylactic or therapeutic purposes, the medical history, the clinical history of the patient and response to the antagonist, as well as the discretion of the attending physician. Typically, the clinician will administer the polypeptide until a dosage is reached that achieves the desired result.

[0285]

[0321] Administration of one or more compositions can be continuous or intermittent, depending, for example, on the physiological state of the recipient, whether the purpose of administration is therapeutic or prophylactic, and other factors known to the skilled practitioner. Administration of the composition can be, for example, either continuously over a preselected period of time, or as a series of spaced doses, either before, during, or after the onset of the disease.

[0286]

[0322] The methods and compositions of the disclosure described herein, including embodiments of the methods and compositions, can be administered with one or more additional therapeutic regimens or agents or treatments that can be co-administered to a mammal. "Co-administration" means administering one or more additional therapeutic regimens or agents or treatments and the compositions of the disclosure at a time sufficiently close to enhance the effect of one or more of the additional therapeutic agents (and vice versa). In this regard, the compositions of the disclosure described herein can be administered simultaneously with, at different times from, or on a completely different treatment schedule from one or more additional therapeutic regimens or agents or treatments (e.g., the first treatment can be daily, while the additional treatment is weekly). For example, in embodiments, a second therapeutic regimen or agent or treatment is administered simultaneously with, prior to, or subsequent to the compositions of the disclosure.

[0287] Pharmaceutical Compositions, Dosage Forms, and Administration

[0323] In one aspect, provided herein is a pharmaceutical composition for epigenetic modification comprising an epigenetic editing system described herein, or one or more nucleic acid sequences encoding components of an epigenetic editing system, such as a nucleic acid encoding an epigenetic editing system fusion protein and / or a guide RNA, and a pharmaceutically acceptable carrier. The compositions for epigenetic modification described herein can be formulated into pharmaceutical compositions. Pharmaceutical compositions are formulated in a conventional manner using one or more pharmaceutically acceptable inactive ingredients that facilitate the processing of the active compounds into preparations that are pharmaceutically useful. Suitable formulations and methods of delivery for use in the present disclosure are generally well known in the art. The appropriate formulation depends on the route of administration chosen. An overview of suitable pharmaceutical compositions described herein can be found, for example, in Remington: The Science and Practice of Pharmacy, 19th Edition (Easton, Pa.: Mack Publishing Company, 1995); Hoover, John E., Remington’s Pharmaceutical Sciences, Mack Publishing Co., Easton, Pennsylvania 1975; Liberman, H.A. and Lachman, L., eds., Pharmaceutical Dosage Forms, Marcel Decker, New York, N.Y., 1980; and Pharmaceutical Dosage Forms and Drug Delivery Systems, 7th Edition (Lippincott Williams & Wilkins 1999), and such disclosures are incorporated herein by reference for such related disclosures.

[0288]

[0324] The pharmaceutical composition can be a mixture of an epigenetic editing system described herein or a nucleic acid encoding the same, and one or more other chemical components (i.e., pharmaceutically acceptable components), such as carriers, excipients, binders, fillers, suspending agents, flavoring agents, sweetening agents, disintegrants, dispersants, surfactants, lubricants, coloring agents, diluents, solubilizers, moistening agents, plasticizers, stabilizers, penetration enhancers, wetting agents, defoaming agents, antioxidants, preservatives, or a combination of one or more thereof. The pharmaceutical composition facilitates the administration of an epigenetic editor, such as a nucleic acid encoding a zinc finger-epigenetic effector fusion protein or a Cas9-epigenetic effector fusion protein and a gRNA or sgRNA as described herein, to an organism or subject in need of administration of the epigenetic editor.

[0289]

[0325] The pharmaceutical compositions of the present disclosure can be administered to a subject using any suitable method known in the art. The pharmaceutical compositions described herein can be administered to a subject in a variety of ways, including parenterally, intravenously, intradermally, intramuscularly, colonic, rectal, or intraperitoneally. In some embodiments, the pharmaceutical composition can be administered by intraperitoneal injection, intramuscular injection, subcutaneous injection, or intravenous injection of the subject. In some embodiments, the pharmaceutical composition can be administered parenterally, intravenously, intramuscularly, or orally.

[0290]

[0326] For administration by inhalation, the adenovirus or LNP described herein can be formulated for use as an aerosol, mist or powder. For buccal or sublingual administration, the pharmaceutical composition can be formulated in the form of tablets, troches or gels formulated in a conventional manner. In some embodiments, the adenovirus or LNP described herein can be prepared as a transdermal dosage form. In some embodiments, the adenovirus or LNP described herein can be formulated into a pharmaceutical composition suitable for intramuscular, subcutaneous or intravenous injection. In some embodiments, the adenovirus or LNP described herein can be topically administered and formulated into various topically administrable compositions, such as solutions, suspensions, lotions, gels, pastes, medicated sticks, balms, creams or ointments. In some embodiments, the adenovirus or LNP described herein can be formulated into an enema composition, such as an enema, enema gel, enema form, enema aerosol, suppository, jelly suppository or retention enema. In some embodiments, the adenovirus or LNP described herein can be formulated for oral administration, such as tablets, capsules, or, without limitation, into a liquid in the form of an aqueous suspension or solution selected from the group comprising aqueous oral dispersions, emulsions, solutions, elixirs, gels and syrups.

