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 / 354,931, filed Jun. 23, 2022, which is incorporated herein by reference in its entirety.

Background Art

[0002]

[0002] Genome editing has been considered a promising therapeutic approach for treating genetic diseases for decades. However, manipulation at the DNA level remains 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 epigenetic editing systems, 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] An epigenetic editing system comprising: (a) a fusion protein comprising a DNA binding domain, a DNA methyltransferase (DNMT) domain, a repressor domain, and two nuclear localization sequences (NLSs), wherein each of the two NLSs is located at the amino (N)-terminal or carboxy (C)-terminal of the fusion protein; or (b) a nucleic acid molecule encoding the fusion protein of (a).

[0005]

[0005] In some embodiments, the fusion protein comprises one or more NLSs at its C-terminus and one or more NLSs at its N-terminus. Optionally, the fusion protein comprises two NLSs at its N-terminus and two NLSs at its C-terminus. In some embodiments, the DNMT domain is from a bacterial species. In some embodiments, the DNMT domain is a mammalian DNMT domain. In some embodiments, the DNMT domain is a mouse DNMT domain. In some embodiments, the DNMT domain is a human DNMT domain. In some embodiments, the DNMT domain is a DNMT3A domain. In some embodiments, the DNMT domain is a DNMT3L domain. In some embodiments, the fusion protein comprises both a DNMT3A domain and a DNMT3L domain.

[0006]

[0006] In some embodiments, the DNMT3L domain is from a species selected from the group consisting of Ailuropoda melanoleuca, Carlito syrichta, Meriones unguiculatus, Ochotona princeps, Neosciurus carolinensis, Bison bison, Equus przewalskii, Mus caroli and Pan troglodytes; optionally, it comprises an amino acid sequence that is identical to one of SEQ ID NOs: 72-80 and 101-109, or is at least 90% identical thereto, optionally at least 95% identical.

[0007]

[0007] In some embodiments, the repressor domain comprises the KRAB domain of the ZFP28, ZN627, KAP1, MeCP2, HP1b, CBX8, CDYL2, TOX, Tox3, Tox4, EED, RBBP4, RCOR1, or SCML2 protein, or a fusion of the N-terminal and C-terminal regions of the ZIM3 KRAB domain and the KOX1 KRAB domain.

[0008]

[0008] (a) A fusion protein comprising a DNA binding domain and a DNA methyltransferase (DNMT) domain derived from a bacterial species, fused to the N-terminus of the DNA binding domain; or (b) A nucleic acid molecule encoding the fusion protein of (a), an epigenetic editing system comprising the same is described herein.

[0009]

[0009] In some embodiments, the fusion protein functions to methylate mammalian target DNA in cells. In some embodiments, the DNMT domain of the fusion protein does not include any one of SEQ ID NOs: 81-93. In some embodiments, the bacterial species is not M. penetrans, S. monbiae, H. parainfluenzae, A. luteus, H. aegyptius, H. haemolyticus, Moraxella, Escherichia coli (E. coli), T. aquaticus, C. crescentus or C. difficile.

[0010]

[0010] In some embodiments, the DNMT domain derived from a bacterial species is (a) optionally, M.Sss1 comprising an amino acid sequence that is optionally at least 90%, optionally at least 95% identical to SEQ ID NO: 40; optionally, NQZ29229 comprising an amino acid sequence that is optionally at least 90%, optionally at least 95% identical to SEQ ID NO: 41; optionally, WP_131599610 comprising an amino acid sequence that is optionally at least 90%, optionally at least 95% identical to SEQ ID NO: 42; or optionally, WP_208057179 comprising an amino acid sequence that is optionally at least 90%, optionally at least 95% identical to SEQ ID NO: 43, and is derived from the same.

[0011]

[0011] (a) a fusion protein comprising a DNA binding domain and being derived from a species selected from the group consisting of Ailuropoda melanoleuca, Carlito syrichta, Meriones unguiculatus, Ochotona princeps, Neosciurus carolinensis, Bison bison, Equus przewalskii, Mus caroli, and Pan troglodytes; optionally, the DNMT3L domain comprising one of SEQ ID NOs: 72 - 80 and 101 - 109, or an amino acid sequence having at least 90%, optionally at least 95% identity thereto; or (b) an epigenetic editing system comprising a nucleic acid molecule encoding the fusion protein of (a), is described herein.

[0012]

[0012] In some embodiments, (i) the fusion protein further comprises a repressor domain, or (ii) the system further comprises an additional fusion protein comprising a DNA binding domain and a repressor domain, or a nucleic acid molecule encoding the additional fusion protein. In some embodiments, the repressor domain comprises a KRAB domain optionally derived from KOX1, ZIM3, ZFP28, or ZN627.

[0013]

[0013] In some embodiments, the KRAB domain is derived from human KOX1 and optionally comprises the amino acid sequence of SEQ ID NO: 94, or an amino acid sequence having at least 90%, optionally at least 95% identity thereto, or comprises the amino acid sequence of SEQ ID NO: 100, or an amino acid sequence having at least 90%, optionally at least 95% identity thereto. In some embodiments, the repressor domain is derived from KAP1, MECP2, HP1a / CBX5, HP1b, CBX8, CDYL2, TOX, TOX3, TOX4, EED, EZH2, RBBP4, RCOR1, or SCML2.

[0014]

[0014] (a) A fusion protein comprising a DNA binding domain and a repressor domain, provided that the repressor domain comprises the KRAB domain of the ZFP28, ZN627, KAP1, MeCP2, HP1b, CBX8, CDYL2, TOX, Tox3, Tox4, EED, RBBP4, RCOR1, or SCML2 protein, or a fusion of the N-terminal region and the C-terminal region of the ZIM3 KRAB domain and the KOX1 KRAB domain; or (b) A nucleic acid molecule encoding the fusion protein of (a), an epigenetic editing system comprising is described herein.

[0015]

[0015] In some embodiments, the repressor domain comprises one of SEQ ID NOs: 44-57, or an amino acid sequence that is at least 90%, optionally at least 95% homologous thereto. In some embodiments, the fusion protein further comprises a DNA methyltransferase (DNMT) domain, or the system further comprises an additional fusion protein comprising a DNA binding domain and a DNMT domain, or a nucleic acid molecule encoding the additional fusion protein.

[0016]

[0016] In some embodiments, the fusion protein or system comprises a human DNMT3A domain and a human DNMT3L domain, or a human DNMT3A domain and a mouse DNMT3L domain, and optionally, (a) the human DNMT3A domain comprises an amino acid sequence that is at least 90%, optionally at least 95% homologous to SEQ ID NO: 12; (b) the human DNMT3L domain comprises an amino acid sequence that is at least 90%, optionally at least 95% homologous to SEQ ID NO: 13, and / or (c) the mouse DNMT3L domain comprises an amino acid sequence that is at least 90%, optionally at least 95% homologous to SEQ ID NO: 15.

[0017]

[0017] In some embodiments, the epigenetic editing system further comprises a fusion protein comprising, from the N-terminus to the C-terminus, one or more nuclear localization signals (NLSs), a DNMT3A domain, a DNMT3L domain, a DNA binding domain, a repressor domain, and one or more NLSs, and optionally, the fusion protein comprises a peptide linker between adjacent domains. In some embodiments, the fusion protein comprises, from the N-terminus to the C-terminus, two NLSs, a DNMT3A domain, an ADD, a DNMT3L domain, a first peptide linker, a DNA binding domain, a second peptide linker, a repressor domain, and two NLSs.

[0018]

[0018] In some embodiments, the DNMT3A domain is a human DNMT3A domain and / or the DNMT3L domain is a human DNMT3L domain. In some embodiments, the repressor domain is a KRAB domain derived from mammalian, optionally human, ZFP28, ZF627 or KOX1.

[0019]

[0019] In some embodiments, the first peptide linker is XTEN80 (SEQ ID NO: 3) and / or the second peptide linker is XTEN16 (SEQ ID NO: 2). In some embodiments, the system comprises an expression construct encoding the fusion protein, provided that the expression construct comprises a WPRE sequence in the 3' non-coding region and upstream of the polyadenylation site.

[0020]

[0020] In some embodiments, the DNA binding domain is a dCas9 domain. In some embodiments, the dCas9 domain comprises an amino acid sequence that is at least 90%, optionally at least 95%, identical to SEQ ID NO: 9. In some embodiments, the epigenetic editing system further comprises one or more guide RNAs (gRNAs) or a nucleic acid molecule encoding a gRNA. In some embodiments, the DNA binding domain is a zinc finger protein (ZFP) domain.

[0021]

[0021] A method for modifying the epigenetic state of a target gene in mammalian cells, the method comprising contacting the cells with the epigenetic editing system of the present disclosure, is described herein. A method for regulating the expression of a target gene in mammalian cells, the method comprising contacting the cells with the epigenetic editing system of the present disclosure, is described herein. A method for treating a disease in a subject in need thereof, the method comprising administering to the subject the epigenetic editing system of the present disclosure, is described herein.

[0022]

[0022] Further aspects and advantages of the present disclosure will become readily apparent to those skilled in the art from the following detailed description, which shows and describes only exemplary embodiments of the present disclosure. As will be recognized, the present disclosure is capable of other different embodiments and that several details thereof are capable of modification in various obvious respects, all without departing from the present disclosure. Accordingly, the drawings and description are to be regarded as illustrative in nature and not as restrictive.

[0023] Incorporation by reference

[0023] All publications, patents, and patent applications mentioned herein 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 any incorporated publication and patent or patent application contradicts the disclosure contained herein, this specification is intended to supersede and / or take precedence over any such conflicting material.

[0024] Brief description of the drawings

[0024] 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 and the accompanying drawings (referred to herein as "figures") that describe exemplary embodiments that utilize the principles of the invention.

Brief description of the drawings

[0025]

Figure 1

[0025] Figure 1A shows a schematic diagram of a fusion protein construct having a variant NLS configuration. Figure 1B shows a schematic diagram of an additional fusion protein construct having a variant KRAB domain.

Figure 2A

[0026] Figures 2A - 2B show the percentages of PCSK9 protein levels measured after treatment with fusion protein constructs having various NLS arrangements using 6.25 ng of RNA (Figure 2A) or 2.5 ng of RNA (Figure 2B) in HeLa cells. The human and mouse DNMT3L sequences are denoted as h3L and m3L, respectively.

Figure 2B

[0026] Figures 2A - 2B show the percentages of PCSK9 protein levels measured after treatment with fusion protein constructs having various NLS arrangements using 6.25 ng of RNA (Figure 2A) or 2.5 ng of RNA (Figure 2B) in HeLa cells. The human and mouse DNMT3L sequences are denoted as h3L and m3L, respectively.

Figure 3

[0027] Figure 3 shows that in mPcsk9 silencing in Hepa1 - 6 cells, the construct having 2×NLS is 3 - fold more efficient than CRISPR off.

Figure 4A

[0028] Figure 4A shows that in mPcsk9 silencing in Huh7 cells, the construct having 2×NLS is more efficient than CRISPR off.

Figure 4B

Figure 4C

Figure 5

[0029] Figure 5 shows that in Huh7 cells, 2×NLS brings about improvements across multiple ZFs in the CRISPR off-like format where dCas9 is replaced with zinc fingers.

Figure 6-1

[0030] Figures 6A - 6D show that methylation of the CTLA4 promoter by a bacterial DNMT protein can induce epigenetic silencing of that locus.

Figure 6-2

[0030] Figures 6A - 6D show that methylation of the CTLA4 promoter by a bacterial DNMT protein can induce epigenetic silencing of that locus.

Figure 7

[0031] Figure 7 shows the methylation profile on day 30 at the VIM3 locus of cells treated with different constructs having a bacterial DNA methyltransferase fused to dCas9. The sample treated with M.SssI is 20% methylated.

Figure 8

[0032] Figure 8 shows the methylation profile on day 29 at the CLTA locus of cells by hybridization capture, comparing M.SssI in the dCas9 fusion with mouse DNMT3A / 3L.

Figure 9A

[0033] Figures 9A - 9D show alternative KRAB domains tested compared to CRISPR off with respect to episilencing activity when using 0.5 ng of effector DNA with CLTA - GFP as a marker (Figure 9A), 3 ng of effector DNA with GFP as a marker (Figure 9B), and 0.5 ng of effector DNA with GFP as a marker (Figure 9C).

Figure 9B

[0033] Figures 9A - 9D show alternative KRAB domains tested compared to CRISPR off with respect to episilencing activity when using 0.5 ng of effector DNA with CLTA - GFP as a marker (Figure 9A), 3 ng of effector DNA with GFP as a marker (Figure 9B), and 0.5 ng of effector DNA with GFP as a marker (Figure 9C).

Figure 9C

[0033] Figures 9A - 9D show alternative KRAB domains tested for epigenetic silencing activity compared to CRISPR off, when using 0.5 ng of effector DNA with CLTA - GFP as a marker (Figure 9A), 3 ng of effector DNA with GFP as a marker (Figure 9B), and 0.5 ng of effector DNA with GFP as a marker (Figure 9C).

