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WO2025030130A1 - Procédés et compositions comprenant des anticorps se liant à dnmt3a - Google Patents

Procédés et compositions comprenant des anticorps se liant à dnmt3a Download PDF

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WO2025030130A1
WO2025030130A1 PCT/US2024/040780 US2024040780W WO2025030130A1 WO 2025030130 A1 WO2025030130 A1 WO 2025030130A1 US 2024040780 W US2024040780 W US 2024040780W WO 2025030130 A1 WO2025030130 A1 WO 2025030130A1
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domain
composition
nucleic acid
seq
fusion protein
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Ari Friedland
Mary Shirley MORRISON
Vic MYER
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Chroma Medicine Inc
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Chroma Medicine Inc
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/10—Transferases (2.)
    • C12N9/1003—Transferases (2.) transferring one-carbon groups (2.1)
    • C12N9/1007—Methyltransferases (general) (2.1.1.)
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/40—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against enzymes
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/62—DNA sequences coding for fusion proteins
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y201/00—Transferases transferring one-carbon groups (2.1)
    • C12Y201/01—Methyltransferases (2.1.1)
    • C12Y201/01037—DNA (cytosine-5-)-methyltransferase (2.1.1.37)
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P3/00—Drugs for disorders of the metabolism
    • A61P3/06—Antihyperlipidemics
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P9/00—Drugs for disorders of the cardiovascular system
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/20—Immunoglobulins specific features characterized by taxonomic origin
    • C07K2317/22—Immunoglobulins specific features characterized by taxonomic origin from camelids, e.g. camel, llama or dromedary
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/56—Immunoglobulins specific features characterized by immunoglobulin fragments variable (Fv) region, i.e. VH and/or VL
    • C07K2317/569—Single domain, e.g. dAb, sdAb, VHH, VNAR or nanobody®
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/80—Fusion polypeptide containing a DNA binding domain, e.g. Lacl or Tet-repressor

Definitions

  • the present disclosure provides systems and compositions for epigenetic modification (“epigenetic editors” or “epigenetic editing systems” herein), and methods of using the same to generate epigenetic modification of target genes, including in host cells and organisms.
  • the present disclosure provides a composition comprising a fusion protein of the formula (from N-terminus to C-terminus): [EBD]x-[Linker]n-[DBD]y (I) or [DBD]y-[Linker]n-[EBD]x (II), wherein [EBD] an endogenous epigenetic effector binding domain, [Linker] is a protein linker or a covalent bond, [DBD] is a DNA binding domain, x is an integer between 1 and 10, y is an integer between 1 and 5, n is an integer between 0 and 1, and optionally wherein [EBD] is [SDA], wherein [SDA] is a single domain antibody or an antigen-binding single domain antibody domain.
  • At least one of the single domain antibody or antigen-binding single domain antibody domains bind to Tetl. In some embodiments, at least one of the single domain antibody or antigen-binding single domain antibody domains bind to Tet2.
  • x is between 1 and 6. In some embodiments, x is 1. In some embodiments, x is 2.
  • the fusion protein comprises at least two single domain antibodies or antigen-binding single domain antibody domains, and at least two of the single domain antibodies or antigen-binding single domain antibody domains are linked via a linker.
  • the linker linking the two single domain antibodies or antigen-binding single domain antibody domains comprises an amino acid sequence comprising between 10 and 100 amino acid residues.
  • the linker comprises an amino acid sequence of SEQ ID NO: 637.
  • the covalent bond is a protein bond.
  • [Linker] or “L” is a protein linker comprising between 10 and 250 amino acid residues. In some embodiments, L comprises between 5 and 100 amino acids. In some embodiments, L comprises between 16 and 80 amino acids. In some embodiments, L comprises 16 amino acids. In some embodiments, L comprises 23 amino acids. In some embodiments, L comprises 27 amino acids. In some embodiments, L comprises 80 amino acids. In some embodiments, L comprises a sequence of any one of SEQ ID NOs: 631-643, 664, or 665. In some embodiments, L comprises a sequence of any one of SEQ ID NOs: 638- 643.
  • the fusion protein further comprises one or more effector domains.
  • the fusion protein comprises the formula (from N-terminus to C-terminus): [SDA]x-[Linker]n- [Effector Domain] -[DBD]y.
  • the fusion protein comprises the formula (from N-terminus to C-terminus): [SDA] x- [Linker] n- [DBD]y -[Effector Domain].
  • the fusion protein comprises the formula (from N-terminus to C-terminus): [Effector Domain]-[SDA]x-[Linker]n-[DBD]y.
  • the fusion protein comprises the formula (from N-terminus to C-terminus): [SDA]x- [Linker] n- [Effector Domain]-[DBD]y-[Effector Domain].
  • the effector domain is linked to the N-terminus of the DNA binding domain. In some embodiments, the effector domain is linked to the N-terminus of the single domain antibody. In some embodiments, the effector domain is linked to the C- terminus of the DNA binding domain. In some embodiments, the effector domain is linked to the C-terminus of the single domain antibody.
  • the effector domain is a Dnmt3L domain. In some embodiments, the effector domain comprises an amino acid sequence of SEQ ID NO: 578.
  • the fusion protein further comprises at least one nuclear localization signal (NLS). In some embodiments, the fusion protein comprises one NLS. In some embodiments, the fusion protein comprises two NLSs. In some embodiments, the fusion protein comprises three NLSs. In some embodiments, the fusion protein comprises four NLSs. In some embodiments, the fusion protein comprises more than four NLSs.
  • NLS nuclear localization signal
  • the fusion protein comprises the structure (from N-terminus to C- terminus): [NLS]-[NLS]-[SDA]-[SDA]-[Linker]-[DNMT3L]-[DNA-binding domain] -[NLS] - [NLS] or [NLS]-[NLS]-[DNA-binding domain]-[DNMT3L]-[Linker]-[SDA]-[SDA]-[NLS]- [NLS],
  • the fusion protein is encoded by a nucleic acid comprising the nucleic acid sequence provided by SEQ ID NO: 1322. In some embodiments, the fusion protein is encoded by a nucleic acid comprising the nucleic acid sequence provided by SEQ ID NO: 1323. In some embodiments, the fusion protein is encoded by a nucleic acid comprising the nucleic acid sequence provided by SEQ ID NO: 1324. In some embodiments, the fusion protein is encoded by a nucleic acid comprising the nucleic acid sequence provided by SEQ ID NO: 1325. In some embodiments, the fusion protein is encoded by a nucleic acid comprising the nucleic acid sequence provided by SEQ ID NO: 1326.
  • the fusion protein is encoded by a nucleic acid comprising the nucleic acid sequence provided by SEQ ID NO: 1327. In some embodiments, the fusion protein is encoded by a nucleic acid comprising the nucleic acid sequence provided by SEQ ID NO: 1328. In some embodiments, the fusion protein is encoded by a nucleic acid comprising the nucleic acid sequence provided by SEQ ID NO: 1329. In some embodiments, the fusion protein is encoded by a nucleic acid comprising the nucleic acid sequence provided by SEQ ID NO: 1330. In some embodiments, the fusion protein is encoded by a nucleic acid comprising the nucleic acid sequence provided by SEQ ID NO: 1331.
  • the fusion protein is encoded by a nucleic acid comprising the nucleic acid sequence provided by SEQ ID NO: 1337. In some embodiments, the fusion protein is encoded by a nucleic acid comprising the nucleic acid sequence provided by SEQ ID NO: 1338. In some embodiments, the fusion protein is encoded by a nucleic acid comprising the nucleic acid sequence provided by SEQ ID NO: 1339. In some embodiments, the fusion protein is encoded by a nucleic acid comprising the nucleic acid sequence provided by SEQ ID NO: 1340. In some embodiments, the fusion protein is encoded by a nucleic acid comprising the nucleic acid sequence provided by SEQ ID NO: 1341.
  • the fusion protein is encoded by a nucleic acid comprising the nucleic acid sequence provided by SEQ ID NO: 1342. In some embodiments, the fusion protein is encoded by a nucleic acid comprising the nucleic acid sequence provided by SEQ ID NO: 1343. In some embodiments, the fusion protein is encoded by a nucleic acid comprising the nucleic acid sequence provided by SEQ ID NO: 1344. In some embodiments, the fusion protein is encoded by a nucleic acid comprising the nucleic acid sequence provided by SEQ ID NO: 1345.
  • the present disclosure provides a method of reducing expression of a target gene in a cell, comprising contacting the cell with a composition or fusion protein of this disclosure. In some embodiments, the present disclosure provides a method of administering a composition or fusion protein of this disclosure to a subject in need thereof. In some embodiments, the subject has or has been diagnosed with a disease or disorder associated with hypercholesterolemia or hypertriglyceridemia.
  • the subject has heart disease, has elevated low-density lipoprotein cholesterol (LDL-C) or hypercholesterolemia, is at risk of developing myocardial infarction, stroke, or unstable angina, and/or has primary hyperlipidemia, optionally heterozygous familial hypercholesterolemia (HeFH), or homozygous familial hypercholesterolemia (HoFH).
  • LDL-C low-density lipoprotein cholesterol
  • hypercholesterolemia is at risk of developing myocardial infarction, stroke, or unstable angina
  • primary hyperlipidemia optionally heterozygous familial hypercholesterolemia (HeFH), or homozygous familial hypercholesterolemia (HoFH).