[0291]

[0327] In some embodiments, the epigenetic editor described herein, or an epigenetic modification pharmaceutical composition comprising a nucleic acid sequence encoding the same, further comprises a therapeutic agent. The additional therapeutic agent can modulate different aspects of the disease, disorder or condition being treated and can provide a greater overall benefit than administration of either the recombinant adenovirus having replication ability or the therapeutic agent alone. Therapeutic agents include, but are not limited to, chemotherapeutic agents, radiation therapy agents, hormone therapy agents, and / or immunotherapy agents. In some embodiments, the therapeutic agent can be a radiation therapy agent. In some embodiments, the therapeutic agent can be a hormone therapy agent. In some embodiments, the therapeutic agent can be an immunotherapy agent. In some embodiments, the therapeutic agent is a chemotherapeutic agent. The preparation and administration schedule of the additional therapeutic agent can be used according to the manufacturer's instructions or as empirically determined by a skilled practitioner. For example, the preparation and administration schedule for chemotherapy is also described in The Chemotherapy Source Book, 4th Edition, 2008, M. C. Perry, editor, Lippincott, Williams & Wilkins, Philadelphia, PA.

[0292]

[0328] A subject that can be treated with the epigenetic modification composition can be any subject having a disease or condition. For example, the subject can be a eukaryotic subject, such as an animal. In some embodiments, the subject is a mammal, such as a human. In some embodiments, the subject is a human. In some embodiments, the subject is a non-human animal. In some embodiments, the subject is a fetus, embryo or child. In some embodiments, the subject is a non-human primate, such as a chimpanzee, as well as other ape and monkey species; livestock, such as cows, horses, sheep, goats, pigs; companion animals, such as rabbits, dogs and cats; laboratory animals, such as rodents, such as rats, mice and guinea pigs.

[0293]

[0329] In some embodiments, the subject is prenatal (e.g., a fetus), a child (e.g., a neonate, infant, toddler, preadolescent), an adolescent, a teenager, an adult (e.g., a young adult, middle-aged adult, elderly). The human subject can be between about 0 months to about 120 years or older after birth. The human subject can be between about 0 months to about 12 months after birth; e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 months after birth. The human subject can be between about 0 years to 12 years; e.g., between about 0 days to 30 days after birth; between about 1 month to 12 months after birth; between about 1 year to 3 years; between about 4 years to 5 years; between about 4 years to 12 years; about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 years old. The human subject can be between about 13 years to about 19 years; e.g., about 13, 14, 15, 16, 17, 18, or 19 years old. The human subject can be between about 20 years to about 39 years; e.g., about 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, or 39 years old. The human subject can be between about 40 years to about 59 years; e.g., about 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, or 59 years old. The human subject can be over 59 years old; e.g., about 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, or 120 years old. The human subject can include male subjects and / or female subjects.

[0294]

[0330] In certain embodiments, kits and products for use with one or more of the methods described herein are also disclosed herein. Such kits include a carrier, package or container that is compartmentalized to receive one or more containers, such as vials, tubes, etc., each of the one or more containers containing one of the distinct elements for use in the methods described herein. Suitable containers include, for example, bottles, vials, syringes and test tubes. In one embodiment, the containers are formed from a variety of materials, such as glass or plastic.

[0295]

[0331] The products provided herein contain a packaging material. Examples of pharmaceutical packaging materials include, but are not limited to, blister packs, bottles, tubes, bags, containers, and any packaging material suitable for the selected formulation and the intended mode of administration and treatment.

[0296]

[0332] For example, the container contains the compositions of the present disclosure, and optionally further contains a treatment plan or therapeutic agent disclosed herein. Such kits optionally include a descriptive label or tag for identification, or instructions related to its use in the methods described herein.

[0297]

[0333] Kits typically include a label and / or instructions listing the contents, and an accompanying document with instructions. A series of instructions is also typically included.

[0334] In embodiments, the label is on the container or associated with the container. In one embodiment, the label is on the container when the letters, numbers or other symbols forming the label are attached, molded or etched within the container itself; the label is associated with the container when it is present within a receptacle or carrier that further holds the container, for example, as an accompanying document. In one embodiment, the label is used to indicate that the contents are to be used for a particular therapeutic application. The label also indicates, for example, the method of use of the contents in the methods described herein.

Examples

[0298]

[0335] The following examples are included for illustrative purposes only and are not intended to limit the scope of the present disclosure.

[0336] Example 1: Development of improved triple ETR combinations

[0337] Several improved triple-engineered transcriptional repressors (ETRs) were developed to have significantly high epigenetic silencing activity.