Figure 9D

Mode for Carrying Out the Invention

[0026] Detailed Description

[0034] 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 by way of example. 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.

[0027]

[0035] 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 described 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.

[0028] Definitions

[0036] 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.

[0029]

[0037] 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.

[0030]

[0038] 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.

[0031]

[0039] 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 the claims.

[0032]

[0040] 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 a medical treatment site, a remote location, a home, a school, or other non-business, informal site.

[0033]

[0041] The terms "determining", "measuring", "evaluating", "assessing", "assaying", and "analyzing" are used interchangeably herein frequently to refer to forms of measurement. These terms include determining whether an element is present or not (e.g., detecting). These terms can include quantitative, qualitative, or both quantitative and qualitative determinations. An evaluation can be relative or absolute. "Detection of the presence of" can, depending on the context, include determining both whether something is present or absent and the amount of what is present.

[0034]

[0042] The terms "subject", "patient" or "individual" are frequently used interchangeably 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 as having a high risk of a disease or suspected of having such a risk. In some cases, a subject is not necessarily diagnosed as having a high risk of a disease or suspected of having such a risk. 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 the 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 the 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.

[0035]

[0043] 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.

[0036]

[0044] The terms "about" or "approximately" mean 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 standard deviation, or more than one standard deviation, of a value in practice. When a particular value is recited in the application and claims, the term "about" is assumed to mean an acceptable error range for the particular value unless otherwise specified.

[0037]

[0045] 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.

[0038]

[0046] 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).

[0039]

[0047] 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 bonded to a phosphate ester at the 5' and 3' carbons of the sugar to form an unbranched alternating polymer. DNA can be in the form of, for example, 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 inter-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 native forms in most cases due to properties such as enhanced cellular uptake, reduced immunogenicity, and increased stability in the presence of nucleases.

[0040]

[0048] The "nucleic acid" described in this specification may include one or more nucleotide variants including 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-thiouridine, beta-D-mannosylqueosine, 5'-methoxycarboxymethyluracil, 5-methoxyuracil, 2-methylthio-N6-isopentenyladenine, uracil-5-oxyacetic acid (v), wybutoxosine, pseudouracil, queosine, 2-thiocytosine, 5-methyl-2-thiouridine, 2-thiouridine, 4-thiouridine, 5-methyluracil, methyl ester of uracil-5-oxyacetic acid, 5-methyl-2-thiouridine, 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 a fairly long phosphate ester chain (e.g., a phosphate ester chain having 4, 5, 6, 7, 8, 9, 10 or more phosphate ester moieties), and a modification including a thiol moiety (e.g., alpha thiotriphosphate ester and beta thiotriphosphate ester).

[0041]

[0049] The "nucleic acids" described herein can 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 can include, but are not limited to, phosphorothioate, phosphorodithioate, phosphorosenoate, phosphorodiselenoate, phosphoranilothioate, phosphoranilidate, phosphoramidate, and phosphorodiamidate linkages. The phosphorothioate linkage delays nuclease degradation of oligonucleotides by substituting a non-bridging oxygen with a sulfur atom in the phosphate ester backbone. The phosphorodiamidate linkage (N3’→P5’) enables prevention of nuclease recognition and degradation. Backbone modifications can 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 a linking group containing linear and cyclic hydrocarbon groups, instead 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 can contain a sugar moiety containing ribose or deoxyribose present in natural 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 linking 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).

[0042]

[0050] Unless otherwise specified, a particular nucleic acid sequence implicitly includes its 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 mixed 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)).

[0043]

[0051] 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 can 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.

[0044]

[0052] 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 may 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 a molecule consisting of two or more amino acids in a specific order determined, for example, 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 part of that protein, for example, a domain, sub - domain, or motif of the 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, deleted, 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 the 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.

[0045]

[0053] 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.

[0046]

[0054] 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.

[0047]

[0055] 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 action.

[0048]

[0056] The term "modulate" refers to a change in amount, degree of function, or range. For example, the epigenetic modification compositions 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 a 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 a transcript. Thus, "modulation" of gene expression includes both gene activation and gene repression.

[0049]

[0057] As used herein, the term "administer" and its grammatical equivalents can refer to the step of providing to a subject or patient one or more replication-competent recombinant adenoviruses or pharmaceutical compositions described herein. 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.

[0050]

[0058] As used herein, the terms "treat", "treating", or "treatment" and grammatical equivalents include the alleviation, reduction, or improvement of at least one symptom of a disease or condition, the prevention of additional symptoms, the inhibition of a disease or condition, e.g., the prevention of the onset of a disease or condition, the reduction of a disease or condition, the regression of a disease or condition, the reduction of a condition caused by a disease or condition, or the arrest, either prophylactically and / or therapeutically, of the symptoms of a disease or condition. "Treating" can relate to the 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 the 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 "managing", and "managing" relates to the reduction of the severity of a particular disease or condition in a patient, or the delay of its recurrence, e.g., the 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 the treatment of any disease in mammals, particularly humans, and includes (a) the prevention of the occurrence of a disease in a subject who is susceptible to, but has not yet been diagnosed as having, the disease; (b) the inhibition of a disease, i.e., the prevention of its onset; or (c) the alleviation of a disease, i.e., the 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.

[0051]

[0059] "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, compared to an equivalent untreated control, 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%. In some embodiments, alleviation of the symptoms of a disorder, compared to an equivalent untreated control, 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.

[0052]

[0060] As used herein, the term "pharmaceutical composition" and its grammatical equivalents refer to a mixture or solution that should be administered to a subject in need of administration, such as a human, containing a therapeutically effective amount of an active pharmaceutical ingredient together with one or more pharmaceutically acceptable excipients, carriers, and / or therapeutic agents.

[0053]

[0061] 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 for the preparation of pharmaceutical compositions acceptable for veterinary and 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 harmful manner with any of the components of the pharmaceutical composition containing the material.

[0054]

[0062] "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 when administered in a dosage sufficient to deliver a therapeutically effective amount of the drug.

[0055]

[0063] "Pharmaceutically acceptable salts" can be salts of acids or bases that are generally considered appropriate in the art for use in contact with human or animal tissues without undue toxicity, inflammation, 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 alkali 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. Those skilled 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 salts of acids or bases 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 forms of those compounds with a stoichiometric amount of the appropriate base or acid in a suitable solvent.

[0056]

[0064] 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 when administered to a subject suffering from or susceptible to that infection, disease, disorder, and / or condition.

[0057]

[0065] The term "repressor domain" or "transcriptional repressor domain" refers to a transcriptional repressor protein or a portion thereof, such as 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 coactivator proteins, repressors, and corepressors.

[0058]

[0066] 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 that have a conserved or improved basic function of inhibiting the transcription of genes. 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 of the KRAB domain, including its function and use, can be found, for example, in Ecco, G., Imbeault, M., Trono, D., KRAB zinc finger proteins, Development 144, (15):2719 - 2717 pages.

[0059]

[0067] The term "DNMT" refers to DNA methyltransferase. As used herein, this term includes enzymes that catalyze the transfer of a methyl group to DNA, such as to standard cytosine-5, DNMTs that catalyze the addition of a methyl group to genomic DNA (e.g., DNMT1, DNMT3A, DNMT3B, and DNMT3C). 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).

[0060]

[0068] The term "DNA binding domain" refers to DNA binding domains from proteins selected from the families of CRISPR proteins, TAL proteins, zinc fingers, and other transcription regulators, homologs, orthologs, and variants thereof that maintain or enhance the basic function of those DNA binding proteins.

[0061]

[0069] The ranges provided herein 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 the above integers, e.g., 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.

[0062]

[0070] 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.

[0063]

[0071] 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.

[0072] As used herein, "Delaying" the expression 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 of "delaying" or alleviating the expression of a disease, or delaying the onset of the 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 deal with a reasonably large number of subjects to yield statistically significant results.

[0064]

[0073] "Expression" or "progression" of a disease means the initial symptoms of the disease and / or subsequent progression. The expression of a disease can 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.

[0065]

[0074] 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.

[0066]

[0075] As used herein, the term "parenteral" includes subcutaneous, intradermal, intravenous, intramuscular, intra-articular, intra-arterial, intra-synovial, intrasternal, intrathecal, intracapsular, and intracranial injection, or infusion techniques. Further, for example, the subject may be administered via an injectable depot administration route using depot injection or biodegradable materials and methods for 1 month, 3 months, or 6 months.

[0067]

[0076] In addition to the specific proteins and nucleotides referred to in this specification, 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 hydrophilic values with uncharged polar head groups include asparagine, glutamine, serine, threonine and tyrosine.

[0068]

[0077] Homologs of any protein or nucleic acid sequence as used herein and intended herein include sequences having a certain degree of homology with the wild-type amino acid and nucleic acid sequences. The homologous sequences can include sequences that are at least 50%, 55%, 65%, 75%, 85% 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.

[0069]

[0078] 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 program). 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.

[0070]

[0079] 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 can, for example, differ in the precursor of the mature protein and in the mature protein itself, and sequence differences between species can affect the numbering. One of ordinary skill in the art can identify each residue in any homologous protein and in its respective coding nucleic acid by methods well known in the art, such as, for example, sequence alignment and determination of homologous residues.

[0071] Epigenetic editing system

[0080] 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 can 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 nuclear localization sequence (NLS). In some embodiments, the fusion protein includes a repressor domain.

[0072]

[0081] 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 a transcription factor, transcriptional activator, transcriptional repressor, or insulator, to a chromatin region, nucleic acid sequence, or histone amino acid residue in or adjacent to the target polynucleotide.

[0073]

[0082] The epigenetic editing system provided herein can include one or more of the effector domains described. In some embodiments, the epigenetic editing system includes multiple 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.

[0074]

[0083] 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.

[0075]

[0084] To achieve the activation or suppression of one or multiple target genes, multiple fusion proteins or constructs can be used. For example, epigenetic editing systems include fusion proteins containing a DNA binding domain (e.g., the 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 can be within the same target gene or within different target genes.

[0076] Effector domain

[0085] The epigenetic editing systems provided herein can 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 containing 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.

[0077]

[0086] 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 modifications. 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.

[0078]

[0087] 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.

[0079]

[0088] 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.

[0080]

[0089] 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. In some embodiments, the change in chemical modification, such as methylation, occurs in a hypomethylated nucleic acid sequence. For example, a chemically modified sequence in a target gene or chromosomal region may 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., at the early stages of neoplasia) relative 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. In some embodiments, the change in chemical modification, such as methylation, occurs in a hypermethylated nucleic acid sequence. In some embodiments, the chemical modification is within a CpG island.

[0081]

[0090] In some embodiments, the protein fusion construct can have 1 effector domain, 2 effector domains, 3 effector domains, 4 effector domains, 5 effector domains, 6 effector domains, 7 effector domains, 8 effector domains, 9 effector domains, or 10 effector domains.

[0082] Methyltransferase domain

[0091] In some embodiments, the effector domain comprises a histone methyltransferase domain. For example, repression (or silencing) can result from repressive chromatin markers, DNA methylation, methylation of histone residues (e.g., H3K9, H3K27), or deacetylation of histone residues on chromatin containing the target nucleic acid sequence. Without being bound by theory, the method can be used to alter the epigenetic state, for example, by closing chromatin via methylation or by introducing repressive chromatin markers on chromatin containing the target nucleic acid sequence (e.g., gene).

[0083]

[0092] 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 associated with various microbiomes of an organism, etc.). Histone modification plays a structural and biochemical role in gene transcription by forming or disrupting the nucleosome structure that binds to histones and inhibits 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 at 92% of the amino acid sequence and differ at only 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 together 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 are modified at their N-terminus, called the histone tail, for example, by acetylation, methylation, phosphorylation, ubiquitination, SUMOylation, etc., to maintain or specifically transform the chromatin structure, and as a result, control responses occurring 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 by modifying 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 for "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 near CpG islands (regions with a high frequency of CpG sites) or have promoter regions that contain CpG islands.

[0084]

[0093] 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 acetyltransferases (HATs) utilize 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 and allows 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.

[0085]

[0094] 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.

[0086]

[0095] 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, provided that H is any one of A, T, or C.

[0087]

[0096] 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 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.

[0088]

[0097] Exemplary DNA methyltransferases (DNMTs) that can be part of an epigenetic effector are presented in Table 1 below. In some embodiments, the epigenetic editing system herein contains one or more epigenetic effector domains selected from Table 1, or functional homologs, orthologs, or variants thereof.

[0089]

Table 1

[0090]

[0098] In some embodiments, the methyltransferase can be a mammalian methyltransferase. In some embodiments, the methyltransferase can be a plant methyltransferase. In some embodiments, the methyltransferase can be a fungal methyltransferase. In some embodiments, the methyltransferase can be a bacterial methyltransferase.