  • HeFH heterozygous familial hypercholesterolemia
  • HoFH homozygous familial hypercholesterolemia
  • FIGs. 2A-2B show epigenetic silencing in GripTite CLTA-GFP cells using single or quad guides to characterize activity of constructs comprising an encoded DNMT3A and 3L (PLA1464) compared to constructs containing DNMT3L only (PLA4213). Empty transfection and constructs provided with off-target guides were used as controls. Data from day 7 (FIG. 2A) and day 14 (FIG. 2B) post-transfection are shown. %GFP+ indicates the percentage of cells expressing CLTA. For each figure, three potential bars are shown. From left to right the bars are single (single guide), quad (quad guide), and off target.
  • FIG. 3 shows the results of a binding ELISA experiment, as described in Example 2.
  • Each single domain antibody clone was tested for binding to full-length DNMT3A (DNMT3A FL), the catalytic domain of DNMT3A (DNMT3A CD), and PBS.
  • R3P1-A1, R3P1-B10, R3P2-F2, R3P2-C3, R3P2-C10, R3P3-D1, R3P3-G3, and R3P3-E8 bind non-CD regions of DNMT3A.
  • R4P2-F7, R5P1-A4, and RP1-C11 have high off-target binding.
  • R5P1- E4 and R5P1-B12 bind the CD only.
  • FIG. 4 shows a tree diagram depicting the similarity between single domain antibodies generated as described in Example 1. Shading indicates if a single domain antibody binds only to the catalytic domain (CD) of DNMT3A or binds to non-CD regions of DNMT3A.
  • CD catalytic domain
  • FIG. 3 shows that R3P2-C10, R3P3-E8, R3P3-G3, R3P3-D1, R3P2-C3, R3P1- Al, R3P1-B10, and R3P2-F2 bind non-CD regions of DNMT3A.
  • R5P1-E4 and R5P1-B12 bind the CD only.
  • FIGs. 5A-5B show diagrams depicting a non-limiting example of a fusion protein comprising two single domain antibody domains (Nbl and Nb2), DNMT3L, and a DNA binding domain (dCas9).
  • FIG. 5A shows a schematic of the example fusion protein.
  • FIG. 5B shows a diagram illustrating the example fusion protein binding to DNA.
  • FIG. 6 shows non-limiting examples of fusion proteins comprising two single domain antibody domains (Nbl and Nb2), DNMT3L, and a DNA binding domain (dCas9 and ZFP152).
  • PLA4303 does not comprise a repressor domain
  • PLA4685 and PLA4752 each further comprise a repressor domain (ZIM3 and ZN627, respectively).
  • FIGs. 7A-7B show dose-response experiments in HeLa PCSK9-TdTomato cells (% TdTomato+ indicates the percentage of PCSK9-expressing cells) using an equal ratio (1:1) of guide: effector protein, using guides 041 and 049. Results from day 7 (FIG. 7A) and day 12 (FIG. 7B) are shown.
  • the present disclosure provides epigenetic editors for repressing or activating expression of a gene designed to recruit at least one endogenous effector with an endogenous epigenetic effector binding domain, or EBD.
  • An EBD is a molecular entity that binds an effector of interest, of which non-limiting examples are provided herein.
  • the epigenetic editor comprises a DNA-binding domain, and at least one EBD that binds an effector of interest, e.g., an effector domain as described herein.
  • the epigenetic editor comprises a DNA-binding domain, and at least one EBD that binds a transcriptional repressor, e.g., a transcriptional repressor as described herein.
  • the epigenetic editor comprises a DNA-binding domain, and at least one EBD that binds a transcriptional activator, e.g., a transcriptional activator as described herein.
  • the epigenetic editor comprises a DNA-binding domain, at least one effector domain, and at least one EBD.
  • the EBD is a single domain antibody or an antigen-binding single domain antibody domain.
  • Fusion proteins comprising a DNA-binding domain, an effector domain, and DNMT3A/3L have been used for epigenetic editing of target genes. Surprisingly, as reported in this application, effective epigenetic editing is achieved using a fusion protein comprising a DNA-binding domain, an effector domain, and a single domain antibody that binds DNMT3A.
  • DNMTs are one example of a class of enzymes that modulate DNA- methylation
  • other enzymes that modulate DNA-methylation may also be recruited by single domain antibodies.
  • An additional non-limiting example is the ten-eleven translocation (Tet) methylcytosine dioxygenase family of enzymes, which includes Tetl and Tet2.
  • Tet ten-eleven translocation
  • a fusion protein described herein recruits Tetl, Tet2, or Tetl and Tet2.
  • recruitment of Tetl, Tet2, or Tetl and Tet2 occurs via one or more single domain antibodies that bind Tetl, Tet2, or Tetl and Tet2.
  • a fusion protein disclosed herein recruits Tetl, Tet2, or Tetl and Tet2 instead of one or more DNMTs. In some embodiments, a fusion protein disclosed herein recruits Tetl, Tet2, or Tetl and Tet2 in addition to one or more DNMTs.
  • the fusion proteins provided herein are useful for targeting of effector domains to specific sequences within a genome without exogenous DNMT3A or other enzyme that modulates DNA methylation.
  • Use of binding domains for recruitment of endogenous effectors allows for smaller fusion proteins, which is advantageous in some embodiments.
  • the fusion proteins provided herein are further useful for multiplex epigenetic editing, e.g., for silencing or activating one or more target genes within a genome.
  • An epigenetic editor described herein may comprise one or more DNA-binding domains that direct the effector domain(s) of the epigenetic editor to target sequences within or close to a target gene locus.
  • a DNA-binding domain as described herein may be, e.g., a polynucleotide guided DNA-binding domain, a zinc finger protein (ZFP) domain, a transcription activator like effector (TALE) domain, a meganuclease DNA-binding domain, and the like. Examples of DNA-binding domains can be found in U.S. Patent 11,162,114, which is incorporated by refence herein in its entirety.
  • a DNA-binding domain described herein is encoded by its native coding sequence. In other embodiments, the DNA-binding domain is encoded by a nucleotide sequence that has been codon-optimized for optimal expression in human cells. Polynucleotide Guided DNA-Binding Domains
  • a DNA-binding domain herein may be a protein domain directed by a guide nucleic acid sequence (e.g., a guide RNA sequence) to a target site in a target gene locus.
  • the protein domain may be derived from a CRISPR-associated nuclease, such as a Class I or II CRIS PR-associated nuclease.
  • the protein domain may be derived from a Cas nuclease such as a Type II, Type IIA, Type IIB, Type IIC, Type V, or Type VI Cas nuclease.
  • the protein domain may be derived from a Class II Cas nuclease selected from Casl, Cas IB, Cas2, Cas3, Cas4, Cas5, Cas6, Cas7, Cas8, Cas9, Cas 10, Cas 14a, Cas 14b, Cas 14c, CasX, CasY, CasPhi, C2c4, C2c8, C2c9, C2cl0, Csyl, Csy2, Csy3, Csel, Cse2, Cscl, Csc2, Csa5, Csn2, Csm2, Csm3, Csm4, Csm5, Csm6, Cmrl, Cmr3, Cmr4, Cmr5, Cmr6, Csbl, Csb2, Csb3, Csxl7, Csxl4, CsxlO, Csxl6, CsaX, Csx3, Csxl, CsxlS, Csfl, Csf
  • “Derived from” is used to mean that the protein domain comprises the full polypeptide sequence of the parent protein, or comprises a variant thereof (e.g., with amino acid residue deletions, insertions, and/or substitutions).
  • the variant retains the desired function of the parent protein (e.g., the ability to form a complex with the guide nucleic acid sequence and the target DNA).
  • the CRISPR-associated protein domain may be a Cas9 domain described herein.
  • Cas9 may, for example, refer to a polypeptide with at least about 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity and/or sequence similarity to a wildtype Cas9 polypeptide described herein.
  • said wildtype polypeptide is Cas9 from Streptococcus pyogenes (NCBI Ref. No. NC_002737.2 (SEQ ID NO: 1)) and/or UniProt Ref. No. Q99ZW2 (SEQ ID NO: 2).
  • said wildtype polypeptide is Cas9 from Staphylococcus aureus (SEQ ID NO: 3).
  • the CRISPR-associated protein domain is a Cpfl domain or protein, or a polypeptide with at least about 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity and/or sequence similarity to a wildtype Cpfl polypeptide described herein (e.g., Cpfl from Francisella novicida (UniProt Ref. No. U2UMQ6 or SEQ ID NO: 4).
  • the CRISPR-associated protein domain may be a modified form of the wildtype protein comprising one or more amino acid residue changes such as a deletion, an insertion, or a substitution; a fusion or chimera; or any combination thereof.
  • Cas9 sequences and structures of variant Cas9 orthologs have been described for various organisms.
  • Exemplary organisms from which a Cas9 domain herein can be derived include, but are not limited to, Streptococcus pyogenes, Streptococcus thermophilus, Streptococcus sp., Staphylococcus aureus, Listeria innocua, Lactobacillus gasseri, Francisella novicida, Wolinella succinogenes, Sutterella wadsworthensis, Gamma proteobacterium, Neisseria meningitidis, Campylobacter jejuni, Pasteurella multocida, Fibrobacter succinogene, Rhodospirillum rubrum, Nocardiopsis rougevillei, Streptomyces pristinaespiralis, Streptomyces viridochromo genes, Streptomyces viridochromogenes , Streptosporangium roseum
  • Nitrosococcus halophilus Nitrosococcus watsoni, Pseudoalteromonas haloplanktis, Ktedonobacter racemifer, Methanohalobium evestigatum, Anabaena variabilis, Nodularia spumigena, Nostoc sp., Arthrospira maxima, Arthrospira platensis, Arthrospira sp., Lyngbya sp., Microcoleus chthonoplastes, Oscillatoria sp., Petrotoga mobilis, Thermosipho africanus, Streptococcus pasteurianus, Neisseria cinerea, Campylobacter lari, Parvibaculum lavamentivorans, Corynebacterium diphtheria, and Acaryochloris marina.