[0299]

[0338] To do this, the relative efficiency of two epigenetic platforms, the triple ETR combinations of Amabile et al., 2016 and WO2016063264, and the CRISPRoff combinations of Nunez et al., 2020 and WO2019204766, were evaluated. Both platforms are based on the following epigenetic effector domains (EDs): KRAB from the human ZNF10 protein, the catalytic domain of human DNMT3A, and human full-length (in the case of the triple ETR combination) or truncated mouse (in the case of CRISPRoff) DNMT3L. In particular, the triple ETR combination consists of three independent constructs in which the above epigenetic effector domains are linked to the C-terminus of catalytically inactivated Cas9 (dCas9) via a short 5 amino acid (aa) spacer sequence (i.e., GSGGG; hereafter referred to as G). Instead, CRISPRoff is an all-in-one fusion protein in which three effector domains are operably linked to a single dCas9 DNA binding domain (DBD). In this fusion, the effector domains were arranged as follows: a previously described heterodimer between the catalytic domain of DNMT3A and mouse Dnmt3L was placed at the N-terminus of dCas9.

[0300]

[0339] The two parts of the DNMT3A-Dnmt3L heterodimer were linked by a 13-amino acid sequence (hereinafter referred to as W), while the heterodimer was linked to dCas9 by an 80-amino acid XTEN sequence (hereinafter referred to as X80). A 16-amino acid XTEN sequence (hereinafter referred to as X16) linked the KRAB domain to the C-terminus of dCas9. Figure 2 shows a schematic diagram of the constructs that make up the two epigenetic silencing platforms.

[0301]

[0340] To properly compare the two epigenetic silencing platforms, CRISPRoff was cloned into the same mammalian expression vector (containing the woodchuck hepatitis virus post-transcriptional response element, WPRE, in the 3’UTR and bovine growth hormone polyA signal sequence) used for ETR, which was already found to improve efficiency two-fold compared to the original CRISPRoff platform (Figures 3A-3B).

[0302]

[0341] In the following cell lines: one with the tdTomato transgene under the transcriptional control of the human B2M gene (hereinafter referred to as K-562 B2M) tdTomato PMID: 27662090, and the other with the same transgene under the transcriptional control of the mouse Pcsk9 gene (hereinafter referred to as Hepa 1-6 Pcsk9) tdTomato these tests were performed by nucleofecting a consistent amount of an unsaturated dose of plasmid encoding either the triple ETR combination or CRISPRoff, along with gRNAs targeting the promoter / enhancer regions of B2M and Pcsk9 (one gRNA for each gene). Then, long-term flow cytometry analysis was used to evaluate the epigenetic silencing efficiency and persistence. Considering these results, CRISPRoff in the ETR plasmid backbone was used as a control for further comparison.

[0303]

[0342] Next, to better understand the final differences in the epigenetic silencing efficiency of the tested constructs, the relative efficacy of two epigenetic silencing platforms (i.e., the triple ETR combination and CRISPRoff in Figure 2) was evaluated using the same experimental conditions as above. These tests showed that both platforms could impose long-term stable epigenetic silencing to different extents (Figures 4A - 4B). In particular, the triple ETR combination outperformed CRISPRoff by 1.4-fold and 1.8-fold in the K-562 B2M tdTomato and Hepa 1-6 Pcsk9 tdTomato cell lines, respectively.

[0304]

[0343] Next, for the purpose of improving efficacy and specificity, the structures and compositions of both epigenetic silencing platforms were improved. For this, initially, attention was paid to the linker sequences and the triple ETR combination was used. Within each ETR, the G linker was replaced with either a protease-resistant XTEN linker (hereinafter referred to as X) or a flexible GGGGS linker (hereinafter referred to as GX4), each in a four-tandem repeat. To better understand the final differences in the epigenetic silencing efficiency of the tested constructs, the epigenetic silencing efficiency of these new constructs was tested in the above cell lines, compared to the parental triple ETR combination (hereinafter referred to as t.ETRv.0) and CRISPRoff, still using a matched amount of unsaturated plasmid dosage (Figures 5A - 5B).

[0305]

[0344] Unexpectedly, the adoption of the XTEN linker reduced the epigenetic silencing efficiency of the triple ETR combination to a value that could be overlaid with or even lower than that obtained using CRISPRoff. On the other hand, the triple ETR combination containing the GX4 linker (i.e., dCas9:GX4:K + dCas9:GX:D3A + dCas9:GX4:hD3; hereinafter referred to as t.ETRv.1) functioned better than its parental counterpart in both cell lines, and reached a maximum 2-fold increase in epigenetic silencing efficiency when compared to CRISPRoff in the Hepa 1-6 Pcsk9 tdTomato cell line. Based on these results, the GX4 linker (i.e., K:GX4:dCas9 + hD3A:GX4:dCas9 + hD3Ls:GX4:dCas9) was still used to test the inversion of the effector domain at the N-terminus of dCas9. This design, hereinafter referred to as t.ETRv.2, further improved the epigenetic silencing efficiency of both genes, where the Hepa 1-6 Pcsk9 tdTomato cell line reached a maximum 2.3-fold increase in epigenetic silencing efficiency compared to CRISPRoff. Based on these results, the inverted GX4 structure was selected for further optimization.