[0091]

[0099] Bacterial DNA methyltransferases can be obtained from bacterial species. The bacterial species can be cocci. The bacterial species can be Bacillus. The bacterial species can be spirilla. The bacteria can be intracellular bacteria, Gram-positive bacteria, or Gram-negative bacteria. Examples of bacterial genera from which suitable DNA methyltransferases can be obtained include, but are not limited to, Acetobacter, Acinetobacter, Actinomyces, Agrobacterium, Anaplasma, Azorhizobium, Azotobacter, Bacillus, Viridans, Bacteroides, Bartonella, Bordetella, Borrelia, Brucella, Brukholderia, Calymmatobacterium, Campylobacter, Chlamydia, Chlamydophila, Clostridium, Corynebacterium, Coxiella, Ehrlichia, Eikenella, Enterobacter, Enterococcus, Escherichia, Fusobacterium, Gardnerella, Haemophilus, Helicobacter, Klebsiella, Lactobacillus, Legionella, Leptospira, Listeria, Methanobacterium, Microbacterium, Micrococcus, Moraxella, Mycobacterium, Mycoplasma, Mycoplasmatales, Neisseria, Pasteurella, Peptostreptococcus, Porphyromonas, Prevotella, Pseudomonas, Rhizobium, Rickettsia, Rochalimaea, Rothia, Salmonella, Shigella, Spirillum, Spiroplasma, Staphylococcus, Stenotrophomonas, Streptococcus, Treponema, Ureaplasma, Vibrio, Wolbachia, and Yersinia.In certain embodiments, the DNMT or DNMT domain is used as an epigenetic effector sequence in the fusion proteins provided herein that are derived from bacterial species, which bacterial species are Mycoplasmatales bacterium, Mycoplasma marinum or Spiroplasma chinense. In certain embodiments, the bacterial species from which the DNMT is derived is not M. penetrans, S. monbiae, H. parainfluenzae, A. luteus, H. aegyptius, H. haemolyticus, Moraxella, Escherichia coli, T. aquaticus, C. crescentus or C. difficile.

[0092]

[0100] In some embodiments, the DNMT can be an animal DNMT. In some embodiments, the DNMT can be a mammalian DNMT. In some embodiments, the DNMT can be a primate DNMT. In some embodiments, the DNMT can be a human DNMT. In some embodiments, the DNMT can be a Pan troglodytes DNMT. In some embodiments, the DNMT can be a Carlito syrichta DNMT. In some embodiments, the DNMT can be a rodent DNMT. In some embodiments, the DNMT can be a mouse DNMT. In some embodiments, the DNMT can be a Mus caroli DNMT. In some embodiments, the DNMT can be a Mus musculus DNMT. In some embodiments, the DNMT can be a Neosciurus carolinensis DNMT. In some embodiments, the DNMT can be a Meriones unguiculatus DNMT. In some embodiments, the DNMT can be a horse DNMT. In some embodiments, the DNMT can be an Equus przewalskii DNMT. In some embodiments, the DNMT can be a bovine DNMT. In some embodiments, the DNMT can be a Bison DNMT. In some embodiments, the DNMT can be an Ochotona princeps DNMT. In some embodiments, the DNMT can be a cat DNMT. In some embodiments, the DNMT can be a dog DNMT. In some embodiments, the DNMT can be a bear DNMT. In some embodiments, the DNMT can be an Ailuropoda melanoleuca DNMT.

[0093]

[0101] In some embodiments, the DNMT can include SEQ ID NO: 12. In some embodiments, the DNMT can be SEQ ID NO: 12. In some embodiments, the DNMT can be at least about 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% similar to SEQ ID NO: 12.

[0094]

[0102] In some embodiments, the DNMT may include SEQ ID NO: 13. In some embodiments, the DNMT may be SEQ ID NO: 13. In some embodiments, the DNMT may be at least 99%, 98%, 97%, 96%, 95%, 90%, 85% or 80% similar to SEQ ID NO: 13.

[0095]

[0103] In some embodiments, the DNMT may include SEQ ID NO: 14. In some embodiments, the DNMT may be SEQ ID NO: 14. In some embodiments, the DNMT may be at least 99%, 98%, 97%, 96%, 95%, 90%, 85% or 80% similar to SEQ ID NO: 14.

[0096]

[0104] In some embodiments, the DNMT may include SEQ ID NO: 15. In some embodiments, the DNMT may be SEQ ID NO: 15. In some embodiments, the DNMT may be at least 99%, 98%, 97%, 96%, 95%, 90%, 85% or 80% similar to SEQ ID NO: 15.

[0097]

[0105] In some embodiments, the DNMT may include SEQ ID NO: 16. In some embodiments, the DNMT may be SEQ ID NO: 16. In some embodiments, the DNMT may be at least 99%, 98%, 97%, 96%, 95%, 90%, 85% or 80% similar to SEQ ID NO: 16.

[0098]

[0106] In some embodiments, the DNMT may include SEQ ID NO: 72. In some embodiments, the DNMT may be SEQ ID NO: 72. In some embodiments, the DNMT may be at least 99%, 98%, 97%, 96%, 95%, 90%, 85% or 80% similar to SEQ ID NO: 72.

[0099]

[0107] In some embodiments, the DNMT may include SEQ ID NO: 73. In some embodiments, the DNMT may be SEQ ID NO: 73. In some embodiments, the DNMT may be at least 99%, 98%, 97%, 96%, 95%, 90%, 85% or 80% similar to SEQ ID NO: 73.

[0100]

[0108] In some embodiments, the DNMT may include SEQ ID NO: 74. In some embodiments, the DNMT may be SEQ ID NO: 74. In some embodiments, the DNMT may be at least 99%, 98%, 97%, 96%, 95%, 90%, 85% or 80% similar to SEQ ID NO: 74.

[0101]

[0109] In some embodiments, the DNMT may include SEQ ID NO: 75. In some embodiments, the DNMT may be SEQ ID NO: 75. In some embodiments, the DNMT may be at least 99%, 98%, 97%, 96%, 95%, 90%, 85% or 80% similar to SEQ ID NO: 75.

[0102]

[0110] In some embodiments, the DNMT may include SEQ ID NO: 76. In some embodiments, the DNMT may be SEQ ID NO: 76. In some embodiments, the DNMT may be at least 99%, 98%, 97%, 96%, 95%, 90%, 85% or 80% similar to SEQ ID NO: 76.

[0103]

[0111] In some embodiments, the DNMT may include SEQ ID NO: 77. In some embodiments, the DNMT may be SEQ ID NO: 77. In some embodiments, the DNMT may be at least 99%, 98%, 97%, 96%, 95%, 90%, 85% or 80% similar to SEQ ID NO: 77.

[0104]

[0112] In some embodiments, the DNMT may include SEQ ID NO: 78. In some embodiments, the DNMT may be SEQ ID NO: 78. In some embodiments, the DNMT may be at least 99%, 98%, 97%, 96%, 95%, 90%, 85% or 80% similar to SEQ ID NO: 78.

[0105]

[0113] In some embodiments, the DNMT may include SEQ ID NO: 79. In some embodiments, the DNMT may be SEQ ID NO: 79. In some embodiments, the DNMT may be at least 99%, 98%, 97%, 96%, 95%, 90%, 85% or 80% similar to SEQ ID NO: 79.

[0106]

[0114] In some embodiments, the DNMT may include SEQ ID NO: 80. In some embodiments, the DNMT may be SEQ ID NO: 80. In some embodiments, the DNMT may be at least 99%, 98%, 97%, 96%, 95%, 90%, 85% or 80% similar to SEQ ID NO: 80.

[0107]

[0115] In some embodiments, the DNMT may include SEQ ID NO: 101. In some embodiments, the DNMT may be SEQ ID NO: 101. In some embodiments, the DNMT may be at least 99%, 98%, 97%, 96%, 95%, 90%, 85% or 80% similar to SEQ ID NO: 101.

[0108]

[0116] In some embodiments, the DNMT may include SEQ ID NO: 102. In some embodiments, the DNMT may be SEQ ID NO: 102. In some embodiments, the DNMT may be at least 99%, 98%, 97%, 96%, 95%, 90%, 85% or 80% similar to SEQ ID NO: 102.

[0109]

[0117] In some embodiments, the DNMT may include SEQ ID NO: 103. In some embodiments, the DNMT may be SEQ ID NO: 103. In some embodiments, the DNMT may be at least 99%, 98%, 97%, 96%, 95%, 90%, 85% or 80% similar to SEQ ID NO: 103.

[0110]

[0118] In some embodiments, the DNMT may include SEQ ID NO: 104. In some embodiments, the DNMT may be SEQ ID NO: 104. In some embodiments, the DNMT may be at least 99%, 98%, 97%, 96%, 95%, 90%, 85% or 80% similar to SEQ ID NO: 104.

[0111]

[0119] In some embodiments, the DNMT may include SEQ ID NO: 105. In some embodiments, the DNMT may be SEQ ID NO: 105. In some embodiments, the DNMT may be at least 99%, 98%, 97%, 96%, 95%, 90%, 85% or 80% similar to SEQ ID NO: 105.

[0112]

[0120] In some embodiments, the DNMT may include SEQ ID NO: 106. In some embodiments, the DNMT may be SEQ ID NO: 106. In some embodiments, the DNMT may be at least 99%, 98%, 97%, 96%, 95%, 90%, 85% or 80% similar to SEQ ID NO: 106.

[0113]

[0121] In some embodiments, the DNMT may include SEQ ID NO: 107. In some embodiments, the DNMT may be SEQ ID NO: 107. In some embodiments, the DNMT may be at least 99%, 98%, 97%, 96%, 95%, 90%, 85% or 80% similar to SEQ ID NO: 107.

[0114]

[0122] In some embodiments, the DNMT may include SEQ ID NO: 108. In some embodiments, the DNMT may be SEQ ID NO: 108. In some embodiments, the DNMT may be at least 99%, 98%, 97%, 96%, 95%, 90%, 85% or 80% similar to SEQ ID NO: 108.

[0115]

[0123] In some embodiments, the DNMT may include SEQ ID NO: 109. In some embodiments, the DNMT may be SEQ ID NO: 109. In some embodiments, the DNMT may be at least 99%, 98%, 97%, 96%, 95%, 90%, 85% or 80% similar to SEQ ID NO: 109.

[0116]

[0124] In some embodiments, the DNMT (e.g., DNMT3) may include an ADD domain. The ADD domain may have the sequence of SEQ ID NO: 98. The ADD domain may inhibit DNMT activity in a specific genomic region (e.g., a region having a specific histone signature).

[0117] Repressor domain

[0125] 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 includes 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 (e.g., a KRAB domain derived from KOX1 (aka ZNF10), ZIM3 (aka ZNF657 or ZNF264), or ZN627). In some embodiments, the effector domain includes a KRAB domain. In some embodiments, the effector domain includes tripartite motif-containing protein 28 (TRIM28, TIF1-beta or KAP1).

[0118]

[0126] In some embodiments, the effector domain includes a protein domain that suppresses the expression of the target gene, which is also referred to herein as a "functional repression domain", a "repression domain", or a "repressor domain". For example, the effector domain may include a functional repression domain derived from a zinc finger repressor protein.

[0119]

[0127] In some embodiments, the effector domain comprises a KOX1 / ZNF10 repression domain, a KOX8 / ZNF708 repression domain, a ZNF43 repression domain, a ZNF184 repression domain, a ZNF91 repression domain, an HPF4 repression domain, an HTF10 repression domain, HTF34, or any combination thereof. In some embodiments, the effector domain comprises a ZIM3 repression domain, a ZNF436 repression domain, a ZNF257 repression domain, a ZNF675 repression domain, a ZNF490 repression domain, a ZNF320 repression domain, a ZNF331 repression domain, a ZNF816 repression domain, a ZNF680 repression domain, a ZNF41 repression domain, a ZNF189 repression domain, a ZNF528 repression domain, a ZNF543 repression domain, a ZNF554 repression domain, a ZNF140 repression domain, a ZNF610 repression domain, a ZNF264 repression domain, a ZNF350 repression domain, a ZNF8 repression domain, a ZNF582 repression domain, a ZNF30 repression domain, a ZNF324 repression domain, a ZNF98 repression domain, a ZNF669 repression domain, a ZNF677 repression domain, a ZNF596 repression domain, a ZNF214 repression domain, a ZNF37A repression domain, a ZNF34 repression domain, a ZNF250 repression domain, a ZNF547 repression domain, a ZNF273 repression domain, a ZNF354A repression domain, a ZFP82 repression domain, a ZNF224 repression domain, a ZNF33A repression domain, a ZNF45 repression domain, a ZNF175 repression domain, a ZNF595 repression domain, a ZNF184 repression domain, a ZNF419 repression domain, a ZFP28-1 repression domain, a ZFP28-2 repression domain, a ZNF18 repression domain, a ZNF213 repression domain, a ZNF394 repression domain, a ZFP1 repression domain, a ZFP14 repression domain, a ZNF416 repression domain, a ZNF557 repression domain, a ZNF566 repression domain, a ZNF729 repression domain, a ZIM2 repression domain, a ZNF254 repression domain, a ZNF764 repression domain, a ZNF785 repression domain, or any combination thereof.In some embodiments, the effector domain comprises a ZIM3 inhibitory domain, a ZNF554 inhibitory domain, a ZNF264 inhibitory domain, a ZNF324 inhibitory domain, a ZNF354A inhibitory domain, a ZNF189 inhibitory domain, a ZNF543 inhibitory domain, ZFP82, ZNF669, a ZNF582 inhibitory domain, or any combination thereof. In some embodiments, the effector domain comprises a ZIM3 inhibitory domain, a ZNF554 inhibitory domain, a ZNF264 inhibitory domain, a ZNF324 inhibitory domain, a ZNF354A inhibitory domain, or any combination thereof. In some embodiments, the effector domain is a ZIM3 inhibitory domain.