  • Cas9 sequences also include those from the organisms and loci disclosed in Chylinski et al., RNA Bio
  • the Cas9 domain is from Streptococcus pyogenes (SpCas9). In some embodiments, the Cas9 domain is from Staphylococcus aureus (SaCas9).
  • Cas domains are also contemplated for use in the epigenetic editors herein. These include, for example, those from CasX (Casl2E) (e.g., SEQ ID NO: 5), CasY (Casl2d) (e.g., SEQ ID NO: 6), Cascp (CasPhi) (e.g., SEQ ID NO: 7), Casl2fl (Casl4a) (e.g., SEQ ID NO: 8), Casl2f2 (Casl4b) (e.g., SEQ ID NO: 9), Casl2f3 (Casl4c) (e.g., SEQ ID NO: 10), and C2c8 (e.g., SEQ ID NO: 11).
  • CasX Casl2E
  • CasY Casl2d
  • Cascp CasPhi
  • Casl2fl Casl4a
  • Casl2f2 Cas
  • the nuclease-derived protein domain may have reduced or no nuclease activity through mutations such that the protein domain does not cleave DNA or has reduced DNA-cleaving activity while retaining the ability to complex with the guide nucleic acid sequence (e.g., guide RNA) and the target DNA.
  • the nuclease activity may be reduced by at least 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 99% compared to the wildtype domain.
  • a CRIS PR-associated protein domain described herein is catalytically inactive (“dead”).
  • dCas9 (“dead” Cas9)
  • dCpfl ddCpfl
  • dCasPhi ddCasl2a
  • dLbCpfl dLbCpfl
  • dFnCpfl dFnCpfl
  • a dCas9 protein domain may comprise one, two, or more mutations as compared to wildtype Cas9 that abrogate its nuclease activity.
  • the DNA cleavage domain of Cas9 is known to include two subdomains: the HNH nuclease subdomain and the RuvCl subdomain.
  • the HNH subdomain cleaves the strand complementary to the gRNA, whereas the RuvCl subdomain cleaves the non-complementary strand. Mutations within these subdomains can silence the nuclease activity of Cas9.
  • the mutations D10A (in RuvCl) and H840A (in HNH) completely inactivate the nuclease activity of SpCas9.
  • SaCas9 similarly, may be inactivated by the mutations D10A and N580A.
  • the dCas9 comprises at least one mutation in the HNH subdomain and/or the RuvCl subdomain that reduces or abrogates nuclease activity.
  • the dCas9 only comprises a RuvCl subdomain, or only comprises an HNH subdomain. It is to be understood that any mutation that inactivates the RuvCl and/or the HNH domain may be included in a dCas9 herein, e.g., insertion, deletion, or single or multiple amino acid substitution in the RuvCl domain and/or the HNH domain.
  • a dCas9 protein herein comprises a mutation at position(s) corresponding to position DIO (e.g., D10A), H840 (e.g., H840A), or both, of a wildtype SpCas9 sequence as numbered in the sequence provided at UniProt Accession No. Q99ZW2 (SEQ ID NO: 2).
  • the dCas9 comprises the amino acid sequence of dSpCas9 (D10A and H840A) (SEQ ID NO: 12).
  • a dCas9 protein as described herein comprises a mutation at position(s) corresponding to position D10 (e.g., D10A), N580 (e.g., N580A), or both, of a wildtype SaCas9 sequence (e.g., SEQ ID NO: 3).
  • the dCas9 comprises the amino acid sequence of dSaCas9 (D10A and N580A) (SEQ ID NO: 13). Additional suitable mutations that inactivate Cas9 will be apparent to those of skill in the art based on this disclosure and knowledge in the field and are within the scope of this disclosure.
  • Such mutations may include, but are not limited to, D839A, N863A, and/or K603R in SpCas9.
  • the present disclosure contemplates any mutations that reduce or abrogate the nuclease activity of any Cas9 described herein (e.g., mutations corresponding to any of the Cas9 mutations described herein).
  • a dCpfl protein domain may comprise one, two, or more mutations as compared to wildtype Cpfl that reduce or abrogate its nuclease activity.
  • the Cpfl 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-terminal of Cpfl does not have the alpha-helical recognition lobe of Cas9.
  • the dCpfl comprises one or more mutations corresponding to position D917A, E1006A, or D1255A as numbered in the sequence of the Francisella novicida Cpfl protein (FnCpfl; SEQ ID NO: 4).
  • the dCpfl protein comprises mutations corresponding to D917A, E1006A, D1255A, D917A/E1006A, D917A/D1255A, E1006A/D1255A, or D917A/ E1006A/D1255A, or corresponding mutation(s) in any of the Cpfl amino acid sequences described herein.
  • the dCpfl comprises a D917A mutation.
  • the dCpfl comprises the amino acid sequence of dFnCpfl (SEQ ID NO: 14).
  • a Cas9 domain described herein may be a high fidelity Cas9 domain, e.g., comprising one or more mutations that decrease electrostatic interactions between the Cas9 domain and the sugar-phosphate backbone of DNA to confer increased target binding specificity.
  • the high fidelity Cas9 domain may be nuclease inactive as described herein.
  • a CRISPR-associated protein domain described herein may recognize a protospacer adjacent motif (PAM) sequence in a target gene.
  • a “PAM” sequence is typically a 2 to 6 bp DNA sequence immediately following the sequence targeted by the CRISPR-associated protein domain. The PAM sequence is required for CRISPR protein binding and cleavage but is not part of the target sequence.
  • the CRISPR-associated protein domain may either recognize a naturally occurring or canonical PAM sequence or may have altered PAM specificity. CRISPR-associated protein domains that bind to non-canonical PAM sequences have been described in the art.
  • Cas9 domains that bind non-canonical PAM sequences have been described in Kleinstiver et al., Nature (2015) 523(7561):481-5 and Kleinstiver et al., Nat Biotechnol. (2015) 33:1293-8.
  • Such Cas9 domains may include, for example, those from “VRER” SpCas9, “EQR” SpCas9, “VQR” SpCas9, “SpG Cas9,” “SpRYCas9,” and “KKH” SaCas9.
  • CRIS PR-associated proteins including nuclease inactive variants and sequences, will be apparent to those of skill in the art based on this disclosure.
  • the DNA-binding domain of an epigenetic editor described herein comprises a zinc finger protein (ZFP) domain (or “ZF domain” as used herein).
  • ZFPs are proteins having at least one zinc finger, and bind to DNA in a sequence-specific manner.
  • a “zinc finger” (ZF) or “zinc finger motif’ (ZF motif) refers to a polypeptide domain comprising a beta-beta-alpha (PPa)-protein fold stabilized by a zinc ion.
  • a ZF binds from two to four base pairs of nucleotides, typically three or four base pairs (contiguous or noncontiguous). Each ZF typically comprises approximately 30 amino acids.
  • ZFP domains may contain multiple ZFs that make tandem contacts with their target nucleic acid sequence.
  • a tandem array of ZFs may be engineered to generate artificial ZFPs that bind desired nucleic acid targets.
  • ZFPs may be rationally designed by using databases comprising triplet (or quadruplet) nucleotide sequences and individual ZF amino acid sequences, in which each triplet or quadruplet nucleotide sequence is associated with one or more amino acid sequences of ZFs that bind the particular triplet or quadruplet sequence. See, e.g., U.S. Patents 6,453,242, 6,534,261, and 8,772,453.
  • ZFPs are widespread in eukaryotic cells, and may belong to, e.g., C2H2 class, CCHC class, PHD class, or RING class.
  • An exemplary motif characterizing one class of these proteins is -Cys-(X)2-4-Cys-(X)i2-His-(X)3-5-His- (SEQ ID NO: 657), where X is any independently chosen amino acid.
  • a ZFP domain herein may comprise a ZF array comprising sequential C2H2-ZFs each contacting three or more sequential nucleotides.
  • a ZFP domain of an epigenetic editor described herein may include 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, or more ZFs.
  • the ZFP domain may include an array of two-finger or three- finger units, e.g., 3, 4, 5, 6, 7, 8, 9 or 10 or more units, wherein each unit binds a subsite in the target sequence.
  • a ZFP domain comprising at least three ZFs recognizes a target DNA sequence of 9 or 10 nucleotides.
  • a ZFP domain comprising at least four ZFs recognizes a target DNA sequence of 12 to 14 nucleotides.
  • a ZFP domain comprising at least six ZFs recognizes a target DNA sequence of 18 to 21 nucleotides.
  • ZFs in a ZFP domain described herein are connected via peptide linkers.
  • the peptide linkers may be, e.g., 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.
  • a linker comprises 5 or more amino acids.
  • a linker comprises 7-17 amino acids.
  • the linker may be flexible or rigid.
  • “XX” in italics may be TR, LR or LK, and “[linker]” represents a linker sequence.
  • the linker sequence is TGSQKP (SEQ ID NO: 651); this linker may be used when sub-sites targeted by the ZFs are adjacent.
  • the linker sequence is TGGGGSQKP (SEQ ID NO: 652); this linker may be used when there is a base between the sub- sites targeted by the zinc fingers.
  • the two indicated linkers may be the same or different.
  • ZFP domains herein may contain arrays of two or more adjacent ZFs that are directly adjacent to one another (e.g., separated by a short (canonical) linker sequence), or are separated by longer, flexible or structured polypeptide sequences.