[0306]

[0345] For this purpose, alternative effector domains from the human ZIM3 protein, such as the KRAB domain and a shortened (short) DNMT3L, both of human and mouse origin, hereinafter referred to as hD3Ls and mD3Ls respectively, were tested (Figures 6A - 6B). All of these new constructs functioned better than CRISPRoff (maximum 2.5-fold increase for the Z:GX4:dCas9 + D3A:GX4:dCas9 + hD3Ls:GX4:dCas9 combination in the Hepa 1-6 Pcsk9 tdTomato cell line) and functioned equivalently to or slightly worse than t.ETRv.2.

[0307]

[0346] Overall, these tests showed that the adoption of the GX4 linker, with or without inversion of the effector domain at the N-terminus of dCas9, resulted in a net improvement in the epigenetic silencing efficiency of the triple ETR combination. Notably, these results were consistent between the two cell types and loci tested.

[0308]

[0347] Example 2: Development of an improved double ETR combination

[0348] ETRs containing the catalytic domain of DNMT3A can exhibit off-target activity (PMID: 31941101, PMID: 29907613). To address this, a double ETR combination composed of KRAB and DNMT3L was tested using the same experimental setup as described in Example 1. In these tests, an ETRv.0 construct (hereinafter referred to as d.ETRv.0) in which the two effector domains were linked at the C-terminus of dCas9 by a G spacer was used. Importantly, these tests showed that d.ETRv.0 functions as efficiently as the parental triple ETR combination in both cell types (Figure 7), again, in K-562 B2M tdTomato and Hepa 1-6 Pcsk9 tdTomato showed performance 1.6-fold and 1.8-fold better than CRISPRoff, respectively.

[0309]

[0349] Based on these results, a similar round of ETR optimization as described in Example 1 was performed. First, XTEN and GX4 linkers were tested. The XTEN linker was found to be detrimental, while the GX4 linker was found to have improved epigenetic silencing efficiency for the platform (Figures 8A - 8B). In particular, the fold change in epigenetic silencing efficiency between CRISPRoff and the double ETR combinations containing dCas9:GX4:K and dCas9:GX4:hD3L (hereinafter referred to as d.ETRv.1) was 1.8 for K-562 B2M tdTomato and 1.8 for Hepa 1-6 Pcsk9 tdTomatoIt was 2.3 for [description in 2.3]. Further inversion of the effector at the N-terminus of dCas9 containing the GX4 linker did not result in any significant increase in the epigenetic silencing efficiency compared to d.ETRv.1, but these constructs still functioned better than CRISPRoff. On the other hand, a significant increase in the epigenetic silencing efficiency was observed, especially, in the Hepa 1-6 Pcsk9 tdTomato cell line when alternative effector domains such as KRAB-type ZIM3 and truncated DNMT3Ls were tested (Figures 9A - 9B). Here, the three double ETR combinations K:GX4:dCas9 + hD3Ls:GX4:dCas9, Z:GX4:dCas9 + mD3Ls:GX4:dCas9, and Z:GX4:dCas9 + hD3Ls:GX4:dCas9 were 3-fold, 3.1-fold, and 3.8-fold more performant than CRISPRoff, respectively.

[0310]

[0350] Contrary to what was shown above for the optimization of the triple ETR combination, the inclusion of shorter human DNMT3L variants gradually improves the epigenetic silencing efficiency when moving from the double ETR combination with KRAB ZNF10 to the double combination with KRAB ZIM3. Overall, these data strengthened the view that the adoption of the GX4 linker is beneficial for dCas9-based ETR combinations. Furthermore, this experiment discovered new double ETR combinations that are more potent than the parental triple ETR combination. Considering the higher specificity profile of the double versus triple ETR combinations and the requirement to produce two versus three constructs, these new double ETR combinations are useful reagents for the clinical translation of the technology.

[0311]

[0351] Example 3: Development of an improved three-component ETR

[0352] This experiment develops an all-in-one fusion construct. To do this, a protein containing three effector domains operably linked to the same dCas9 DNA binding domain (DBD) of the three-element ETR was developed. In this regard, a three-element ETR was constructed in which the KRAB domain derived from ZNF10 was linked to the N-terminus of dCas9 by either the X16 or GX4 spacer. At the C-terminus of dCas9, a DNMT3A-DNMT3L heterodimer containing either the N-terminus of human full-length DNMT3L's shortened counterpart (hD3Ls) or the mouse equivalent of the human shortened protein (mD3Ls) was placed via either the G or X80 linker. A schematic diagram of these constructs is given in Figure 10A. Similar to Examples 1 and 2, the relative efficiency of these new structures was evaluated by plasmid nucleofection in the above cell model using cells treated with CRISPRoff as a reference (Figure 10B).