[0120]

[0128] In some embodiments, the repression domain is a KRAB domain. In some embodiments, the effector domain comprises or is derived from a functional repression domain comprising the KOX1 / ZNF10 KRAB domain, the KOX8 / ZNF708 KRAB domain, the ZNF43 KRAB domain, the ZNF184 KRAB domain, the ZNF91 KRAB domain, the HPF4 KRAB domain, the HTF10 KRAB domain, or the HTF34 KRAB domain, or any combination thereof.In some embodiments, the effector domain comprises a functional inhibitory domain derived from a ZIM3 KRAB domain, a ZNF436 KRAB domain, a ZNF257 KRAB domain, a ZNF675 KRAB domain, a ZNF490 KRAB domain, a ZNF320 KRAB domain, a ZNF331 KRAB domain, a ZNF816 KRAB domain, a ZNF680 KRAB domain, a ZNF41 KRAB domain, a ZNF189 KRAB domain, a ZNF528 KRAB domain, a ZNF543 KRAB domain, a ZNF554 KRAB domain, a ZNF140 KRAB domain, a ZNF610 KRAB domain, a ZNF264 KRAB domain, a ZNF350 KRAB domain, a ZNF8 KRAB domain, a ZNF582 KRAB domain, a ZNF30 KRAB domain, a ZNF324 KRAB domain, a ZNF98 KRAB domain, a ZNF669 KRAB domain, a ZNF677 KRAB domain, a ZNF596 KRAB domain, a ZNF214 KRAB domain, a ZNF37A KRAB domain, a ZNF34 KRAB domain, a ZNF250 KRAB domain, a ZNF547 KRAB domain, a ZNF273 KRAB domain, a ZNF354A KRAB domain, a ZFP82 KRAB domain, a ZNF224 KRAB domain, a ZNF33A KRAB domain, a ZNF45 KRAB domain, a ZNF175 KRAB domain, a ZNF595 KRAB domain, a ZNF184 KRAB domain, a ZNF419 KRAB domain, a ZFP28-1 KRAB domain, a ZFP28-2 KRAB domain, a ZNF18 KRAB domain, a ZNF213 KRAB domain, a ZNF394 KRAB domain, a ZFP1 KRAB domain, a ZFP14 KRAB domain, a ZNF416 KRAB domain, a ZNF557 KRAB domain, a ZNF566 KRAB domain, a ZNF729 KRAB domain, a ZIM2 KRAB domain, a ZNF254 KRAB domain, a ZNF764 KRAB domain, a ZNF785 KRAB domain, or any combination thereof.In some embodiments, the domain is the ZIM3 KRAB domain, the ZNF554 KRAB domain, the ZNF264 KRAB domain, the ZNF324 KRAB domain, the ZNF354A KRAB domain, the ZNF189 KRAB domain, the ZNF543 KRAB domain, the ZFP82 KRAB domain, the ZNF669 KRAB domain, or the ZNF582 KRAB domain, or any combination thereof. In some embodiments, the domain is the ZIM3 KRAB domain, the ZNF554 KRAB domain, the ZNF264 KRAB domain, the ZNF324 KRAB domain, or the ZNF354A KRAB domain, or any combination thereof. In some embodiments, the domain is the ZIM3 KRAB domain.

[0121]

[0129] Exemplary functional inhibitory domain sequences that reduce or silence target gene expression are presented in Table 2 below. In some embodiments, the epigenetic editing system herein includes one or more inhibitory domains selected from Table 2, or functional homologs, orthologs or variants thereof. Further examples of suitable repressors and repressor domains can be found in PCT / US2021 / 030643; and Tycko et al., High-Throughput Discovery and Characterization of Human Transcriptional Effectors., Cell, December 23, 2020;183(7):2020 - 2035, each of which is incorporated herein by reference in its entirety.

[0122]

Table 2

[0123]

[0130] In some embodiments, the effector domain may include SEQ ID NO: 9. In some embodiments, the DNMT may be SEQ ID NO: 9. In some embodiments, the DNMT may be at least 99%, 98%, 97%, 96%, 95%, 90%, 85% or 80% similar to SEQ ID NO: 9.

[0124]

[0131] In some embodiments, the effector domain may include SEQ ID NO: 10. In some embodiments, the DNMT may be SEQ ID NO: 10. In some embodiments, the DNMT may be at least 99%, 98%, 97%, 96%, 95%, 90%, 85% or 80% similar to SEQ ID NO: 10.

[0125]

[0132] In some embodiments, the effector domain may include SEQ ID NO: 11. In some embodiments, the DNMT may be SEQ ID NO: 11. In some embodiments, the DNMT may be at least 99%, 98%, 97%, 96%, 95%, 90%, 85% or 80% similar to SEQ ID NO: 11.

[0126]

[0133] In some embodiments, the effector domain may include SEQ ID NO: 100. In some embodiments, the DNMT may be SEQ ID NO: 100. In some embodiments, the DNMT may be at least 99%, 98%, 97%, 96%, 95%, 90%, 85% or 80% similar to SEQ ID NO: 100.

[0127]

[0134] In some embodiments, the effector domain may include SEQ ID NO: 44. In some embodiments, the DNMT may be SEQ ID NO: 44. In some embodiments, the DNMT may be at least 99%, 98%, 97%, 96%, 95%, 90%, 85% or 80% similar to SEQ ID NO: 44.

[0128]

[0135] In some embodiments, the effector domain may comprise SEQ ID NO: 45. In some embodiments, the DNMT may be SEQ ID NO: 45. In some embodiments, the DNMT may be at least 99%, 98%, 97%, 96%, 95%, 90%, 85% or 80% similar to SEQ ID NO: 45.

[0129]

[0136] In some embodiments, the effector domain may comprise SEQ ID NO: 46. In some embodiments, the DNMT may be SEQ ID NO: 46. In some embodiments, the DNMT may be at least 99%, 98%, 97%, 96%, 95%, 90%, 85% or 80% similar to SEQ ID NO: 46.

[0130]

[0137] In some embodiments, the effector domain may comprise SEQ ID NO: 47. In some embodiments, the DNMT may be SEQ ID NO: 47. In some embodiments, the DNMT may be at least 99%, 98%, 97%, 96%, 95%, 90%, 85% or 80% similar to SEQ ID NO: 47.

[0131]

[0138] In some embodiments, the effector domain may comprise SEQ ID NO: 48. In some embodiments, the DNMT may be SEQ ID NO: 48. In some embodiments, the DNMT may be at least 99%, 98%, 97%, 96%, 95%, 90%, 85% or 80% similar to SEQ ID NO: 48.

[0132]

[0139] In some embodiments, the effector domain may comprise SEQ ID NO: 49. In some embodiments, the DNMT may be SEQ ID NO: 49. In some embodiments, the DNMT may be at least 99%, 98%, 97%, 96%, 95%, 90%, 85% or 80% similar to SEQ ID NO: 49.

[0133]

[0140] In some embodiments, the effector domain may comprise SEQ ID NO: 50. In some embodiments, the DNMT may be SEQ ID NO: 50. In some embodiments, the DNMT may be at least 99%, 98%, 97%, 96%, 95%, 90%, 85% or 80% similar to SEQ ID NO: 50.

[0134]

[0141] In some embodiments, the effector domain may comprise SEQ ID NO: 51. In some embodiments, the DNMT may be SEQ ID NO: 51. In some embodiments, the DNMT may be at least 99%, 98%, 97%, 96%, 95%, 90%, 85% or 80% similar to SEQ ID NO: 51.

[0135]

[0142] In some embodiments, the effector domain may comprise SEQ ID NO: 52. In some embodiments, the DNMT may be SEQ ID NO: 52. In some embodiments, the DNMT may be at least 99%, 98%, 97%, 96%, 95%, 90%, 85% or 80% similar to SEQ ID NO: 52.

[0136]

[0143] In some embodiments, the effector domain may comprise SEQ ID NO: 53. In some embodiments, the DNMT may be SEQ ID NO: 53. In some embodiments, the DNMT may be at least 99%, 98%, 97%, 96%, 95%, 90%, 85% or 80% similar to SEQ ID NO: 53.

[0137]

[0144] In some embodiments, the effector domain may comprise SEQ ID NO: 54. In some embodiments, the DNMT may be SEQ ID NO: 54. In some embodiments, the DNMT may be at least 99%, 98%, 97%, 96%, 95%, 90%, 85% or 80% similar to SEQ ID NO: 54.

[0138]

[0145] In some embodiments, the effector domain may include SEQ ID NO: 55. In some embodiments, the DNMT may be SEQ ID NO: 55. In some embodiments, the DNMT may be at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% similar to SEQ ID NO: 55.

[0139]

[0146] In some embodiments, the effector domain may include SEQ ID NO: 56. In some embodiments, the DNMT may be SEQ ID NO: 56. In some embodiments, the DNMT may be at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% similar to SEQ ID NO: 56.

[0140]

[0147] In some embodiments, the effector domain may include SEQ ID NO: 57. In some embodiments, the DNMT may be SEQ ID NO: 57. In some embodiments, the DNMT may be at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% similar to SEQ ID NO: 57.

[0141]

[0148] In some embodiments, the effector domain may include SEQ ID NO: 94. In some embodiments, the DNMT may be SEQ ID NO: 94. In some embodiments, the DNMT may be at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% similar to SEQ ID NO: 94.

[0142]

[0149] In some embodiments, the effector domain includes a fusion of two effector domains (e.g., KOX1KRAB and ZIM3). In some embodiments, the effector domain includes a fusion of two, three, four, five, six, seven, eight, nine, or ten effector domains. In some embodiments, the effector domain includes a fusion of a truncated form of an effector domain and a second effector domain. In some embodiments, the effector domain includes a fusion of truncated forms of two effector domains. In some embodiments, the fusion effector domain includes at least one truncated form of an effector domain.

[0143]

[0150] 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 those that suppress or upregulate gene expression, can be identified from larger proteins using known methods and the methods provided herein. For example, functional effector domains that can reduce or silence target gene expression can be identified based on the sequences of repressor or activator proteins. The amino acid sequences of proteins having a function of regulating gene expression can be obtained from available genome browsers, such as the UCSD Genome Browser or the Ensembl Genome Browser.

[0144] Linker

[0151] The epigenetic editing systems 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.

[0145]

[0152] In certain embodiments, the 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 of many atoms in length. In certain embodiments, the linker is a polypeptide or amino acid-based. In other embodiments, the linker is not peptidomimetic. 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 a 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 attachment of a nucleophile (e.g., thiol, amino) from a peptide to the linker. Any electrophile can be used as part of the linker. Exemplary electrophiles include, but are not limited to, activated esters, activated amides, Michael acceptors, alkyl halides, aryl halides, acyl halides and isothiocyanates.

[0146]

[0153] 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.

[0147]

[0154] In certain embodiments, the linker is a polymer (e.g., polyethylene, polyethylene glycol, polyamide, polyester, etc.). In certain embodiments, the linker includes monomers, dimers, or polymers of aminoalkanoic acids. In certain embodiments, the linker includes 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 includes 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 includes a polyethylene glycol moiety (PEG). In other embodiments, the linker includes an amino acid. In certain embodiments, the linker includes a peptide. In certain embodiments, the linker includes 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 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.

[0148]

[0155] 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 between 1 and 200 amino acids. In some embodiments, a DNA binding domain, such as a zinc finger array, and an effector domain are fused via a linker comprising from 1 to 5, 1 to 10, 1 to 20, 1 to 30, 1 to 40, 1 to 50, 1 to 60, 1 to 80, 1 to 100, 1 to 150, 1 to 200, 5 to 10, 5 to 20, 5 to 30, 5 to 40, 5 to 60, 5 to 80, 5 to 100, 5 to 150, 5 to 200, 10 to 20, 10 to 30, 10 to 40, 10 to 50, 10 to 60, 10 to 80, 10 to 100, 10 to 150, 10 to 200, 20 to 30, 20 to 40, 20 to 50, 20 to 60, 20 to 80, 20 to 100, 20 to 150, 20 to 200, 30 to 40, 30 to 50, 30 to 60, 30 to 80, 30 to 100, 30 to 150, 30 to 200, 40 to 50, 40 to 60, 40 to 80, 40 to 100, 40 to 150, 40 to 200, 50 to 60, 50 to 80, 50 to 100, 50 to 150, 50 to 200, 60 to 80, 60 to 100, 60 to 150, 60 to 200, 80 to 100, 80 to 150, 80 to 200, 100 to 150, 100 to 200, or 150 to 200 amino acids in length. 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.