  • directly adjacent fingers bind to contiguous nucleic acid sequences, i.e., to adjacent trinucleotides/triplets.
  • adjacent fingers cross-bind between each other’s respective target triplets, which may help to strengthen or enhance the recognition of the target sequence, and leads to the binding of overlapping sequences.
  • distant ZFs within the ZFP domain may recognize (or bind to) non-contiguous nucleotide sequences.
  • the ZFP domain of the present epigenetic editor binds to a target sequence selected from any one of SEQ ID NOs: 700-747.
  • the ZFP domain comprises, in order, the F1-F6 amino acid sequences of any one of ZF001- ZF048 as shown in Table 1.
  • the F1-F6 amino acid sequences may be placed within the ZF framework sequence of SEQ ID NO: 650, or within any other ZF framework known in the art.
  • the DNA-binding domain of an epigenetic editor described herein comprises a transcription activator-like effector (TAEE) domain.
  • the DNA-binding domain of a TAEE comprises a highly conserved sequence of about 33-34 amino acids, with a repeat variable di-residue (RVD) at positions 12 and 13 that is central to the recognition of specific nucleotides.
  • RVD repeat variable di-residue
  • TAEEs can be engineered to bind practically any desired DNA sequence. Methods for programming TALEs are known in the art. For example, such methods are described in Carroll et al., Genet Soc Amer. (2011) 188(4):773-82; Miller et al., Nat Biotechnol.
  • the DNA-binding domain comprises an argonaute protein domain, e.g., from Natronobacterium gregoryi (NgAgo).
  • NgAgo is a ssDNA-guided endonuclease that is guided to its target site by 5' phosphorylated ssDNA (gDNA), where it produces double-strand breaks.
  • gDNA 5' phosphorylated ssDNA
  • the NgAgo-gDNA system does not require a protospacer- adjacent motif (PAM).
  • PAM protospacer- adjacent motif
  • NgAgo The characterization and use of NgAgo have been described, e.g., in Gao et al., Nat Biotechnol. (2016) 34(7):768-73; Swarts et al., Nature (2014) 507(7491):258-61; and Swarts et al., Nucl Acids Res. (2015) 43(10):5120-9.
  • the DNA-binding domain comprises an inactivated nuclease, for example, an inactivated meganuclease.
  • DNA-binding domains include tetracycline-controlled repressor (tetR) DNA-binding domains, leucine zippers, helix-loop-helix (HLH) domains, helix-turn-helix domains, P-sheet motifs, steroid receptor motifs, bZIP domains homeodomains, and AT-hooks.
  • Epigenetic editors described herein that comprise a polynucleotide guided DNA- binding domain may also include a guide polynucleotide that is capable of forming a complex with the DNA-binding domain.
  • the guide polynucleotide may comprise RNA, DNA, or a mixture of both.
  • the guide polynucleotide may be a guide RNA (gRNA).
  • gRNA guide RNA
  • a “guide RNA” or “gRNA” refers to a nucleic acid that is able to hybridize to a target sequence and direct binding of the CRISPR-Cas complex to the target sequence.
  • a guide polynucleotide sequence may comprises two parts: 1) a nucleotide sequence comprising a “targeting sequence” that is complementary to a target nucleic acid sequence (“target sequence”), e.g., to a nucleic acid sequence comprised in a genomic target site; and 2) a nucleotide sequence that binds a polynucleotide guided DNA- binding domain (e.g., a CRISPR-Cas protein domain).
  • the nucleotide sequence in 1) may comprise a targeting sequence that is 100% complementary to a genomic nucleic acid sequence, e.g., a nucleic acid sequence comprised in a genomic target site, and thus may hybridize to the target nucleic acid sequence.
  • the nucleotide sequence in 1) may be referred to as, e.g., a crispr RNA, or crRNA.
  • the nucleotide sequence in 2) may be referred to as a scaffold sequence of a guide nucleic acid, e.g., a tracrRNA, or an activating region of a guide nucleic acid, and may comprise a stem-loop structure.
  • Parts 1) and 2) as described above may be fused to form one single guide (e.g., a single guide RNA, or sgRNA), or may be on two separate nucleic acid molecules.
  • a guide polynucleotide comprises parts 1) and 2) connected by a linker.
  • a guide polynucleotide comprises parts 1) and 2) connected by a non-nucleic acid linker, for example, a peptide linker or a chemical linker.
  • the targeting domain of the gRNA thus may base pair (in full or partial complementarity) with the sequence of the double- stranded target site that is complementary to the target sequence, and thus with the strand complementary to the strand that comprises the PAM sequence. It will be understood that the targeting domain of the gRNA typically does not include a sequence that resembles the PAM sequence. It will further be understood that the location of the PAM may be 5’ or 3’ of the target sequence, depending on the nuclease employed. For example, the PAM is typically 3’ of the target sequence for Cas9 nucleases, and 5’ of the target sequence for Casl2a nucleases.
  • the targeting domain sequence comprises between 17 and 30 nucleotides and corresponds fully to the target sequence (i.e., without any mismatch nucleotides). In some embodiments, however, the targeting domain sequence may comprise one or more, but typically not more than 4, mismatches, e.g., 1, 2, 3, or 4 mismatches. As the targeting domain is part of gRNA, which is an RNA molecule, it will typically comprise ribonucleotides, while the DNA targeting domain will comprise deoxyribonucleotides.
  • target domain DNA
  • PAM PAM
  • Casl2a target site comprising a 22 nucleotide target domain, and a TTN PAM sequence, as well as of a gRNA comprising a targeting domain that fully corresponds to the target sequence (and thus base pairs with full complementarity with the DNA strand complementary to the strand comprising the target sequence and PAM) is provided below:
  • binding domain [ binding domain ] [ target ing domain ( RNA) ]
  • the length and complementarity of the targeting domain with the target sequence contributes to specificity of the interaction of the gRNA/Cas9 molecule complex with a target nucleic acid.
  • the targeting domain of a gRNA provided herein is 5 to 50 nucleotides in length. In some embodiments, the targeting domain is 15 to 25 nucleotides in length. In some embodiments, the targeting domain is 18 to 22 nucleotides in length. In some embodiments, the targeting domain is 19-21 nucleotides in length. In some embodiments, the targeting domain is 15 nucleotides in length. In some embodiments, the targeting domain is 16 nucleotides in length.
  • the targeting domain fully corresponds, without mismatch, to a target sequence provided herein, or a part thereof.
  • the targeting domain of a gRNA provided herein comprises 1 mismatch relative to a target sequence provided herein. In some embodiments, the targeting domain comprises 2 mismatches relative to the target sequence. In some embodiments, the target domain comprises 3 mismatches relative to the target sequence.
  • gRNA design tools include the ones described in Bae et al., Bioinformatics (2014) 30:1473-5.
  • Guide polynucleotides may be of various lengths.
  • the length of the spacer or targeting sequence depends on the CRISPR- associated protein component of the epigenetic editor system used.
  • Cas proteins from different bacterial species have varying optimal targeting sequence lengths.
  • the spacer sequence may comprise, e.g., 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.
  • the spacer comprises 10-24, 11-20, 11-16, 18-24, 19-21, or 20 nucleotides in length.
  • a guide polynucleotide e.g., gRNA
  • gRNA is from 15-100 (e.g., 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50) nucleotides in length and comprises a spacer sequence of at least 10 (e.g., 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50) contiguous nucleotides complementary to the target sequence.
  • a guide polynucleotide described herein may be truncated, e.g., by 1, 2,
  • the 3’ end of the PCSK9 target sequence is immediately adjacent to a PAM sequence (e.g., a canonical PAM sequence such as NGG for SpCas9).
  • a PAM sequence e.g., a canonical PAM sequence such as NGG for SpCas9
  • the degree of complementarity between the targeting sequence of the guide polynucleotide (e.g., the spacer sequence of a gRNA) and the target sequence may be at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%.
  • the targeting and the target sequence may be 100% complementary.
  • the targeting sequence and the target sequence may contain, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 mismatches.
  • a guide polynucleotide may be modified with, for example, chemical alterations and synthetic modifications.
  • a modified gRNA can include an alteration or replacement of one or both of the non-linking phosphate oxygens and/or of one or more of the linking phosphate oxygens in the phosphodiester backbone linkage, an alteration of the ribose sugar (e.g., of the 2’ hydroxyl on the ribose sugar), an alteration of the phosphate moiety, modification or replacement of a naturally occurring nucleobase, modification or replacement of the ribose-phosphate backbone, modification of the 3’ end and/or 5’ end of the oligonucleotide, replacement of a terminal phosphate group or conjugation of a moiety, cap, or linker, or any combination thereof.
  • one or more ribose groups of the gRNA may be modified.
  • chemical modifications to the ribose group include, but are not limited to, 2’-O- methyl (2’-0Me), 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-acetalester, or a bicyclic nucleotide such as locked nucleic acid (LNA), 2’-(5-constrained ethyl (S-cEt)), constrained MOE, or 2’-0,4’-C- aminomethylene bridged nucleic acid (2’,4’-BNANC).
  • 2’-O-methyl modification and/or 2’- fluoro modification may increase binding affinity and/or nuclease stability of the gRNA
  • one or more phosphate groups of the gRNA may be chemically modified.
  • chemical modifications to a phosphate group include, but are not limited to, a phosphorothioate (PS), phosphonoacetate (PACE), thiophosphonoacetate (thioPACE), amide, triazole, phosphonate, and phosphotriester modification.