[0312]

[0353] K-562 B2M tdTomato In cells, all variants except K:X16:dCas9:X80:D3A:W:h3DL functioned approximately 1.5-fold better than CRISPRoff. Hepa 1-6 Pcsk9 tdTomato In cells, the ETR with the X linker functioned better than the ETR with the G-based linker, reaching a maximum 2.2-fold increase in the episilencing efficiency compared to CRISPRoff. Among the ETRs based on the X linker, the K:X16:dCas9:X80:D3A:W:hD3L construct was lower but still better than CRISPRoff. Then, the KRAB domain from XNF10 was replaced with its equivalent from ZIM3 in each of the above three-element ETR structures (Figure 11A). This substitution was found to overall improve the activity of the constructs (Figure 11B). K-562 B2M tdTomato In cells, all variants except Z:X16:dCas9:X80:D3A:W:mD3L functioned approximately 2-fold better than CRISPRoff, but Hepa 1-6 Pcsk9 tdTomatoIn cell lines, the increase in epigenetic silencing efficiency ranged from 2-fold to 5.6-fold, and all X-linker-based ETRs achieved improvements at ratios higher than 4.3-fold. Collectively, these data indicate that for this effector configuration (i.e., KRAB at the N-terminus and DNMT3A-DNMT3L at the C-terminus of dCas9), KRAB from ZIM3 functions better than its equivalent from ZNF10. With very few exceptions, the adoption of these new three-element ETR variants resulted in a significant increase in epigenetic silencing efficiency (from 2-fold to 5.6-fold) compared to CRISPRoff.

[0313]

[0354] Furthermore, the inversion of the effector domain with respect to dCas9 was tested, still comparing the DNMT3A-DNMT3L heterodimers containing either KRAB from ZNF10 and ZIM3, and either full-length or truncated DNMT3Ls (Figure 12A). These tests showed that almost all mirror ETR configurations functioned better than their corresponding, non-inverted counterparts (Figure 12B). This effect was more evident in the Hepa 1-6 Pcsk9 tdTomato cell line, and effector inversion made all constructs function better than CRISPRoff by more than 4.9-fold. These tests also showed that the new three-element ETRs (both full-length and truncated) with KRAB and human-derived DNMT3Ls are significantly more efficient than CRISPRoff, and the unexpected finding was obtained that these new three-element ETRs and CRISPRoff share the same effector domain structure and linker, but differ from each other only by the human vs. mouse DNMT3L domain.

[0314]

[0355] Example 4: Development of improved two-element ETR

[0356] In this example, a de novo two - element ETR containing the KRAB and DNMT3L domains is constructed. This experiment follows an optimization strategy similar to that described in Example 4. First, the KRAB domain from ZNF10 was compared for two - element ETRs where it was linked to the N - terminus of dCas9 by either X or GX4. At the C - terminus of dCas9, a full - length or truncated DNMT3L domain was placed using either G, GX4, or X linker. Schematic diagrams of these constructs are given in Figure 13A. These experiments showed that some of the variants tested functioned significantly better than CRISPRoff in both cell types, with the epigenetic silencing efficiency exceeding 2.4 - fold that of CRISPRoff and reaching 4 - fold for K:X16:dCas9:X80:mD3Ls in the Hepa 1 - 6 Pcsk9 tdTomato cell line (Figure 13B).

[0315]

[0357] For these constructs, the adoption of the X linker was detrimental to what was observed for two - element ETRs with G - based linkers, except for K:X16:dCas9:X80:mD3Ls which functioned better than the others, for the K - 562 B2M tdTomato cell line), or resulted in comparable epigenetic silencing efficiency (for the Hepa 1 - 6 Pcsk9 tdTomato cell line).

[0316]

[0358] Similar experiments were conducted using a panel of two - element ETRs with the KRAB domain from ZIM3 (Figure 14A). The adoption of this effector, especially in the Hepa 1 - 6 Pcsk9 tdTomatoIt is particularly beneficial in cell lines (Figure 14B). Here, the two-element ETR Z:GX4:dCas9:GX4:hD3Ls, Z:GX4:dCas9:GX4:mD3ls, and Z:GX4:dCas9:GX4:mD3ls function 5.6-fold, 6-fold, and 6.1-fold better than CRISPRoff, respectively. Regarding the three-element ETR, inversion of the effector domain was also tested to compare the two-element ETR with KRAB derived from ZNF10 (Figure 15A). Hepa 1-6 Pcsk9 tdTomato In cell lines, except for mD3Ls:X18:dCas9:X16:K, domain inversion nearly doubles the efficiency of episilencing compared to the corresponding non-inverted two-element ETR and achieves a 4.2-fold to 5-fold improvement compared to CRISPRoff (Figure 15B).