[0149]

[0156] 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.

[0150]

[0157] In some embodiments, the peptide linker comprises the amino acid sequences of SEQ ID NOs: 1-5.

[0158] 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.

[0151]

[0159] 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 utilized (e.g., ranging from very flexible linkers such as glycine / serine-rich linkers to more rigid linkers to achieve an optimal length for effector domain activity for a particular application). In some embodiments, a flexible linker is a glycine / serine-rich linker (GS-rich linker) in which more than 45% (e.g., more than 48, 50, 55, 60, 70, 80, or 90%) of its residues are glycine residues or serine residues. Non-limiting examples of GS-rich linkers are (GGGGS)n (SEQ ID NO: 4) and (G)n. In some embodiments, more rigid linkers include (EAAAK)n, (SGGS)n, and (XP)n. In the above formulas for flexible linkers and rigid linkers, n is any integer between 1 and 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 includes a (GGS)n motif, where n is 1, 3, or 7. In some embodiments, the linker includes a (GGGGS)n motif, where n is 4 (SEQ ID NO: 4).

[0152]

[0160] In some embodiments, the linker in the epigenetic editing system includes, for example, a nuclear localization signal of peptide sequence SEQ ID NO: 7. In some embodiments, the linker in the epigenetic editing system includes an expression tag, such as a detectable tag like green fluorescent protein.

[0153]

[0161] 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 may 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 may 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.

[0154]

[0162] 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 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.

[0155]

[0163] In some embodiments, the linker comprises a repeat peptide array. In some embodiments, the linker comprises an epitope tag, such as 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, such as 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 to 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.

[0156] Nuclear localization sequence

[0164] In some embodiments, the epigenetic editing system provided herein includes one or more nuclear targeting arrays. In some embodiments, the epigenetic editing system provided herein includes at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more nuclear targeting arrays. For example, the zinc finger-repressor fusion protein 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 translocation 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.

[0157]

[0165] In some embodiments, the epigenetic editing system provided herein includes two NLS sequences. In some embodiments, the fusion protein of the epigenetic editing system includes one NLS at the N-terminus and one NLS at the C-terminus. In some embodiments, the fusion protein includes two NLSs at the N-terminus. In some embodiments, the fusion protein includes two NLSs at the C-terminus. In some embodiments, one NLS is located at the N-terminus and one NLS is embedded in the middle of the fusion protein. In some embodiments, one NLS is located at the C-terminus and one NLS is embedded in the middle of the fusion protein. In some embodiments, both NLSs are embedded in the middle of the fusion protein.

[0158]

[0166] In some embodiments, the fusion protein of the epigenetic editing system includes two NLS sequences adjacent to the DNMT domain. In some embodiments, the fusion protein includes two NLS sequences adjacent to the fused DNMT domain. In some embodiments, the fusion protein includes two NLS sequences adjacent to the DNA binding domain. In some embodiments, the fusion protein includes two NLS sequences adjacent to the effector domain.

[0159]

[0167] In some embodiments, the epigenetic editing system provided herein includes four NLS sequences. In some embodiments, the fusion protein of the epigenetic editing system includes at least two NLSs at the N-terminus. In some embodiments, the fusion protein of the epigenetic editing system includes at least two NLSs at the C-terminus. In some embodiments, the fusion protein of the epigenetic editing system includes two NLSs at the N-terminus and two NLSs at the C-terminus. In some embodiments, at least one NLS is embedded in the middle of the fusion protein.

[0160]

[0168] In some embodiments, the NLS comprises the amino acid sequence of SEQ ID NO: 7. In some embodiments, the NLS sequence is an endogenous NLS sequence. Additional nuclear localization sequences are known in the art and will be apparent to those of skill in the art. Examples of suitable NLS sequences for inclusion in the fusion proteins provided herein include, but are not limited to, those described in Lu et al., Types of nuclear localization signals and mechanisms of protein import into the nucleus, Cell Commun Signal. 2021; 19:60, 2021, the entire contents of which are incorporated herein by reference.

[0161]

[0169] In some embodiments, a fusion protein comprising two NLSs at the N-terminus and two NLSs at the C-terminus can increase the efficiency of an epigenetic editor system by at least 5%, at least 10%, at least 15%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 100%, at least 200%, at least 300%, at least 400%, at least 500%, at least 600%, at least 700%, at least 800%, at least 900%, at least 1,000%, at least 5,000%, at least 10,000%, at least 50,000%, at least 100,000% or more compared to an epigenetic editor system that does not include two NLSs at the N-terminus and two NLSs at the C-terminus. In some embodiments, a fusion protein comprising two NLSs at the N-terminus and two NLSs at the C-terminus can increase the efficiency of an epigenetic editor system by up to 100,000%, up to 50,000%, up to 10,000%, up to 5,000%, up to 1,000%, up to 900%, up to 800%, up to 700%, up to 600%, up to 500%, up to 400%, up to 300%, up to 200%, up to 100%, up to 90%, up to 80%, up to 70%, up to 60%, up to 50%, up to 40%, up to 30%, up to 20%, up to 15%, up to 10%, up to 5% or less compared to an epigenetic editor system that does not include two NLSs at the N-terminus and two NLSs at the C-terminus.

[0162] Tag

[0170] The epigenetic editing systems provided herein may include one or more additional sequences, domains, tags to track, detect, and localize 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.

[0163]

[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 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, but are not limited thereto. Further suitable sequences will be apparent to those skilled in the art.

[0164]

[0172] In some embodiments, the epigenetic editing system includes 1 to 2 detectable tags. In an aspect, the fusion protein includes 1 detectable tag. In an aspect, the fusion protein includes 2 detectable tags. In an aspect, the fusion protein includes 3 detectable tags. In an aspect, the fusion protein includes 4 detectable tags. In an aspect, the fusion protein includes 5 detectable tags.

[0165]

[0173] Epigenetic editing system structure

[0174] Some aspects of the present disclosure provide an epigenetic editing system. Exemplarily, a non-limiting structure of such an editing system is described in more detail herein. Further suitable structures will be apparent to those skilled in the art based on the present disclosure, which is not limited in that regard.

[0166]

[0175] 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. In this structural example, N’ represents the N-terminus, C’ represents the C-terminus, and ]-[ represents a linking element or a linker.

[0167]

[0176] 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.

[0168]

[0177] 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.

[0169]

[0178] 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.

[0170]

[0179] 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 KRAB 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.

[0171]

[0180] The components of an epigenetic editing system can be structured in different configurations. For example, the 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 at both ends. In some embodiments, the epigenetic editing system has the configuration of N’]-[epigenetic effector domain 1]-[DNA binding domain]-[epigenetic effector domain 2]-[C’. In some embodiments, the epigenetic editing system has the 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 has the 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 has the 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 has the configuration of N’]-[KRAB]-[DNA binding domain]-[Dnmt3A]-[C’. In some embodiments, the epigenetic editing system has the configuration of N’]-[KRAB]-[DNA binding domain]-[Dnmt3A]-[Dnmt3L]-[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.

[0172]

[0181] 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, or each may be a different linker, tag, NLS or peptide bond.

[0173]

[0182] 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 purposes 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.

[0174]

[0183] 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’]-[suppression 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]-[suppression 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.

[0175]

[0184] In some embodiments, the epigenetic editing system comprises a DNA binding domain (DBD) and a DNA methyltransferase domain that suppresses or silences the expression of a target gene by placing one or more methylation marks on the target gene. 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.

[0176]

[0185] 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.

[0177]

[0186] 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.

[0178]

[0187] 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 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 suppression or silencing of 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. In some embodiments, the repressor domain includes any one of the domains in Table 2 or Table 3, or any combination or homolog thereof.

[0179]

[0188] The DNA methyltransferase domain in an epigenetic editing system may include any one of the DNA methyltransferase proteins described herein and known to those of ordinary skill in the art, or any homolog or combination thereof. In some embodiments, the effector domain includes a DNMT3 domain. In some embodiments, the DNA methyltransferase domain includes a DNMT3A domain. In some embodiments, the DNA methyltransferase domain includes a DNMT3B domain. In some embodiments, the DNA methyltransferase domain includes a DNMT3C domain. In some embodiments, the DNA methyltransferase domain includes a DNMT3L domain. In some embodiments, the DNA methyltransferase domain includes a fusion of the DNMT3A-DNMT3L domains. The DNMT3A-DNMT3L fusion domain described herein may 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.

[0180]

[0189] In some embodiments, the epigenetic editing system includes a DNA binding domain (DBD) and an effector domain that increases 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.

[0181]

[0190] 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 has the configuration of N’]-[DNA demethylase domain]-[DBD]-[-C’, where 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 has the configuration of N’]-[DBD]-[DNA demethylase domain]-[-C’, where 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.

[0182]

[0191] 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 has the configuration of N’]-[DNA demethylase domain]-[DBD]-[activation domain]-[-C’, where 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 has the configuration of N’]-[activation domain]-[DBD]-[DNA demethylase domain]-[-C’, where 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.

[0183]

[0192] 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.

[0184]

[0193] 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.

[0185]

[0194] 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 can 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.

[0186]

[0195] 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 against a DNA methyltransferase domain, and an antibody against 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, a MECP2 affinity domain, and a Dnmt3A affinity domain. In some embodiments, the epigenetic editing system includes a DBD, a 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.

[0187]

[0196] In some embodiments, the DNA methyltransferase may comprise any one of the DNMT domains provided herein, or any combination or homolog thereof. In certain embodiments, the DNA methyltransferase domain comprises DNMT3A or a truncated version thereof, DNMT3L or a truncated version thereof, or both. In certain embodiments, the DBD is a catalytically inactive polynucleotide-induced DNA binding domain (e.g., dCas9) or a ZFP domain. In certain embodiments, the repressor domain comprises any one of the repressor domains provided herein, or any combination or homolog thereof. For example, in some embodiments, the repressor domain can be a KRAB domain. In certain embodiments, the repressor domain is a ZFP28, ZN627, KAP1, MeCP2, HP1b, CBX8, CDYL2, TOX, Tox3, Tox4, EED, RBBP4, RCOR1, or SCML2 KRAB domain, or a fusion of two of the foregoing domains (e.g., a fusion of the N-terminal and C-terminal regions of the ZIM3 KRAB domain and the KOX1 KRAB domain). In certain embodiments, the repressor domain is a KRAB domain derived from ZFP28, ZF627, ZIM3 or KOX1.

[0188]

[0197] Specific constructs contemplated herein include the following.

[0198] DNMT3A-DNMT3L-XTEN80-NLS-dCas9-NLS-XTEN16-KOX1 KRAB (Construct 1),

[0199] DNMT3A-DNMT3L-XTEN80-NLS-ZFP domain-NLS-XTEN16-KOX1 KRAB (Construct 2),

[0200] NLS-DNMT3A-DNMT3L-XTEN80-dCas9-XTEN16-KOX1 KRAB-NLS (Construct 3),

[0201] NLS-DNMT3A-DNMT3L-XTEN80-ZFP domain-XTEN16-KOX1 KRAB-NLS (Construct 4),

[0202] NLS-NLS-DNMT3A-DNMT3L-XTEN80-dCas9-XTEN16-KOX1 KRAB-NLS-NLS (Configuration 5), and

[0203] NLS-NLS-DNMT3A-DNMT3L-XTEN80-ZFP domain-XTEN16-KOX1 KRAB-NLS-NLS (Configuration 6)

[0204] In certain embodiments, the fusion construct may have the following configurations.

[0189]

[0205] NLS-NLS-hDNMT3A-hDNMT3L-XTEN80-dCas9-XTEN16-KOX1 KRAB-NLS-NLS (Configuration 7),

[0206] NLS-NLS-DNMT3A-DNMT3L-XTEN80-ZFP domain-XTEN16-KOX1 KRAB-NLS-NLS (Configuration 8),

[0207] NLS-NLS-hDNMT3A-hDNMT3L-XTEN80-dCas9-XTEN16-ZFP28 KRAB-NLS-NLS (Configuration 9),

[0208] NLS-NLS-DNMT3A-DNMT3L-XTEN80-ZFP domain-XTEN16-ZFP28 KRAB-NLS-NLS (Configuration 10),

[0209] NLS-NLS-hDNMT3A-hDNMT3L-XTEN80-dCas9-XTEN16-ZN627 KRAB-NLS-NLS (Configuration 11), or

[0210] NLS-NLS-DNMT3A-DNMT3L-XTEN80-ZFP domain-XTEN16-ZN627 KRAB-NLS-NLS (Configuration 12)

[0211] NLS-NLS-DNMT3A-hDNMT3L-XTEN80-dCas9-NLS-XTEN16-ZIM3 KRAB-NLS-NLS (Configuration 13).

[0190]

[0212] NLS-NLS-DNMT3A-hDNMT3L-XTEN80-dCas9-NLS-XTEN16-ZN627 KRAB-NLS-NLS (Constitution 14).