  • a guide polynucleotide described herein may comprise one, two, three, or more PS linkages at or near the 5’ end and/or the 3’ end; the PS linkages may be contiguous or noncontiguous.
  • the gRNA herein comprises a mixture of ribonucleotides and deoxyribonucleotides and/or one or more PS linkages.
  • one or more nucleobases of the gRNA may be chemically modified.
  • chemically modified nucleobases include, but are not limited to, 2- thiouridine, 4-thiouridine, N6-methyladenosine, pseudouridine, 2,6-diaminopurine, inosine, thymidine, 5-methylcytosine, 5-substituted pyrimidine, isoguanine, isocytosine, and nucleobases with halogenated aromatic groups.
  • Chemical modifications can be made in the spacer region, the tracr RNA region, the stem loop, or any combination thereof.
  • Table 2 lists exemplary gRNA target sequences for epigenetic modification of a non-limiting example gene: human PCSK9, as well as the coordinates of the start and end positions of the targeted site on human chromosome 1 (“SEQ” means SEQ ID NO).
  • SEQ means SEQ ID NO.
  • the Table also shows the distance from the start coordinate to the TSS coordinate of the PCSK9 gene.
  • the gRNA herein is provided to the cell directly (e.g., through an RNP complex together with the CRIS PR-associated protein domain).
  • the gRNA is provided to the cell through an expression vector (e.g., a plasmid vector or a viral vector) introduced into the cell, where the cell then expresses the gRNA from the expression vector.
  • an expression vector e.g., a plasmid vector or a viral vector
  • Epigenetic editors described herein include one or more effector protein domains (also “epigenetic effector domains,” or “effector domains,” as used herein) that effect epigenetic modification of a target gene.
  • An epigenetic editor with one or more effector domains may modulate expression of a target gene without altering its nucleobase sequence.
  • an effector domain described herein may provide repression or silencing of expression of a target gene such as PCSK9, e.g., by repressing transcription or by modifying or remodeling chromatin.
  • effector domains are also referred to herein as “repression domains,” “repressor domains,” or “epigenetic repressor domains.”
  • compression domains include methylation, demethylation, acetylation, deacetylation, phosphorylation, SUMOylation and/or ubiquitination of DNA or histone residues.
  • an effector domain of an epigenetic editor described herein may make histone tail modifications, e.g., by adding or removing active marks on histone tails.
  • an effector domain of an epigenetic editor described herein may comprise or recruit a transcription-related protein, e.g., a transcription repressor.
  • the transcription-related protein may be endogenous or exogenous.
  • an effector domain of an epigenetic editor described herein may, for example, comprise a protein that directly or indirectly blocks access of a transcription factor to the gene of interest harboring the target sequence.
  • An effector domain may be a full-length protein or a fragment thereof that retains the epigenetic effector function (a “functional domain”).
  • Functional domains that are capable of modulating (e.g., repressing) gene expression can be derived from a larger protein.
  • functional domains that can reduce target gene expression may be identified based on sequences of repressor proteins.
  • Amino acid sequences of gene expression-modulating proteins may be obtained from available genome browsers, such as the UCSD genome browser or Ensembl genome browser.
  • Protein annotation databases such as UniProt or Pfam can be used to identify functional domains within the full protein sequence. As a starting point, the largest sequence, encompassing all regions identified by different databases, may be tested for gene expression modulation activity. Various truncations then may be tested to identify the minimal functional unit.
  • variants of effector domains described herein are also contemplated by the present disclosure.
  • a variant may, for example, refer to a polypeptide with at least about 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity and/or sequence similarity to a wildtype effector domain described herein.
  • the variant retains at least about 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% of the epigenetic effector function of the wildtype effector domain.
  • an effector domain described herein may comprise a fusion of two or more effector domains (e.g., KOX1 KRAB and ZIM3).
  • the effector domain may, for example, comprise a fusion of 2, 3, 4, 5, 6, 7, 8, 9, or 10 effector domains, such as effector domains described herein.
  • an effector domain comprises a fusion of a truncated form of an effector domain and a second effector domain.
  • an effector domain comprises a fusion of the truncated forms of two effector domains (e.g., fusions of the N- and C-terminal portions of the two effector domains).
  • an epigenetic editor described herein may comprise 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, 10 effector domains, or more.
  • the epigenetic editor comprises one or more fusion proteins (e.g., one, two, or three fusion proteins), each with one or more effector domains (e.g., one, two, or three effector domains) linked to a DNA-binding domain.
  • the effector domains may induce a combination of epigenetic modifications, e.g., transcription repression and DNA methylation, DNA methylation and histone deacetylation, DNA methylation and histone demethylation, DNA methylation and histone methylation, DNA methylation and histone phosphorylation, DNA methylation and histone ubiquitylation, DNA methylation, and histone SUMOylation.
  • epigenetic modifications e.g., transcription repression and DNA methylation, DNA methylation and histone deacetylation, DNA methylation and histone demethylation, DNA methylation and histone methylation, DNA methylation and histone phosphorylation, DNA methylation and histone ubiquitylation, DNA methylation, and histone SUMOylation.
  • an effector domain described herein (e.g., DNMT3L) is encoded by a nucleotide sequence as found in the native genome (e.g., human or murine) for that effector domain.
  • an effector domain described herein is encoded by a nucleotide sequence that has been codon-optimized for optimal expression in human cells.
  • Effector domains described herein may include, for example, transcriptional repressors, DNA methyltransferases, and/or histone modifiers, as further detailed below.
  • an epigenetic effector domain described herein mediates repression of a target gene’s expression (e.g., transcription).
  • the effector domain may comprise, e.g., a Kriippel-associated box (KRAB) repressor domain, a Repressor Element Silencing Transcription Factor (REST) repressor domain, a KRAB -associated protein 1 (KAP1) domain, a MAD domain, a FKHR (forkhead in rhabdosarcoma 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), a WRPW motif of the hairy-related basic helix-loop-helix (bHLH) repressor proteins, an HP1 alpha chromo-shadow repressor domain, an HP1 beta repressor proteins
  • the effector domain may recruit one or more protein domains that repress expression of the target gene, e.g., through a scaffold protein.
  • the effector domain may recruit or interact with a scaffold protein domain that recruits a PRMT protein, a HD AC protein, a SETDB 1 protein, or a NuRD protein domain.
  • the effector domain comprises a functional domain derived from a zinc finger repressor protein, such as a KRAB domain.
  • KRAB domains are found in approximately 400 human ZFP-based transcription factors. Descriptions of KRAB domains may be found, for example, in Ecco et al., Development (2017) 144(15):2719-29 and Lambert et al., Cell (2018) 172:650-65.
  • the effector domain comprises a repressor domain (e.g., KRAB) derived from ZIM3, ZNF436, ZNF257, ZNF675, ZNF490, ZNF320, ZNF331, ZNF816, ZNF680, ZNF41, ZNF189, ZNF528, ZNF543, ZNF554, ZNF140, ZNF610, ZNF264, ZNF350, ZNF8, ZNF582, ZNF30, ZNF324, ZNF98, ZNF669, ZNF677, ZNF596, ZNF214, ZNF37, ZNF34, ZNF250, ZNF547, ZNF273, ZNF354, ZFP82, ZNF224, ZNF33, ZNF45, ZNF175, ZNF595, ZNF184, ZNF419, ZFP28-1, ZFP28-2, ZNF18, ZNF213, ZNF394, ZFP1, ZFP14, ZNF416, ZNF557, ZNF566, ZNF729, ZIM2, ZNF254, ZNF
  • a functional analog of any one of the above-listed proteins i.e., a molecule having the same or substantially the same biological function (e.g., retaining 70% or more, 80% or more, 90% or more, 95% or more, or 98% or more) of the protein’s transcription factor function) is encompassed by the present disclosure.
  • the functional analog may be an isoform or a variant of the above-listed protein, e.g., containing a portion of the above protein with or without additional amino acid residues and/or containing mutations relative to the above protein.
  • the functional analog has a sequence identity that is at least 75, 80, 85, 90, 95, 98, or 99% to one of the sequences listed in Table 4. Homologs, orthologs, and mutants of the above-listed proteins are also contemplated.
  • an epigenetic editor described herein may comprise a CDYL2, e.g., a human CDYL2, and/or a TOX domain (e.g., a human TOX domain) in combination with a KOX1 KRAB domain (e.g., a human KOX1 KRAB domain).
  • a CDYL2 e.g., a human CDYL2
  • a TOX domain e.g., a human TOX domain
  • a KOX1 KRAB domain e.g., a human KOX1 KRAB domain
  • the repressor domain may comprise the amino acid sequence of SEQ ID NO: 565, or a sequence at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 565.
  • the repressor domain may comprise the amino acid sequence of SEQ ID NO: 566, or a sequence at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 566.
  • the repressor domain may comprise the amino acid sequence of SEQ ID NO: 567, or a sequence at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 567.
  • the repressor domain may comprise the amino acid sequence of SEQ ID NO: 568, or a sequence at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 568.
  • the repressor domain may comprise the amino acid sequence of SEQ ID NO: 570, or a sequence at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 570.
  • the repressor domain may comprise the amino acid sequence of SEQ ID NO: 571, or a sequence at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 571.
  • the repressor domain may comprise the amino acid sequence of SEQ ID NO: 572, or a sequence at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 572.
  • an epigenetic effector domain described herein mediates activation of a target gene’s expression (e.g., transcription).