[0317]

[0359] Finally, the inclusion of the KRAB domain derived from ZIM3 in these constructs is beneficial, especially for Pcsk9 tdTomato In cell lines, it brings an episilencing efficiency worthy of note. In fact, in this cell model, all except the hD3L:X80:dCas9:X16:Z two-element ETR can induce an episilencing efficiency 5.9-fold higher than CRISPRoff, and mD3ls:X80:dCas9:X16:Z was the best construct. Overall, these tests show that the best structure for the two-element ETR contains the ZIM3 KRAB domain, preferentially for the shorter DNMT3L version and independently of its relative position regarding dCas9 and the linker used.

[0318]

[0360] Example 5: Linker substitution

[0361] In this example, substitution of the W linker with the GX4 linker in the two-element ETR and three-element ETR constructs increases the episilencing efficiency of the constructs.

[0319]

[0362] Example 6: Verification of ETR in human primary T cells

[0363] In this example, the best improved ETR, as either a combination or an all-in-one fusion protein, is selected from screening in human primary T cells, which are cell types appropriate for cancer immunotherapy.

[0320]

[0364] This experiment uses nucleofection of purified human T cells with mRNA encoding combinations of ETR and gRNA to target three clinically relevant genes, namely, B2M, TET2, and TGFBR2.

[0321]

[0365] The treated cells are analyzed by flow cytometry or ddPCR to measure the efficiency of epigenetic silencing of the three genes in real time. These analyses show that all ETRs (both combinations and all-in-one fusion proteins) function better than their parental counterparts (in the case of the triple ETR combination) and CRISPRoff. In this regard, the three- and two-element ETRs hD3A:hD3L:X80:dCas9:X16:K and Z:GX4:dCas9:G:hD3L function 6-fold and 10-fold better than CRISPRoff, respectively, in the silencing of B2M (Figures 16A - 16C). Similar results are obtained for the other two genes.

[0322]

[0366] Example 7: Development of ETR Combinations with ZFP-DBD

[0367] In this example, an optimization strategy similar to that described in Examples 1 and 2 was performed for the ZFP DNA binding domain (DBD). Here, different linkers were tested (including X80, X16, and GX4). The inversion of the effector and the use of ZIM3 KRAB domains and truncated DNMT3Ls of human and mouse origin were also tested.

[0323]

[0368] Based on three previously validated ZFPs, an ETR was used to target the mouse Pcsk9 gene (Figure 17). These ETRs contained ZNF10 KRAB, the catalytic domain of DNMT3A, and human full-length DNMT3L linked to the C-terminus of the ZFP by a G linker sequence. Hepa 1-6 Pcsk9 TdTomato Using the cell line, it was shown that all ZFP-ETRs, as either triple or double combinations, functioned better than their parental counterparts.

[0324]

[0369] Example 8: Comparison of double ETR and triple ETR

[0370] In this example, the described double and triple ETR combinations are evaluated for off-target activity. The double and triple ETR combinations exhibit a lower off-target effect or no off-target effect in the double ETR combination compared to the triple ETR combination.

[0325]

[0371] Example 9: Development of two-element and three-element ETRs with ZFP-DBD

[0372] In this example, the best two-element and three-element ETR structures from Examples 4-6 are tested using ZFP DBD technology by replacing dCas9 with ZFP8. The effectiveness of these new ETRs was determined using cells treated with a standard triple ZFP-based ETR combination (i.e., having a G linker and effector at the C-terminus of the ZFP) as a reference for epigenetic silencing efficiency. Hepa 1-6 Pcsk9 tdTomatoTest in cell lines. These experiments show that many new ETRs function as efficiently as, or better than, standard triple ZFP-based ETR combinations that include the two- and three-element combinations mD3ls:X80:ZFP:X16:K, hD3A:hD3L:X80:ZFP:X16:K, and hD3A:hD3L:X80:ZFP:X16:Z (Figures 18A - 18B). The enhanced activity of these ETRs is also shown in vivo upon their LNP-mediated delivery to mice. The improvement in the observed episilencing efficiency for these new ZFP-based ETRs is comparable to that observed for the dCas9-based parental counterparts, indicating that optimization efforts are transferable to different DBD structures.

[0326]

[0373] Example 10: Selection of ETR Structures for Pcsk9

[0374] In this example, to rapidly select ETR structures for Pcsk9, the inventors developed an engineered mouse liver cancer cell line that reports on the transcriptional activity of this gene at the single cell level (hereinafter, Hepa 1-6 Pcsk9 tdTomato cells; Figure 19A). Using this system, the inventors separately tested and nominated the most effective triple ETR combinations for each of the following programmable DBD platforms: dCas9 (8 target sites), TALE (16 target sites), and ZFP (16 target sites) (Figures 19A and 20A - 20F). To identify the best of these platforms, the inventors then used RNAs encoding different ETR components to Hepa 1-6 Pcsk9 tdTomatoExperiments were conducted in cells (Figure 19B). Both the ZFP-based ETR and the dCas9-based ETR were superior to the TALE-based construct and achieved more than 80% Pcsk9 silencing (Figure 19C). These values were equivalent, if not higher, than those observed upon gene inactivation of Pcsk9 using an equivalent amount of RNA encoding catalytically active CRISPR-Cas9. Both gene disruption and epigenetic silencing of Pcsk9 were demonstrated to be stable over the long term (Figure 20G). The inventors then selected the ZFP-based ETR for subsequent testing.