[0213] NLS-NLS-DNMT3A-hDNMT3L-XTEN80-dCas9-NLS-XTEN16-KOX1 KRAB-NLS-NLS (Constitution 15).

[0191]

[0214] NLS-DNMT3A-hDNMT3L-XTEN80-dCas9-NLS-XTEN16-KOX1-KRAB (Constitution 16). DNA binding domain

[0215] The epigenetic editing systems and epigenetic editing complexes described herein may include one or more nucleic acid binding protein domains, such as DNA binding domains, that can direct the epigenetic editing system to a target gene associated with specific conditions.

[0192]

[0216] The target gene as used herein 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.

[0193]

[0217] The epigenetic editing systems described herein 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.

[0194]

[0218] The nucleic acid binding domain of the epigenetic editing system described herein can 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 a copy of one or both of the wild-type DNA sequences.

[0195]

[0219] The DNA binding domain can be modular and / or programmable. In some embodiments, the DNA binding domain includes a zinc finger domain, a transcription activator-like effector (TALE) domain, a meganuclease DNA binding domain, or a polynucleotide-induced 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.

[0196]

[0220] 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.

[0197]

[0221] 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 may 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.

[0198]

[0222] In some embodiments, the epigenetic editing system provided herein comprises a Cas protein, such as 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 with one or more of the effector protein domains described herein. In some embodiments, any of the Cas proteins provided herein can be fused with two or more 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 the Cas9 protein that can be wild-type or contain amino acid changes, such as deletions, insertions, substitutions, variants, mutations, fusions, chimeras, or any combination thereof.

[0199]

[0223] 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 species, 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 species, Crocosphaera watsonii, Cyanothece species, Microcystis aeruginosa, Synechococcus species, 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, Oscillator 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.

[0200]

[0224] 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 the organisms and loci disclosed in Chylinski et al. (2013) RNA Biology 10:5, pages 726-737, which are incorporated herein by reference.

[0201]

[0225] Epigenetic editing systems 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 its 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.

[0202]

[0226] 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.

[0203]

[0227] In some embodiments, the epigenetic editing system includes a high-fidelity Cas9 domain. For example, a high-fidelity Cas9 domain containing 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, e.g., 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.

[0204]

[0228] 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 non-standard PAM sequences are described in the art and will be apparent to those of skill in the art. For example, Cas9 domains that bind 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.

[0205]

[0229] In some embodiments, the Cas9 domain is the Cas9 domain from S. pyogenes (SpCas9). In some embodiments, SpCas9 recognizes the standard NGG PAM sequence, provided that "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, provided that 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, provided that 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, provided that 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, provided that N is any one of the nucleotides A, G, C or T, R is nucleotide A or G, and Y is nucleotide C or T.

[0206]

[0230] 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, where 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, where N is any one of the nucleotides A, G, C, or T.

[0207]

[0231] In some embodiments, any Cas9 domain 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.

[0208]

[0232] In some embodiments, the epigenetic editing systems provided herein comprise 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 the alpha-helical recognition lobe of Cas9.

[0209]

[0233] 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 Cpf1s from other bacterial species can also be used in accordance with the present disclosure.

[0210]

[0234] In some embodiments, Cpf1 is the Cpf1 protein from Lachnospiraceae bacterium (LbCpf1). LbCpf1 can 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 species (AsCpf1). AsCpf1 can 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.

[0211]

[0235] 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.

[0212]

[0236] 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 can be inactivated in terms of their nuclease activity. In some embodiments, the epigenetic editing system includes a Cas protein derived from a type II, type II-A, type II-B, type II-C, type V or type 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.

[0213]

[0237] 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 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 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 reduced or abolished nuclease activity (dCasφ).

[0214]

[0238] 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 Cas protein of interest). 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, 1-499 following amino acid numbers 500-1000 from the N-terminus to the C-terminus, 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 the amino acids will have the same local order in the specific region of the circular permutant, although the overall amino acid primary sequence has changed.

[0215]

[0239] 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.

[0216]

[0240] 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

[0241] 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.

[0217]

[0242] 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.

[0218]

[0243] 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 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 each incorporated herein by reference in their entirety.

[0219]

[0244] 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, such as a peptide linker or a chemical linker.

[0220]

[0245] 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, for example, 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.

[0221]

[0246] 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 different optimal targeting array lengths. Thus, the spacer sequence can include nucleotides that are 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, or more than 50 nucleotides in length. In some embodiments, the spacer includes 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 have 100% complementarity.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.

[0222]

[0247] In some embodiments, the target sequence is a sequence in the mammalian genome. In some embodiments, the target sequence is a sequence in the human genome. In some embodiments, the 3' end of the target sequence is immediately adjacent to the 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.

[0223]

[0248] In some embodiments, the guide RNA is a truncated form. The truncation may include any number of nucleotide deletions. For example, the truncation 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.

[0224]

[0249] The guide polynucleotide can be modified. The modifications can include chemical changes, synthetic modifications, nucleotide addition, and / or nucleotide removal. Modified nucleosides or nucleotides can be present in the gRNA. For example, the gRNA can contain 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 RNA 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 to the ribose sugar, such as changes to the 2'-hydroxyl on the ribose sugar (an exemplary sugar modification), changes to the phosphate ester moiety, modification or substitution of the natural nucleobases, substitution or modification of the ribose phosphate backbone, modification of the 3'- or 5'-terminus of the oligonucleotide, or substitution of the terminal phosphate group, or conjugation of a moiety, cap, or linker, or one or more of any combination thereof.

[0225]

[0250] In some embodiments, the ribose group (or sugar) can be modified. In some embodiments, the modified ribose can control the oligonucleotide binding affinity to the 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-constrained ethyl (S-cEt)), constrained 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.

[0226]

[0251] 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 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 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 3'-end. In some embodiments, the gRNA or sgRNA can include three phosphorothioate (PS) bonds at the 5'-end or 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 3'-end. In some embodiments, the gRNA or sgRNA can include three consecutive phosphorothioate (PS) bonds at the 5'-end or 3'-end. In some embodiments, the gRNA or sgRNA can include the sequence 5'-UsUsU-3' at the 3'-end or 5'-end, where U represents uridine and s represents a phosphorothioate (PS) bond.

[0227]

[0252] 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.

[0228]

[0253] 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

[0254] In some embodiments, the epigenetic editing system described herein includes a nucleic acid binding domain that includes a zinc finger domain.

[0229]

[0255] 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 a nucleic acid triplet or overlapping quadruplet within a double-stranded DNA target sequence. In certain embodiments, the ZF can also bind RNA and proteins.

[0230]

[0256] 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.

[0231]

[0257] 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 two invariant histidine residues with two cysteines of a single beta turn via zinc.

[0232]

[0258] In some embodiments, the amino acid sequence of a zinc finger protein, such as the Zif268 protein, can be altered 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-natural DNA recognition specificity in which a suitable 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.

[0233]

[0259] In some embodiments, a zinc finger includes a C2H2 finger. In some embodiments, a zinc finger protein includes a ZF array containing 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 modularly by a one-to-one interaction between the zinc finger and a triplet nucleotide sequence in the DNA sequence.

[0234]

[0260] In some embodiments, the epigenetic editing system comprises a zinc finger motif having 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 comprises a zinc finger motif having 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.

[0235]

[0261] In some embodiments, two or more zinc fingers are linked together in a tandem array to achieve specific recognition and binding of a continuous DNA sequence. The zinc finger or zinc finger array in the epigenetic editing system may be native or may be 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 by use of a selection method such as phage display to identify desirable variants that can bind to the target DNA site of interest.

[0236]

[0262] An engineered zinc finger binding domain can have a novel binding specificity compared to a native zinc finger protein. Zinc fingers with desired 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, e.g., 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, e.g., 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 that express a protein capable of binding 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, e.g., 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 fingers to the target site activates transcription to recruit the RNA polymerase-Gal4 fusion and enable cell survival on selective media.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.

[0237]

[0263] In some embodiments, the zinc finger is evolvable and selectable using a phage-assisted continuous evolution (PACE) system that includes a "helper phagemid" that is present in a host cell, e.g., an E. coli cell, that encodes all phage proteins except one phage protein (e.g., the g3p protein), an "accessory plasmid" that expresses the g3p protein in response to an active library member that is present in all host cells, 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. An adaptive selection phagemid encoding a library member that induces g3p expression from the accessory plasmid can be packaged into phage particles containing g3p. The g3p-containing phage particles can infect new cells and further replicate the adaptive selection phagemid, but the g3p-deficient phage particles are non-infectious, so the low fitness selection phagemids cannot proliferate. A selection system combined with a continuous flow of host cells through a lagoon that permits phagemid replication but not host cell replication 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.

[0238]

[0264] 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.

[0239]

[0265] 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 a target site. Then, three selected populations can be randomly shuffled to generate a library of multi-finger proteins, and subsequently, that 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 pre-selected zinc finger pools, each containing a mixture of fingers that target different 3-base pair sub-sites of the DNA sequence at a defined 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.

[0240]

[0266] 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. This is because the first library contains randomization at all base-contact positions of finger 1 and specific 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 phages, and a set of "one-and-a-half" fingers can be paired to yield a recombinant 3-finger DNA-binding domain.

[0241]

[0267] 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 the N-terminal and C-terminal fingers 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.

[0242]

[0268] 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.

[0243]

[0269] Thus, 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.

[0244]

[0270] 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 to form 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.

[0245]

[0271] In some embodiments, the zinc finger or zinc finger array targeting a specific DNA sequence is designed using a modular assembly approach. For example, two or more preselected zinc fingers can be fused in tandem.

[0246]

[0272] In some embodiments, the zinc finger array comprises multiple zinc fingers fused via peptide bonds. In some embodiments, the zinc finger array comprises multiple zinc fingers, one or more of which are linked by a peptide linker. For example, the zinc fingers in the multiple finger array can be linked by a peptide linker having a length of 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or more amino acids. In some embodiments, the zinc fingers in the multiple finger array are linked by a 5 - amino acid - long peptide linker. In some embodiments, the zinc fingers in the multiple finger array are linked by a 6 - amino acid - long peptide linker.

[0247]

[0273] In some embodiments, the ZF - containing protein can 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 triplets / nucleotides. 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 - contiguous nucleic acid sequences and may also bind to different molecules (e.g., proteins rather than nucleic acids).

[0248]

[0274] In some embodiments, the epigenetic editing system includes zinc fingers that include more than three fingers. In some embodiments, the epigenetic editing system includes at least six zinc fingers in the DNA binding domain. In some embodiments, the epigenetic editing system includes 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 epigenetic editing system includes zinc fingers that include 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 enable binding to DNA. Without wishing to be bound by any theory, zinc finger proteins that include multiple two-finger units or three-finger units linked by an extended linker may confer higher DNA binding specificity compared to fewer fingers, or an array where the same number of fingers are simply linked via peptide bonds. In some embodiments, the epigenetic editing system includes at least three two-finger units linked by a peptide linker, each of those two-finger units binding to one sub-site in the target DNA sequence. In some embodiments, the epigenetic editing system includes at least four two-finger units linked by a peptide linker, each of those two-finger units binding to one sub-site in the target DNA sequence. In some embodiments, the epigenetic editing system includes at least five two-finger units linked by a peptide linker, each of those two-finger units binding to one sub-site in the target DNA sequence. In some embodiments, the epigenetic editing system includes at least six, seven, eight, nine, ten or more two-finger units linked by a peptide linker, each of those two-finger units binding 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 six, seven, eight, nine, ten 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.

[0249]

[0275] In some embodiments, multiple zinc fingers, each recognizing three specific DNA nucleotides, or trinucleotide "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 subsite gaps 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 contains 5 or more amino acids. In some embodiments, the linker contains 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 containing one or more prolines.

[0250]

[0276] A zinc finger array having sequence-specific DNA binding activity can be fused to a functional effector domain, e.g., 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.

[0251]

[0277] In some embodiments, the programmable DNA-binding protein includes an Argonaute protein. An example of such a nucleic acid programmable DNA-binding protein is the Argonaute protein (NgAgo) from Natronobacterium gregoryi. NgAgo is a ssDNA-guided endonuclease. NgAgo binds to a 5'-phosphorylated ssDNA (gDNA) of -24 nucleotides, directs 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 significantly broaden 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.

[0252]

[0278] In some embodiments, the nucleic acid binding domain includes 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 includes 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.

[0253]

[0279] 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.

[0254] Target sequence

[0280] As used herein, a “target polynucleotide sequence” can be a nucleic acid sequence present in a gene of interest. The target sequence may be within the genome of a cell or may 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.

[0255]

[0281] 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.

[0282] 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.

[0256]

[0283] In some embodiments, the target polynucleotide sequence is within about 100 base pairs (bp) adjacent to the transcription start site.

[0284] 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 at, near, or within a promoter sequence. In some embodiments, the target polynucleotide sequence is at, 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.

[0257] Regulation of target gene expression

[0285] In some embodiments, the present disclosure provides an epigenetic editing system, composition, and method for epigenetic modification in a target polynucleotide in a target gene encoding a protein. In some embodiments, the epigenetic editing system effects an 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 an 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 an 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 an 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.