  • Some exemplary and nonlimiting activator effector domains, or combinations of effector domains, that may activate target gene expression contemplated by the present disclosure are OCT4 (POU5F1), GATA4, PU.l (SPI1), EOMES, PAX6 (FVH1), FOXA1 (HNF3A), FOXA2 (HNF3B), FOXD3 (HFH2), CEBPalpha, CEBPbeta, Satbl, KLF4, HNF4 alpha, HNF1 alpha, GATA2, GATA6, PBX1, EBF1, HOXB2, FOXN1, FOXR2, KLF14, SOX7, SPDYE4, CSRNP1, ATF6, CITED2-TAD, CITED1-TAD, C3orf62-TAD, CBX-C (CBX2), FAM22F-23, KLF6-1
  • An effector domain described herein may be, e.g., a DNA methyltransferase (DNMT), or a catalytic domain thereof, or may be capable of recruiting a DNA methyltransferase.
  • DNMTs encompass enzymes that catalyze the transfer of a methyl group to a DNA nucleotide, such as canonical cytosine-5 DNMTs that catalyze the addition of methyl groups to genomic DNA (e.g., DNMT3A, DNMT3B, and DNMT3C).
  • DNMT3L a non-limiting example of such a DNMT.
  • a DNMT domain may refer to a polypeptide domain derived from a catalytically active DNMT (e.g., DNMT3A, and DNMT3B) or from a catalytically inactive DNMT (e.g., DNMT3L).
  • a DNMT may repress expression of the target gene through the recruitment of repressive regulatory proteins.
  • the methylation is at a CG (or CpG) dinucleotide sequence.
  • the methylation is at a CHG or CHH sequence, where H is any one of A, T, or C.
  • a DNMT described herein can be an animal DNMT (e.g., a mammalian DNMT), a plant DNMT, a fungal DNMT, or a bacterial DNMT.
  • a bacterial DNMT can be obtained from a bacterial species (e.g., a coccus bacterium, bacillus bacterium, spiral bacterium, or an intracellular, gram-positive, or gram-negative bacterium.
  • the bacterial species is Mycoplasmatales bacterium, Mycoplasma marinum, or Spiroplasma chinense.
  • the bacterial species is not M. penetrans, S. monbiae, H.
  • an epigenetic editor described herein recruits a DNMT comprising a DNMT3A domain comprising SEQ ID NO: 574, or a sequence at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 574.
  • an epigenetic editor described herein recruits a DNMT comprising a DNMT3A domain comprising SEQ ID NO: 575, or a sequence at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 575
  • an epigenetic editor described herein comprises a DNMT3L domain comprising SEQ ID NO: 578, or a sequence at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 578.
  • an epigenetic editor herein comprises a DNMT3L domain comprising SEQ ID NO: 579, or a sequence at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 579.
  • the DNMT3L domain may have, e.g., a mutation corresponding to that at position D226 (such as D226V), Q268 (such as Q268K), or both (numbering according to SEQ ID NO: 578).
  • an epigenetic editor for repression of a target as described herein may recruit a DNMT domain, or a protein or polypeptide comprising a DNMT domain.
  • the DNMT domain, or protein or polypeptide comprising a DNMT domain is a naturally occurring DNMT domain, or protein or polypeptide comprising a DNMT domain.
  • the epigenetic editor as described herein comprises an antigen binding protein or antigen binding domain that binds to a DNMT domain or protein or polypeptide comprising a DNMT domain.
  • the epigenetic editor comprises a single domain antibody or antigen-binding single domain antibody domain that binds to a DNMT domain, or protein or polypeptide comprising a DNMT domain.
  • a single domain antibody or antigen-binding single domain antibody domain that binds to a DNMT domain, or protein or polypeptide comprising a DNMT domain.
  • Nonlimiting single domain antibodies and antigen-binding single domain antibody domains are described later in this application.
  • an epigenetic editor as described herein may recruit a DNMT3 domain, or a protein or polypeptide comprising a DNMT3 domain.
  • the DNMT3 domain, or protein or polypeptide comprising a DNMT3 domain is a naturally occurring DNMT3 domain, or protein or polypeptide comprising a DNMT3 domain.
  • the epigenetic editor as described herein comprises an antigen binding protein or antigen binding domain that binds to a DNMT3 domain or protein or polypeptide comprising a DNMT3 domain.
  • the epigenetic editor comprises a single domain antibody or antigen-binding single domain antibody domain that binds to a DNMT3 domain, or protein or polypeptide comprising a DNMT3 domain.
  • an epigenetic editor for activation of a target as described herein may recruit a domain that is associated with changes in DNA methylation.
  • a nonlimiting example of a domain that is associated with changes in DNA methylation is the ten eleven translocation (Tet) enzyme family.
  • Tet enzymes oxidize 5- methylcytosines (5mCs) and promote locus-specific reversal of DNA methylation.
  • Nonlimiting examples of Tet enzymes are human Tetl (UniProt ID: Q8NFU7) and human Tet2 (UniProt ID: Q6N021).
  • an epigenetic editor as described herein may recruit a Tet domain, or a protein or polypeptide comprising a Tet domain.
  • the Tet domain, or protein or polypeptide comprising a Tet domain is a naturally occurring Tet domain, or protein or polypeptide comprising a Tet domain.
  • the epigenetic editor as described herein comprises an antigen binding protein or antigen binding domain that binds to a Tet domain or protein or polypeptide comprising a Tet domain.
  • the epigenetic editor comprises a single domain antibody or antigenbinding single domain antibody domain that binds to a Tet domain, or protein or polypeptide comprising a Tet domain.
  • an epigenetic editor as described herein may recruit a Tetl domain, or a protein or polypeptide comprising a Tetl domain.
  • the Tetl domain, or protein or polypeptide comprising a Tetl domain is a naturally occurring Tetl domain, or protein or polypeptide comprising a Tetl domain.
  • the epigenetic editor as described herein comprises an antigen binding protein or antigen binding domain that binds to a Tetl domain or protein or polypeptide comprising a Tetl domain.
  • the epigenetic editor comprises a single domain antibody or antigenbinding single domain antibody domain that binds to a Tetl domain, or protein or polypeptide comprising a Tetl domain.
  • an epigenetic editor as described herein may recruit both DNMT and DNMT-like effector domains.
  • the epigenetic editor may comprise one or more single domain antibodies or antigen-binding single domain antibody domains that bind a DNMT3A domain and a DNMT3L domain, wherein the single domain antibodies or antigen-binding single domain antibody domains may be covalently linked.
  • Non-limiting linkers are described later in this application.
  • an epigenetic editor described herein may comprise a DNMT-like effector domain (such as a DNMT3L domain) and a single domain antibody or antigen-binding single domain antibody domain that binds a DNMT domain (e.g., human DNMT3A).
  • the DNMT-like domain such as a DNMT3L domain
  • DNMT3L is a human or mouse DNMT3L.
  • Table 6 below provides exemplary DNMTs that may be part of an epigenetic effector domain described herein, may be recruited by an epigenetic editor comprising, for example, a single domain antibody binding the DNMT, or from which an effector domain of an epigenetic editor described herein may be derived.
  • a functional analog of any one of the above-listed proteins i.e., a molecule having the same or substantially the same biological function (e.g., retaining 70% or more, 80% or more, 90% or more, 95% or more, or 98% or more) of the protein’s DNA methylation function or recruiting function) is encompassed by the present disclosure.
  • the functional analog may be an isoform or a variant of the above-listed protein, e.g., containing a portion of the above protein with or without additional amino acid residues and/or containing mutations relative to the above protein.
  • the functional analog has a sequence identity that is at least 75, 80, 85, 90, 95, 98, or 99% to one of the sequences listed in Table 6.
  • the effector domain herein comprises only the functional domain (or functional analog thereof), e.g., the catalytic domain or recruiting domain, of an abovelisted protein.
  • the effector domain herein comprises one or more epigenetic effector domains selected from Table 6, or functional homologs, orthologs, or variants thereof.
  • An epigenetic editor herein may effect methylation at, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500, 600, 700, 800, 900, or 1000 or more CpG dinucleotide sequences in the target gene or chromosome.
  • the CpG dinucleotide sequences may be located within or near the target gene in CpG islands, or may be located in a region that is not a CpG island.
  • a CpG island generally refers to a nucleic acid sequence or chromosome region that comprises a high frequency of CpG dinucleotides.
  • a CpG island may comprise at least 50% GC content.
  • the CpG island may have a high observed-to-expected CpG ratio, for example, an observed-to-expected CpG ratio of at least 60%.
  • an observed-to-expected CpG ratio is determined by Number of CpG * (sequence length) / (Number of C * Number of G).
  • the CpG island has an observed-to-expected CpG ratio of at least 60%, 70%, 80%, 90% or more.
  • an epigenetic editor herein effects methylation at a hypomethylated nucleic acid sequence, i.e., a sequence that may lack methyl groups on the 5- methyl cytosine nucleotides (e.g., in CpG) as compared to a standard control.
  • Hypomethylation may occur, for example, in aging cells or in cancer (e.g., early stages of neoplasia) relative to a younger cell or non-cancer cell, respectively.
  • methylation may be introduced by the epigenetic editor at a site other than a CpG dinucleotide.
  • the target gene sequence may be methylated at the C nucleotide of CpA, CpT, or CpC sequences.
  • an epigenetic editor comprises a DNMT3A domain and effects methylation at CpG, CpA, CpT, CpC sequences, or any combination thereof.
  • an epigenetic editor comprises a DNMT3A domain that lacks a regulatory subdomain and only maintains a catalytic domain.
  • the epigenetic editor comprising a DNMT3A catalytic domain effects methylation exclusively at CpG sequences.