[0327]

[0375] Example 11: Delivery of ZFP-ETR to the Liver of Mice

[0376] In parallel with Example 10, Example 11 describes the delivery of ZFP-ETR to the liver of mice. Since the ETR technology involves the use of transient gene delivery modalities, the inventors performed in vivo screening to identify lipid nanoparticles (LNPs) that are compatible with the efficient introduction of the editing machinery into the liver (K. Paunovska, D. Loughrey, J. E. Dahlman, Drug delivery systems for RNA therapeutics. Nat. Rev. Genet. 23, pages 265 - 280 (2022)). Here, the inventors used CRISPR-Cas9-mediated inactivation of Pcsk9 as a surrogate readout for LNP-mediated delivery of the editor's RNA. Of the 10 LNPs tested, 7 induced a robust decrease in the circulating levels of Pcsk9 (more than 60%) and efficient editing of the gene (Figure 21A). When mRNA encoding ZFP-ETR was packaged in an LNP formulation and its performance was first tested in cultured primary mouse hepatocytes, near-complete disappearance of Pcsk9 was observed (Figure 22A).

[0328]

[0377] The inventors then intravenously administered LNP loaded with ETR to adult C57BL / 6 mice and monitored the circulating levels of Pcsk9 over a maximum of 330 days when the experiment was terminated (Figure 22B). LNP loaded with vehicle and eGFP mRNA (hereinafter, mock) was used as a control. Initial analysis showed a rapid and significant decrease in Pcsk9, which then stabilized at approximately 50% of the vehicle-treated levels until the last time point analyzed (Figure 22C). Consistent with these data, at day 30 after LNP injection, the levels of LDL-bound cholesterol (LDL-C) decreased in ETR-treated mice compared to vehicle-treated mice (by approximately 35%; Figure 21B). Equivalent efficiency and kinetics of Pcsk9 and LDL-C reduction were observed in mice treated with LNP loaded with CRISPR-Cas9 RNA targeting Pcsk9 (Figure 22C, Figure 21B). Treatment-related toxicity was self-limiting, with a transient increase in the serum levels of the liver enzymes ALT and AST at comparable levels between the CRISPR-Cas9-treated group and the ZFP-ETR-treated group (Figure 21C). Since previous studies showed no significant hepatotoxicity for any CRISPR-Cas9 components targeting the same Pcsk9, although loaded in different LNP formulations (H. Yin, C. Q. Song, S. Suresh, Q. Wu, S. Walsh, L. H. Rhym, E. Mintzer, M. F. Bolukbasi, L. J. Zhu, K. Kauffman, H. Mou, A. Oberholzer, J. Ding, S. Y. Kwan, R. L. Bogorad, T. Zatsepin, V. Koteliansky, S. A. Wolfe, W. Xue, R. Langer, D. G. Anderson, Structure-guided chemical modification of guide RNA enables potent non-viral in vivo genome editing. Nat. Biotechnol. 35, pp. 1179-1187 (2017)), the inventors concluded that the enzyme elevation observed here is likely due to their LNP formulation.To confirm and extend the inventors' findings, the inventors treated a second cohort of mice with LNP loaded with an ETR targeting Pcsk9 (Figure 21D) and, three months later, subjected four of them to partial hepatectomy (PH), a surgical procedure that induces robust waves of hepatocyte proliferation to regenerate excised liver lobes (26.Y. Nevzorova, R. Tolba, C. Trautwein, C. Liedtke, Partial hepatectomy in mice. Lab. Anim. 49, pp. 81-88 (2015)) (Figure 22D). Notably, no significant difference in the circulating levels of Pcsk9 was observed between pre-hepatectomy and post-hepatectomy mice (Figure 22E), which further supports the stability of epigenetic silencing even during active cell replication. Similar results were obtained in mice treated for gene inactivation of Pcsk9. Finally, the inventors compared the CpG methylation profiles of the Pcsk9 gene promoter in pre-hepatectomy mice treated with ETR or vehicle and found a net increase in DNA methylation in the former group (Figure 22F). These levels remained stable even during PH (up to three months), further supporting the persistence of epigenetic silencing during liver regeneration.