[0258]

[0286] In some embodiments, the target gene and / or the encoded protein is associated with a disease, disorder or pathological condition.

[0287] 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 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.

[0259]

[0288] Epigenetic modifications can be performed on a target gene in any genome of interest, e.g., a prokaryotic genome, a plant 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, 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 tracked 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, e.g., 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.

[0260]

[0289] Target genes also include plant genes whose suppression or activation results in the improvement of 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 an advantageous increase in oleic acid and a decrease in linoleic acid and linolenic acid.

[0261]

[0290] In some embodiments, the epigenetic editing system provided herein results in epigenetic modification in a gene containing a target sequence. In some embodiments, the epigenetic editing system regulates the expression of the protein encoded by that gene. In some embodiments, the epigenetic editing system decreases 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.

[0262]

[0291] 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 histone proteins associated with the target gene.

[0263]

[0292] 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.

[0264]

[0293] 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.

[0265]

[0294] 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 an in vivo subject. In some embodiments, the target polynucleotide sequence is a locus in the genomic DNA of the 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 can come into contact with 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.

[0266]

[0295] In some embodiments, the epigenetic editing system described herein measures by transcription of the 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 as 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 the 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 as 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 reduces or abolishes 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 as compared to a control cell, control tissue, or control subject.

[0267]

[0296] In some embodiments, the epigenetic editing system described herein measures by transcription of the 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 about 100%, at least about 110%, at least about 120%, at least about 130%, at least about 140%, at least about 150%, at least about 160%, at least about 170%, at least about 180%, at least about 190%, at least about 200%, at least about 250%, at least about 300%, at least about 350%, at least about 400%, at least about 450%, at least about 500% 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 the 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 6%, at least about 70%, at least about 80%, at least about 90%, at least about 95%, at least about 99%, at least about 100%, at least about 110%, at least about 120%, at least about 130%, at least about 140%, at least about 150%, at least about 160%, at least about 170%, at least about 180%, at least about 190%, at least about 200%, at least about 250%, at least about 300%, at least about 350%, at least about 400%, at least about 450%, at least about 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 a protein encoded by a 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.

[0268]

[0297] Methods for determining the expression level of a gene, for example, 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, for example, total messenger ribonucleic acid (mRNA), or the expression level of a specific gene, for example, a housekeeping gene.

[0269]

[0298] 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 encoding 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.

[0270]

[0299] 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 for 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 for 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.

[0271]

[0300] 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.

[0272]

[0301] 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.

[0273]

[0302] To determine the level of gene expression regulation by ZFP, the degree of inhibition or activation is examined in cells contacted with ZFP as compared to control cells, for example, cells without zinc finger protein or cells with 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.

[0274] Delivery

[0303] In one aspect, provided herein is a composition for regulating gene expression, comprising an epigenetic editing system provided herein that generates epigenetic modifications 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 ways known in the art. For example, in some embodiments, the epigenetic editing system is delivered into a host cell, integrated into the genome of the host cell, or for transient expression within the host cell.

[0275]

[0304] 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).

[0276]

[0305] 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 a viral vector. As used herein, the term "vector" refers to a nucleic acid molecule capable of transporting other nucleic acids linked thereto. In some examples, the vector is an expression vector and can direct the expression of nucleic acids operably linked thereto. Examples of expression vectors include plasmid vectors, 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 leukemia virus, lentivirus, human immunodeficiency virus, myeloproliferative sarcoma virus, and mammary tumor virus), and other recombinant vectors, but are not limited thereto. In some embodiments, the vector is a virus-like particle (VLP).

[0277]

[0306] 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 to a target cell. Typical 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).

[0278]

[0307] In some embodiments, the epigenetic editing system is delivered to a host cell for transient expression, for example, 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 more 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.

[0279]

[0308] In some embodiments, the nucleic acid sequence encoding the epigenetic editing system or a component thereof 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.

[0280]

[0309] The nucleic acid encoding the epigenetic editing system can be delivered directly to the cell as naked DNA or RNA, for example, by transfection or electroporation, or can be conjugated to a molecule that facilitates uptake by the target cell (e.g., N-acetylgalactosamine). Nucleic acid vectors, such as vectors, can also be used. In certain embodiments, a polynucleotide encoding the epigenetic editing system or a functional component thereof, such as mRNA, can be co-electroporated with a combination of multiple guide RNAs described herein.

[0281]

[0310] The nucleic acid vector can contain one or more sequences encoding domains of the fusion protein or epigenetic editing system described herein. The vector can also contain a sequence encoding a signal peptide (e.g., nuclear localization, nucleosome localization, or mitochondrial localization) associated with (e.g., inserted or fused to) the protein-encoding sequence. As an example, the nucleic acid vector can contain one or more nuclear localization sequences (e.g., nuclear localization sequences from SV40), and one or more effector domains, e.g., a Cas9-encoding sequence containing a repression domain.

[0282]

[0311] 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 an appropriate capsid protein of any viral vector. Thus, in some aspects, the 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), herpes viral vectors (e.g., HSV-2).

[0283]

[0312] 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 a 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, given 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.

[0284]

[0313] 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.

[0285]

[0314] 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, a chimeric AAV can include an inverted terminal repeat (ITR) that is 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 includes amino acid sequences from 2, 3, 4, 5 or more different AAV serotypes.

[0286]

[0315] 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 intracellularly. The epigenetic editing system can be packaged within an AAV viral vector according to any method known to those of skill in the art. An example of a useful method is described in McClure et al., J Vis Exp, 2001, 57:3378.

[0287]

[0316] 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.

[0288]

[0317] 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. Furthermore, 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.

[0289]

[0318] In addition to viral vectors, non-viral vectors can be used to deliver nucleic acids encoding the genome editing system according to the present disclosure. One category of non-viral nucleic acid vectors is 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 can be suitable for use as delivery vehicles in certain embodiments of the present disclosure.

[0290]

[0319] In other aspects, lipid nanoparticles (LNPs) comprising the compositions provided herein are provided herein. As used herein, "lipid nanoparticle (LNP) composition" or "nanoparticle composition" is a composition comprising one or more of the described lipids. LNP compositions are typically on the order of micrometers or less in size and can include a lipid bilayer. Nanoparticle compositions include lipid nanoparticles (LNPs), liposomes (e.g., lipid vesicles), and lipoplexes. In some embodiments, the 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.

[0291]

[0320] 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, for producing LNP, lipids can be combined in any number of molar ratios. In some embodiments, for producing LNP, polynucleotides can be combined with lipids over a wide range of molar ratios.

[0292] Method of treatment

[0321] 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, such that the disease, condition or disorder is treated or prevented as a result.

[0293]

[0322] The epigenetic modification achieved by the epigenetic editing system described herein is 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, e.g., decrease or increase, of the expression of one copy of the target gene containing the specific allele, while the other copy of the target gene does not. In some embodiments, the specific allele is associated with a disease, condition or disorder.

[0294]

[0323] In some embodiments, the epigenetic editing system decreases the expression of a target gene associated with a disease, condition or disorder.

[0324] The epigenetic editing system described herein can be administered in a therapeutically effective amount to a subject in need thereof for treating a disease, condition or disorder.

[0295]

[0325] In another aspect, provided herein is a method of treating or preventing a condition in a subject in need of treatment or prevention of the condition, the method comprising administering to the subject an epigenetic editing complex, vector, nucleic acid, protein, 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 a cell of the subject, thereby regulating the expression of a target gene and treating or preventing the condition.

[0296]

[0326] In some embodiments, the modification decreases the expression of a functional protein encoded by a target gene in the subject.

[0327] 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 onset of a condition may or may not have received 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).

[0297]

[0328] For the purpose of curing, healing, alleviating, reducing, modifying, 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.

[0298]

[0329] 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 of "delaying" or alleviating disease onset 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 as compared to not using the method. Such comparisons are typically based on clinical trials that deal with a sufficient number of subjects to yield statistically significant results.

[0299]

[0330] "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.

[0300]

[0331] 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.

[0301]

[0332] 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.

[0302]

[0333] 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 daily, 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.

[0303]

[0334] 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 the population) and ED50 (the therapeutically effective dose in 50% of the 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.

[0304]

[0335] 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 the 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 composition of the present disclosure used in the treatment may increase or decrease during the course of a particular treatment. Changes in dosage may be derived from and apparent from the results of the diagnostic assays described herein. The therapeutically effective dosage will generally depend on 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 the disease and adjusting the treatment accordingly.

[0305]

[0336] The frequency of administration can be determined and adjusted during the course of treatment and will generally, but not necessarily, be based on the treatment and / or suppression and / or amelioration and / or retardation of the disease. Alternatively, sustained release formulations 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 third day, every fourth day, every fifth day, or every sixth day. 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.

[0306]

[0337] The dosing schedule (including the compositions disclosed herein) can vary over time. In some embodiments, for an adult subject of standard weight, 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 medical history of that subject, 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).

[0307]

[0338] 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 being administered for prophylactic or therapeutic purposes, the medical history, the clinical history of the patient and response to the antagonist, and the discretion of the attending physician. Typically, the clinician will administer the polypeptide until a dosage is reached that achieves the desired result.

[0308]

[0339] 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 essentially continuously over a preselected period of time, or a series of spaced doses, either before, during, or after the manifestation of the disease.

[0309]

[0340] The methods and compositions of the disclosure described herein, including embodiments of the methods and compositions, can be administered with one or more additional treatment regimens or agents or procedures that can be co-administered to a mammal. "Co-administered" means administering one or more additional treatment regimens or agents or procedures and a composition 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 than one or more additional treatment regimens or agents or procedures (e.g., the first treatment can be daily, while the additional treatment is weekly). For example, in embodiments, a second treatment regimen or agent or procedure is administered simultaneously with, prior to, or subsequent to a composition of the disclosure.

[0310] Pharmaceutical Compositions, Dosage Forms, and Administration

[0341] 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 inert ingredients that facilitate the processing of the active compound into a pharmaceutically usable preparation. Suitable formulations and methods of delivery for use in the present disclosure are generally well known in the art. Suitable formulations depend on the route of administration chosen. An overview of 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 hereby incorporated herein by reference.

[0311]

[0342] The pharmaceutical composition can be a mixture of the 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 to an organism or subject in need of 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.

[0312]

[0343] The pharmaceutical compositions of the 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, colonically, rectally, 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.

[0313]

[0344] For administration by inhalation, the adenoviruses 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 adenoviruses described herein can be prepared as a transdermal dosage form. In some embodiments, the adenoviruses described herein can be formulated into a pharmaceutical composition suitable for intramuscular, subcutaneous or intravenous injection. In some embodiments, the adenoviruses described herein can be administered topically and formulated into various topically administrable compositions, such as solutions, suspensions, lotions, gels, pastes, medicated sticks, balms, creams or ointments. In some embodiments, the adenoviruses 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 adenoviruses described herein can be formulated for oral administration, for example, in the form of tablets, capsules, or, without limitation, aqueous suspensions or solutions selected from the group comprising aqueous oral dispersions, emulsions, solutions, elixirs, gels and syrups.

[0314]

[0345] In some embodiments, an 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, radiotherapy agents, hormonal therapy agents, and / or immunotherapy agents. In some embodiments, the therapeutic agent can be a radiotherapy agent. In some embodiments, the therapeutic agent can be a hormonal therapy agent. In some embodiments, the therapeutic agent can be an immunotherapy agent. In some embodiments, the therapeutic agent is a chemotherapeutic agent. Preparations and dosing schedules of the additional therapeutic agent can be used according to the manufacturer's instructions or as empirically determined by a skilled practitioner. For example, preparations and dosing schedules for chemotherapy are also described in The Chemotherapy Source Book, 4th Edition, 2008, M.C. Perry, Editor, Lippincott, Williams & Wilkins, Philadelphia, PA.

[0315]

[0346] A subject that can be treated with an 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.

[0316]

[0347] In some embodiments, the subject is prenatal (e.g., a fetus), a child (e.g., a neonate, infant, toddler, pre - adolescent child), an adolescent, an adolescent, an adult (e.g., a young adult, middle - aged adult, elderly). The human subject can be between about 0 months after birth and about 120 years or older. The human subject can be between about 0 months after birth and 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 and 12 years; e.g., between about 0 days and 30 days after birth; between about 1 month and 12 months after birth; between about 1 year and 3 years; between about 4 years and 5 years; between about 4 years and 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 and 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 and 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 and 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.

[0317]

[0348] 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 container is formed from a variety of materials, such as glass or plastic.

[0318]

[0349] 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.

[0319]

[0350] 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 statement or label for identification, or instructions related to its use in the methods described herein.

[0320]

[0351] 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.

[0352] In embodiments, the label is on 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, for example as an accompanying document, within a receptacle or carrier that further holds the container. 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 using the contents in the methods described herein.

Example

[0321]

[0353] The following examples are included for illustrative purposes only and are not intended to limit the scope of the disclosure.

[0354] Example 1: Fusion proteins with variant NLS constructs

[0355] Using variant nuclear localization sequence (NLS) constructs, several fusion protein constructs were developed to have significantly high epigen silencing activity.