  • an epigenetic editor comprising a DNMT3A domain that comprises a mutation e.g. a R836A or R836Q mutation (numbering according to SEQ ID NO: 574), has higher methylation activity at CpA, CpC, and/or CpT sequences as compared to an epigenetic editor comprising a wildtype DNMT3A domain.
  • an effector domain of an epigenetic editor herein mediates histone modification.
  • Histone modifications play a structural and biochemical role in gene transcription, such as by formation or disruption of the nucleosome structure that binds to the histone and prevents gene transcription.
  • Histone modifications may include, for example, acetylation, deacetylation, methylation, phosphorylation, ubiquitination, SUMOylation and the like, e.g., at their N-terminal ends (“histone tails”). These modifications maintain or specifically convert chromatin structure, thereby controlling responses such as gene expression, DNA replication, DNA repair, and the like, which occur on chromosomal DNA.
  • Post-translational modification of histones is an epigenetic regulatory mechanism and is considered essential for the genetic regulation of eukaryotic cells.
  • chromatin remodeling factors such as SWI/SNF, RSC, NURF, NRD, and the like, which facilitate transcription factor access to DNA by modifying the nucleosome structure; histone acetyltransferases (HATs) that regulate the acetylation state of histones; and histone deacetylases (HDACs), act as important regulators.
  • HATs histone acetyltransferases
  • HDACs histone deacetylases
  • the unstructured N-termini of histones may be modified by acetylation, deacetylation, methylation, ubiquitylation, phosphorylation, SUMOylation, ribosylation, citrullination O-GlcNAcylation, crotonylation, or any combination thereof.
  • histone acetyltransferases utilize acetyl-CoA as a cofactor and catalyze the transfer of an acetyl group to the epsilon amino group of the lysine side chains.
  • lysine This neutralizes the lysine’s positive charge and weakens the interactions between histones and DNA, thus opening the chromosomes for transcription factors to bind and initiate transcription.
  • Acetylation of K14 and K9 lysines of histone H3 by histone acetyltransferase enzymes may be linked to transcriptional competence in humans. Lysine acetylation may directly or indirectly create binding sites for chromatin-modifying enzymes that regulate transcriptional activation.
  • histone methylation of lysine 9 of histone H3 may be associated with heterochromatin, or transcriptionally silent chromatin.
  • an effector domain of an epigenetic editor described herein comprises a histone methyltransferase domain.
  • the effector domain may comprise, for example, a D0T1L 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.
  • the effector domain comprises a histone-lysine-N- methyltransferase SETDB1 domain.
  • the effector domain comprises a histone deacetylase protein domain.
  • the effector domain comprises a HD AC family protein domain, for example, a HDAC1, HDAC3, HDAC5, HDAC7, or HDAC9 protein domain.
  • the effector domain comprises a nucleosome remodeling and deacetylase complex (NURD), which removes acetyl groups from histones.
  • NURD nucleosome remodeling and deacetylase complex
  • the effector domain comprises a tripartite motif containing protein (TRIM28, TIFl-beta, or KAP1).
  • the effector domain comprises one or more KAP1 proteins.
  • a KAP1 protein in an epigenetic editor herein may form a complex with one or more other effector domains of the epigenetic editor or one or more proteins involved in modulation of gene expression in a cellular environment.
  • KAP1 may be recruited by a KRAB domain of a transcriptional repressor.
  • a KAP1 protein domain may interact with or recruit one or more protein complexes that reduces or silences gene expression.
  • KAP1 interacts with or recruits a histone deacetylase protein, a histone-lysine methyltransferase protein, a chromatin remodeling protein, and/or a heterochromatin protein.
  • a KAP1 protein domain may interact with or recruit a heterochromatin protein 1 (HP1) protein, a SETDB1 protein, an HD AC protein, and/or a NuRD protein complex component.
  • a KAP1 protein domain interacts with or recruits a ZFP90 protein (e.g., isoform 2 of ZFP90), and/or a FOXP3 protein.
  • An exemplary KAP1 amino acid sequence is shown in SEQ ID NO: 629.
  • the effector domain comprises a protein domain that interacts with or is recruited by one or more DNA epigenetic marks.
  • the effector domain may comprise a methyl CpG binding protein 2 (MECP2) protein that interacts with methylated DNA nucleotides in the target gene (which may or may not be at a CpG island of the target gene).
  • MECP2 protein domain in an epigenetic editor described herein may induce condensed chromatin structure, thereby reducing or silencing expression of the target gene.
  • an MECP2 protein domain in an epigenetic editor described herein may interact with a histone deacetylase (e.g. HD AC), thereby repressing or silencing expression of the target gene.
  • a histone deacetylase e.g. HD AC
  • an MECP2 protein domain in an epigenetic editor described herein may block access of a transcription factor or transcriptional activator to the target sequence, thereby repressing or silencing expression of the target gene.
  • An exemplary MECP2 amino acid sequence is shown in SEQ ID NO: 630.
  • effector domains for the epigenetic editors described herein are, e.g., a chromoshadow domain, a ubiquitin-2 like Rad60 SUMO-like (Rad60-SLD/SUMO) domain, a chromatin organization modifier domain (Chromo) domain, a Y af2/RYBP C- terminal binding motif domain (YAF2_RYBP), a CBX family C-terminal motif domain (CBX7_C), a zinc finger C3HC4 type (RING finger) domain (ZF-C3HC4_2), a cytochrome b5 domain (Cyt-b5), a helix-loop-helix domain (HLH), a helix-hairpin-helix motif domain (e.g., HHH_3), a high mobility group box domain (HMG-box), a basic leucine zipper domain (e.g., bZIP_l or bZIP_2), a Myb_DNA-bind
  • the effector domain is a protein domain comprising a YAF2_RYBP domain or homeodomain or any combination thereof.
  • the homeodomain of the YAF2_RYBP domain is a PRD domain, an NKL domain, a HOXL domain, or a LIM domain.
  • the YAF2_RYBP domain may comprise a 32 amino acid Yaf2/RYBP C-terminal binding motif domain (32 aa RYBP).
  • the effector domain comprises a protein domain selected from a group consisting of SUMO3 domain, Chromo domain from M phase phosphoprotein 8 (MPP8), chromoshadow domain from Chromobox 1 (CBX1), and SAM_1/SPM domain from Scm Polycomb Group Protein Homolog 1 (SCMH1).
  • MPP8 Chromo domain from M phase phosphoprotein 8
  • CBX1 Chromobox 1
  • SCMH1 Scm Polycomb Group Protein Homolog 1
  • the effector domain comprises an HNF3 C-terminal domain (HNF_C).
  • HNF_C domain may be from FOXA1 or FOXA2.
  • the HNF_C domain comprises an EH1 (engrailed homology 1) motif.
  • the effector domain may comprise an interferon regulatory factor 2-binding protein zinc finger domain (IRF-2BP1_2), a Cyt-b5 domain from DNA repair factor HERC2 E3 ligase, a variant SH3 domain (SH3_9) from Bridging Integrator 1 (BINI), an HMG-box domain from transcription factor TOX or ZF-C3HC4_2 RING finger domain from the polycomb component PCGF2, a Chromodomain-helicase-DNA binding protein 3 (CHD3) domain, or a ZNF783 domain.
  • IRF-2BP1_2 interferon regulatory factor 2-binding protein zinc finger domain
  • BINI Bridging Integrator 1
  • HMG-box domain from transcription factor TOX or ZF-C3HC4_2 RING finger domain from the polycomb component PCGF2
  • CHD3 Chromodomain-helicase-DNA binding protein 3
  • the epigenetic editors provided herein comprise a DNA binding domain that specifically binds a target sequence in a gene of interest, and an EBD specifically binding an epigenetic effector domain.
  • the EBD recruits the epigenetic effector domain to the target sequence bound by the DNA binding domain.
  • Exemplary EBDs are provided herein, e.g., antibodies and antigen-binding fragments thereof that bind to epigenetic effector domains. Additional suitable EBDs will be apparent to the skilled artisan based on the present disclosure and the disclosure is not limited in this respect.
  • the present disclosure relates to epigenetic editors capable of recruiting naturally-occurring one or more DNMT domain (e.g., DNMT3A) or other enzyme capable of modulating DNA methylation (e.g., Tetl, or Tet2).
  • the epigenetic editors comprise antigen-binding proteins capable of binding DNMT3A, Tetl, or Tet2.
  • an antigen-binding protein is an antibody or antibody fragment capable of binding DNMT3A, Tetl, or Tet2.
  • the antibody fragment is a single chain variable fragment (scFv).
  • the antibody fragment is a single domain antibody (sdAb) comprising only a heavy chain variable domain of a camelid heavy chain antibody (VHH) or a heavy chain variable domain of a four-chain antibody (VH).
  • the single domain antibody is a NANOBODY® (Ablynx N.V., Belgium). See, e.g., WO2012/175741 and WO 2015/173325, each of which is incorporated herein in its entirety.
  • the single domain antibody is a camelid single domain antibody.
  • the single domain antibody is a humanized single domain antibody.
  • the single domain antibody is a human single domain antibody.
  • the single domain antibody or antigen-binding single domain antibody domain has a sequence set out in Table 7.
  • Non-limiting single domain antibody or antigen-binding single domain antibody domain sequences are determined by the complementarity-determining regions (CDRs) contained in the antigen-binding protein or antibody.
  • CDRs are hypervariable regions of the variable domain or domains of an antigen-binding protein or antibody and form part of the binding interface between the antibody and an antigen.
  • a single domain antibody disclosed herein comprises one or more CDRs disclosed in Table 8 below. Table 8.