[0329]

[0378] Conclusions from Examples 10-11

[0379] Examples 10 - 11 demonstrate that LNP-mediated delivery of mRNA encoding ETR to the mouse liver can result in stable (almost 1-year follow-up) epigenetic silencing of Pcsk9. Notably, epigenetic silencing was shown to be stable even during PH, which further confirms the genetic nature of the epigenetic marks abolished by the ETR technology and indicates that epigenetically silenced hepatocytes still possess the ability to regenerate the liver. When compared to RNAi, which requires multiple administrations (K. K. Ray, R. S. Wright, D. Kallend, W. Koenig, L. A. Leiter, F. J. Raal, J. A. Bisch, T. Richardson, M. Jaros, P. L. J. Wijngaard, J. J. P. Kastelein, Two phase 3 trials of inclisiran in patients with elevated LDL cholesterol. N. Engl. J. Med. 382, pp. 1507 - 1519 (2020)), the applicant's approach constitutes a feature shared only with other genome editing technologies as a single treatment. However, unlike these latter, the ETR technology does not require the induction of potentially genotoxic DNA cleavage to edit the desired gene (M. L. Leibowitz, S. Papathanasiou, P. A. Doerfler, L. J. Blaine, L. Sun, Y. Yao, C. Z. Zhang, M. J. Weiss, D. Pellman, Chromothripsis as an on-target consequence of CRISPR-Cas9 genome editing. Nat. Genet. 53, pp. 895 - 905 (2021), G. Turchiano, G. Andrieux, J. Klermund, G. Blattner, V. Pennucci, M. el Gaz, G. Monaco, S. Poddar, C. Mussolino, T. I. Cornu, M. Boerries, T.Cathomen, Quantitative evaluation of chromosomal rearrangements in gene-edited human stem cells by CAST-Seq. Cell Stem Cell 28, 1136 - 1147.e5 pages (2021), H. A....

Claims

1. (a) A fusion protein comprising, from the N-terminus to the C-terminus, a first DNA methyltransferase (DNMT) domain, a first linker (linker DD), a second DNMT domain, a second linker (linker BD), a DNA binding domain, a third linker (linker RB), and a transcriptional repressor domain; or (b) Nucleic acid molecule encoding the fusion protein of (a) An epigenetic editing system, including [this].

2. The epigenetic editing system according to claim 1, wherein linker RB, linker BD, and / or linker DD comprises flexible or unstructured peptide linkers.

3. The epigenetic editing system according to claim 1, wherein linker RB, linker BD, and / or linker DD comprise a glycine-rich and / or serine-rich polypeptide sequence, and optionally the glycine-rich and / or serine-rich polypeptide sequence comprises the sequence (G x S y) z (wherein x is an integer from 1 to 10, y is an integer from 1 to 10, and z is an integer from 1 to 10) (SEQ ID NO: 135).

4. Linker RB, (GGGGS) ４ (Sequence ID 4) and / or linker DD, (GGGGS) ４ An epigenetic editing system according to any one of claims 1 to 3, comprising (SEQ ID NO: 4) or the amino acid sequence SSGNSNANSRGPSFSSGLVPLSLRGSH (SEQ ID NO: 5).

5. The epigenetic editing system according to any one of claims 1 to 3, wherein linker RB, linker BD, and / or linker DD include an XTEN linker, and optionally the XTEN linker is an XTEN16 or XTEN80 linker.

6. An epigenetic editing system according to any one of claims 1 to 3, wherein linker RB comprises an XTEN16 linker, linker BD comprises an XTEN80 linker, and linker DD comprises the amino acid sequence SSGNSNANSRGPSFSSGLVPLSLRGSH (SEQ ID NO: 5).

7. The epigenetic editing system according to any one of claims 1 to 3, wherein the transcriptional repressor domain is a KRAB domain, and optionally the KRAB domain is a KRAB domain derived from ZIM3.

8. The epigenetic editing system according to claim 7, wherein the KRAB domain comprises an amino acid sequence that is at least 90% homologous to SEQ ID NO:

16.

9. The epigenetic editing system according to any one of claims 1 to 3, wherein the first DNMT domain or the second DNMT domain is a DNMT3A domain or a DNMT3L domain.

10. The epigenetic editing system according to claim 9, wherein the DNMT3A domain is a human DNMT3A domain or a mouse DNMT3A domain.

11. The epigenetic editing system according to claim 9, wherein the DNMT3A domain comprises an amino acid sequence that is at least 90% homologous to SEQ ID NO:

13.

12. The epigenetic editing system according to claim 9, wherein the DNMT3L domain is a human DNMT3L (hD3L) domain or a mouse DNMT3L (mD3L) domain.

13. The hD3L domain contains an amino acid sequence that is at least 90% homologous to SEQ ID NO: 14; or The epigenetic editing system according to claim 12, wherein the mD3L domain comprises an amino acid sequence that is at least 90% homologous to SEQ ID NO:

69.

14. The first DNMT domain is the DNMT3A domain, and the second DNMT domain is the DNMT3L domain; or The epigenetic editing system according to any one of claims 1 to 3, wherein the first DNMT domain is a DNMT3L domain and the second DNMT domain is a DNMT3A domain.

15. An epigenetic editing system according to any one of claims 1 to 3, wherein at least one of the DNA-binding domains comprises a DNA-binding domain of a CRISPR-Cas protein, and optionally the CRISPR-Cas protein comprises nuclease-inactive Cas9 (dCas9), nuclease-inactive Cas12a (dCas12a), or nuclease-inactive CasX (dCasX).

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