[0322]

[0356] Several constructs with variant configurations of the NLS domain (Figure 1) were constructed and tested at the Pcsk9 locus in HeLa cells (Figures 2A - 2B). Those constructs were further tested at the Pcsk9 locus in Hepa1 - 6 (Figure 3) and HuH7 (Figures 4 - 5). The DNA sequences and amino acid sequences of exemplary fusion protein constructs are found in SEQ ID NOs: 17 - 24, 28 - 35, 110 - 117, and 160 - 167. The sequences from Figure 4A can be found in SEQ ID NOs: 118 - 130.The sequences from Figure 5 can be found in SEQ ID NOs: 131 - 150.

[0323]

[0357] Cell culture and transfection

[0358] HeLa (ATCC-CRM-CCL-2), Hepa1-6 (PCSK9-IRES-TdTomato), Huh7 (Sekisui XenoTech, LLC), and HEK293T Griptite (CLTA-GFP) cells were cultured in DMEM containing 10% FBS. All experiments in HeLa and Huh7 cells were performed using chemically synthesized guide RNAs and in vitro transcribed effector constructs. HeLa cells were reverse transfected using the TransIT-X2 transfection reagent (catalog number MIR6003) from Mirus. Huh7 cells were reverse transfected using the MessengerMAX reagent (catalog number LMRNA003) from Invitrogen. Secreted PCSK9 levels were measured at the indicated time points using the LEGEND MAX™ Human PCSK9 ELISA Kit (catalog number 443107) from Biolegend. All ELISA data were normalized to cell number using the CellTiter-Glo kit (catalog number G7571) from Promega.

[0324]

[0359] HEK293T Griptite cells containing GFP knocked-in at the CLTA locus as an in-frame CLTA fusion were co-transfected with an effector construct and a plasmid encoding human CLTA guide RNA using the TransIT-X2 transfection reagent (catalog number MIR6003) from Mirus. GFP was measured by FACS for GFP expression as an alternative to CLTA expression.

[0325]

[0360] Hepa1-6 was co-transfected with an effector construct and a plasmid encoding mouse PCSK9 guide RNA in an Amaxa 4D nucleofector device from Lonza using the SF cell line 96-well Nucleofector kit (catalog number V4SC-2096, program code: CM-138). At the time of presentation, as an alternative to PCSK9 levels, cells were FACS analyzed for TdTomato expression.

[0326]

[0361] In Vitro Transcription of Effector Construct and Synthetic gRNA

[0362] To obtain RNA with a cap1 structure on the 5' end and 3' polyadenylated, 1 μg of linearized effector template was used to set up an in vitro transcription reaction according to the manufacturer's instructions using the T7 mScript™ Standard mRNA Production System from CellScript (catalog number C-MSC100625). A terminal-modified sgRNA with three 2'O-methyl modified nucleotides, including phosphorothioate bonds at both the 5' and 3' ends, was obtained from Integrated DNA technologies.

[0327]

[0363] Methylation Profiling

[0364] Genomic DNA was extracted from each well of a 96-well culture plate using the DNAdvance DNA extraction kit from Beckman Coulter. After quantification of the genomic DNA using the Quant-IT High Sensitivity DNA 1X kit, each genomic DNA sample was bisulfite-converted using the EZ-96 DNA Methylation-Gold MagPrep kit from Zymo Research according to the manufacturer's instructions. For the hybridization capture experiment, a DNA library was prepared using the xGen Methyl-Seq DNA Library Prep kit from IDT, and hybridization capture was performed using the xGen hybridization capture kit from IDT. For the amplicon sequencing experiment, a DNA library was prepared using the xGen Methyl-Seq DNA Library Prep kit from IDT, and hybridization capture was performed using the xGen hybridization capture from IDT. To set up PCR corresponding to each of the two VIM amplicons, Platinum Taq kit from Invitrogen was used with bisulfite-converted DNA from each sample. The product pool was purified using the AMPure XP kit from Beckman Coulter, and after fragment size assessment using the D1000 ScreenTape on a Tapestation 4200 from Agilent, sequencing was performed by a commercial service (Azenta).

[0328]

[0365] Example 2: Bacterial DNA methyltransferase

[0366] In this experiment, a panel of bacterial proteins was screened for DNA methyltransferase activity in mammalian cells. Those bacterial DNA methyltransferases (Table 3) were tested for epigenetic silencing activity by fusion to the N-terminus of the dCas9 domain using the experimental procedure of Example 1. Then, those constructs were transfected into a reporter cell line expressing GFP under the control of the mammalian promoter of CTLA4.

[0329]

Table 3

[0330]

[0367] M.SssI DNA methyltransferase can efficiently methylate DNA in mammalian cells (Figures 6A - 6D), with stable silencing for up to 30 days. The sequences from Figures 6A - 6D can be found in SEQ ID NOs: 151 - 158. The methylation profiles of those cells were also analyzed at the 29-day time point, confirming 20% methylation of the target gene (Figures 7 - 8).

[0331]

[0368] Three other orthologous DNA methyltransferases predicted to be related to M.SssI were identified and tested for epigenetic silencing activity using the experimental procedure of Example 1 (Table 4).

[0332]

Table 4

[0333]

[0369] The DNA methyltransferases in Table 4 are predicted to have functions similar to or improved from M.SssI. Instead of mouse DNMT3A / DNMT3L, their functions are compared to that of M.SssI DNA methyltransferase in terms of silencing of the Pcsk9 locus in the HeLa TdTomato system to identify new features and improved functions.

[0334]

[0370] Example 3: Alternative KRAB Domain

[0371] In this example, when a fusion protein was constructed using an alternative KRAB domain (Table 5) and tested using the experimental procedure of Example 1, improved activity was shown compared to CRISPR off (Figure 9).

[0335]

Table 5

[0336]

[0372] Example 4: ZIM3 Fusion Construct

[0373] Generate novel fusions of ZIM3 and KOX1 KRAB. Both ZIM3 and KOX1 KRAB are KRAB family proteins with extensive homology. Therefore, design an array at the midpoint between ZIM3 and KOX1 KRAB. Their KOX1 KRAB and ZIM3 constructs encode small regions of KOX1 KRAB and ZIM3 that were noted around the zinc finger domains of those proteins. The regions of KOX1 KRAB and ZIM3 used are quite similar within the first approximately 75 bp of their sequences, but ZIM3 further has a small alpha-helical region at the C-terminus that is not present in KOX1 KRAB. The KOX1 KRAB-FL sequence contains the KOX1 KRAB sequence equivalent to its special piece, but the ZIM3 truncation removed that special piece from the ZIM3 sequence. The ZIM3 / KOX1 KRAB chimera is a fusion of the N-terminal and C-terminal pieces of those two proteins. The ZIM3-like KOX1 KRAB variants are both first assembled by BLAST of ZIM3 or KOX1 KRAB proteins from non-human species to assemble the closest 100 homologs (the "family") of each gene; second, identify 3 members of the KOX1 KRAB family that most closely resemble ZIM3, and 3 members of the ZIM3 family that most closely resemble KOX1 KRAB; third, rationally modify the KOX1 KRAB-FL sequence to mimic each of the three sets (Table 6).

[0337]

Table 6

[0338]

[0374] Preferred embodiments of the present disclosure have been shown and described herein, but it will be apparent to those skilled in the art that such examples are provided merely by way of example. Numerous variations, modifications, and substitutions will be apparent 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 in the practice of the present disclosure. The following claims define the scope of the present disclosure and are intended to cover methods and structures within the scope of those claims and their equivalents.

[0339]

Table 7-1

[0340]

Table 7-2

[0341]

Table 7-3

[0342]

Table 7-4

[0343]

Table 7-5

[0344]

Table 7-6

[0345]

Table 7-7

[0346]

Table 7-8

[0347]

Table 7-9

[0348]

Table 7-10

[0349]

Table 7-11

[0350]

Table 7-12

[0351]

Table 7-13

[0352]

Table 7-14

[0353]

Table 7-15

[0354]

Table 7-16

[0355]

Table 7-17

[0356]

Table 7-18

[0357]

Table 7-19

[0358]

Table 7-20

[0359]

Table 7-21

[0360]

Table 7-22

[0361]

Table 7-23

[0362]

Table 7-24

[0363]

Table 7-25

[0364]

Table 7-26

[0365]

Table 7-27

[0366]

Table 7-28

[0367]

Table 7-29

[0368]

Table 7-30

[0369]

Table 7-31

[0370]

Table 7-32

[0371]

Table 7-33

[0372]

Table 7-34

[0373]

Table 7-35

[0374]

Table 7-36

[0375]

Table 7-37

[0376]

Table 7-38

[0377]

Table 7-39

[0378]

Table 7-)40

[0379]

Table 7-41

[0380]

Table 7-42

[0381]

Table 7-43

[0382]

Table 7-44

[0383]

Table 7-45

[0384]

Table 7-46

[0385]

Table 7-47

[0386]

Table 7-48

[0387]

Table 7-49

[0388]

Table 7-50

[0389]

Table 7-51

[0390]

Table 7-52

[0391]

Table 7-53

[0392]

Table 7-54

[0393]

Table 7-55

[0394]

Table 7-56

[0395]

Table 7-57

[0396]

Table 7-58

[0397]

Table 7-59

[0398]

Table 7-60

[0399]

Table 7-61

[0400]

Table 7-62

[0401]

Table 7-63

[0402]

Table 7-64

[0403]

Table 7-65

[0404]

Table 7-66

[0405]

Table 7-67

[0406]

Table 7-68

[0407]

Table 7-69

[0408]

Table 7-70

[0409]

Table 7-71

[0410]

Table 7-72

[0411]

Table 7-73

[0412]

Table 7-74

[0413]

Table 7-75

[0414]

Table 7-76

[0415]

Table 7-77

[0416]

Table 7-78

[0417]

Table 7-79

[0418]

Table 7-80

[0419]

Table 7-81

[0420]

Table 7-82

[0421]

Table 7-83

[0422]

Table 7-84

[0423]

Table 7-85

[0424]

Table 7-86

[0425]

Table 7-87

[0426]

Table 7-88

[0427]

Table 7-89

[0428]

Table 7-90

[0429]

Table 7-91

[0430]

Table 7-92

Claims

1. (a) DNA-binding domain, and The DNMT3L domain is derived from a species selected from the group consisting of Equus przewalskii, Ailuropoda melanoleuca, Carlito syrichta, Meriones unguiculatus, Ochotona princeps, Neosciurus carolinensis, Bison bison, Mus caroli, and Pan troglodytes. A fusion protein containing, or (b) Nucleic acid molecule encoding the fusion protein of (a) An epigenetic editing system that includes [this].

2. The epigenetic editing system according to claim 1, wherein the DNMT3L domain comprises an amino acid sequence that is at least 90% homologous to any one of SEQ ID NOs. 72-80 and 101-109, or any one of SEQ ID NOs. 72-80 and 101-109.

3. The epigenetic editing system according to claim 2, wherein the DNMT3L domain comprises an amino acid sequence that is at least 90% homologous to SEQ ID NO: 107 or the amino acid sequence of SEQ ID NO:

107.

4. The epigenetic editing system according to any one of claims 1 to 3, wherein the fusion protein further comprises a repressor domain, and the repressor domain comprises a KRAB domain.

5. The epigenetic editing system according to claim 4, wherein the KRAB domain is derived from KOX1, ZIM3, ZFP28, or ZN627.

6. The epigenetic editing system according to claim 5, wherein the KRAB domain is derived from ZIM3.

7. The epigenetic editing system according to claim 6, wherein ZIM3 comprises an amino acid sequence that is at least 90% homologous to SEQ ID NO: 60 or 100.

8. The epigenetic editing system according to any one of claims 1 to 3, wherein the fusion protein further comprises a DNMT3A domain.

9. The epigenetic editing system according to claim 8, wherein the DNMT3A domain comprises an amino acid sequence that is at least 90% homologous to SEQ ID NO: 96 or the amino acid sequence of SEQ ID NO:

96.

10. The epigenetic editing system according to any one of claims 1 to 3, wherein the fusion protein comprises two or more nuclear localization sequences (NLS).

11. The epigenetic editing system according to claim 10, wherein the first of the two NLSs is positioned at the amino (N) terminus of the fusion protein, and the second of the two NLSs is positioned at the carboxy (C) terminus of the fusion protein.

12. The epigenetic editing system according to any one of claims 1 to 3, wherein the fusion protein comprises one or more peptide linkers, and the one or more peptide linkers comprises XTEN80 or XTEN16.

13. The epigenetic editing system according to any one of claims 1 to 3, wherein the DNA-binding domain is a nuclease-inactive Cas9 (dCas9) domain.

14. The epigenetic editing system according to claim 13, wherein the dCas9 domain comprises an amino acid sequence that is at least 90% homologous to SEQ ID NO:

8.

15. The epigenetic editing system according to claim 13, further comprising one or more guide RNAs (gRNAs), or nucleic acid molecules encoding the gRNAs.

Images