  • an epigenetic editor described herein comprises one single domain antibody or antigen-binding single domain antibody domain. In some embodiments, an epigenetic editor described herein comprises two or more single domain antibodies or antigen-binding single domain antibody domains. In some embodiments, an epigenetic editor comprises between 1 and 6 single domain antibodies or antigen-binding single domain antibody domains. In some embodiments, an epigenetic editor comprises two single domain antibodies or antigen-binding single domain antibody domains. In some embodiments, an epigenetic editor described herein comprises one or more single domain antibodies or antigen-binding single domain antibody domains and one or more protein tags for signal amplification (e.g., SunTag).
  • SunTag protein tags for signal amplification
  • the epigenetic editor described herein comprises two single domain antibodies encoded by the nucleic acid sequences disclosed in Table 9 below.
  • the two or more single domain antibodies or antigen-binding single domain antibody domains bind the same antigen. In some embodiments, the two or more single domain antibodies or antigen-binding single domain antibody domains bind the same epitope on the same antigen. In some embodiments, the two or more single domain antibodies or antigen-binding single domain antibody domains bind different epitopes on the same antigen. In some embodiments, the two or more single domain antibodies or antigenbinding single domain antibody domains bind different antigens.
  • an epigenetic editor comprises two or more single domain antibodies or antigen-binding single domain antibody domains wherein one binds DNMT3A and one binds Tetl.
  • an epigenetic editor comprises two or more single domain antibodies or antigen-binding single domain antibody domains wherein one binds DNMT3A and one binds Tet2. In other embodiments, an epigenetic editor comprises two or more single domain antibodies or antigen-binding single domain antibody domains wherein one binds Tetl and one binds Tet2.
  • the present disclosure provides epigenetic editors (also referred to herein as epigenetic editing systems) for repressing or activating expression of a gene of interest, e.g., by directing epigenetic modification(s) to a target sequence in the gene of interest.
  • the epigenetic editors provided herein comprise a fusion protein that comprises a DNA binding domain and an effector binding domain (EBD), wherein the EBD binds at least one endogenous effector, e.g., an epigenetic effector domain.
  • EBD effector binding domain
  • an epigenetic editor provided herein comprises a DNA binding domain and an EBD that binds a transcriptional repressor, a transcriptional activator, a DNA methyltransferase, a DNA demethylase (e.g., a DNA methylcytosine dioxygenase), and/or a histone modifier.
  • an epigenetic editor provided herein comprises a DNA binding domain and an EBD that binds one or more transcriptional repressors.
  • an epigenetic editor provided herein comprises a DNA binding domain and an EBD that binds a transcriptional activator.
  • an epigenetic editor provided herein comprises a DNA binding domain and an EBD that binds a DNA methyltransferase. In some embodiments, an epigenetic editor provided herein comprises a DNA binding domain and an EBD that binds a DNA demethylase. In some embodiments, an epigenetic editor provided herein comprises a DNA binding domain and an EBD that binds a histone modifier.
  • the DNA-binding domain (in concert with a guide polynucleotide such as one described herein, where the DNA-binding domain is a polynucleotide guided DNA-binding domain) directs the effector domain to epigenetically modify a target sequence in the gene of interest.
  • the epigenetic editor comprises an EBD that binds an effector domain that represses gene expression, thus recruiting the effector domain to the target sequence in the gene of interest, resulting in repression of gene expression (silencing) of the gene of interest.
  • the epigenetic editor comprises an EBD that binds an effector domain that activates gene expression or relieves gene repression, e.g., by removing DNA methyl marks, thus recruiting the effector domain to the target sequence in the gene of interest, resulting in activation of gene expression (activation) of the gene of interest.
  • the repression or activation of the gene of interest is durable and inheritable across cell generations.
  • the repression or activation of the gene of interest is temporary.
  • the repression or activation of the gene of interest is reversible.
  • an epigenetic editor provided herein comprises one or more fusion proteins that comprises (1) a DNA-binding domain; (2) an EBD; and (3) an effector domain, e.g., and epigenetic effector domain.
  • an epigenetic editor provided herein comprises one or more fusion proteins that comprises (1) a DNA-binding domain; (2) an EBD; and (3) two or more effector domains, e.g., two or more epigenetic effector domains.
  • an epigenetic editor comprises one or more fusion proteins that comprises (1) a DNA-binding domain; (2) an EBD; and (3) two or more effector domains, e.g., two or more epigenetic effector domains, wherein the two or more epigenetic effector domains are different effector domains, e.g., a DNA methyltransferase domain and a histone modifier.
  • the epigenetic editor comprises a single fusion protein. In some embodiments, the epigenetic editor consists of a single fusion protein. In some embodiments, the epigenetic editor comprises two or more fusion proteins, wherein each fusion protein comprises, independently, (1) a DNA-binding domain; (2) an EBD; and, optionally, (3) an effector domain.
  • the epigenetic editor comprises a single EBD. In some embodiments, the epigenetic editor comprises two or more EBDs. In some embodiments, the epigenetic editor comprises two or more EBDs that comprise the same structure (e.g., the same amino acid sequence or chemical structure). In some embodiments, the epigenetic editor comprises two or more EBDs that comprise a different structure (e.g., each of the two or more EBDs comprises a different amino acid sequence or chemical structure). In some embodiments, the epigenetic editor comprises two or more EBDs that bind the same epigenetic effector domain. In some embodiments, the epigenetic editor comprises two or more EBDs that bind different epigenetic effector domains.
  • the epigenetic editor comprises two or more EBDs that bind the same epigenetic effector domain, wherein the two or more EBDs comprise a different structure (e.g., each of the two or more EBDs comprises a different amino acid sequence or chemical structure). In some embodiments, the epigenetic editor comprises two or more EBDs that bind the same epigenetic effector domain, wherein the two or more EBDs bind a different antigen or binding domain of the epigenetic effector domain. In some embodiments, the epigenetic editor comprises an EBDs that binds an epigenetic effector domain, wherein the epigenetic effector domain comprises a catalytic domain, and wherein the EBD does not bind the catalytic domain.
  • the EBD comprises an antibody or an antigen-binding fragment thereof that specifically binds an epigenetic effector. In some embodiments, the EBD comprises a single domain antibody or an antigen-binding single domain antibody domain.
  • a fusion protein described herein may further comprise one or more linkers (e.g., peptide linkers), detectable tags, nuclear localization signals (NLSs), or any combination thereof.
  • linkers e.g., peptide linkers
  • detectable tags e.g., detectable tags
  • NLSs nuclear localization signals
  • fusion protein refers to a chimeric protein in which two or more coding sequences (e.g., for a DNA-binding domain, an EBD, and/or an effector domain) are covalently joined, typically via a peptide bond.
  • an epigenetic editor described herein comprises a DNA binding domain and an EBD. In some embodiments, an epigenetic editor described herein comprises 2, 3, 4, 5, 6, 7, 8, 9, 10, or more EBDs, which may be identical or different. In certain embodiments, two or more of said EBDs function synergistically. In some embodiments, an epigenetic editor comprises an EBD or a combination of two or more EBDs that bind to a transcriptional repressor, a transcriptional activator, a DNA methyltransferase, a DNA demethylase, and/or a histone modifier.
  • an epigenetic editor described herein may comprise an EBD or a combination of two or more EBDs that bind to one or more transcriptional repressor domains (e.g., a KRAB domain such as K0X1, ZIM3, ZFP28, or ZN627 KRAB), one or more DNA methylation domains (e.g., a DNMT3A domain).
  • a KRAB domain such as K0X1, ZIM3, ZFP28, or ZN627 KRAB
  • DNA methylation domains e.g., a DNMT3A domain
  • Such an epigenetic editor may comprise, for instance, a DNA binding domain, a KRAB domain, and an EBD binding to an endogenous DNMT3A protein.
  • the epigenetic editor may further comprise a DNMT3L domain.
  • the epigenetic editor further comprises an additional effector domain, or an EBD binding to an additional effector domain.
  • the additional effector domain is a KAP1, MECP2, HPlb, CBX8, CDYL2, TOX, T0X3, T0X4, EED, RBBP4, RCOR1, or SCML2 domain.
  • the additional effector domain is a CDYL2, TOX, T0X3, T0X4, or HP la domain.
  • an epigenetic editor described herein may comprise a CDYL2 and/or a TOX domain in combination with a KRAB domain (e.g., a K0X1 KRAB domain).
  • an epigenetic editor described herein comprises a DNA binding domain, an EBD, and an epigenetic effector domain.
  • an epigenetic editor described herein comprises 2, 3, 4, 5, 6, 7, 8, 9, 10, or more effector (e.g., repression/repressor) domains, which may be identical or different.
  • effector e.g., repression/repressor
  • two or more of said effector domains function synergistically.
  • Combinations of effector domains may comprise DNA methylation domains, histone deacetylation domains, histone methylation domains, and/or scaffold domains that recruit any of the above.
  • an epigenetic editor described herein may comprise one or more transcriptional repressor domains (e.g., a KRAB domain such as KOX1, ZIM3, ZFP28, or ZN627 KRAB) in combination with one or more DNA methylation domains (e.g., a DNMT domain) and/or recruiter domain (e.g., a DNMT3L domain).
  • a KRAB domain such as KOX1, ZIM3, ZFP28, or ZN627 KRAB
  • DNA methylation domains e.g., a DNMT domain
  • recruiter domain e.g., a DNMT3L domain
  • the epigenetic editor further comprises an additional effector domain (e.g., a KAP1, MECP2, HPlb, CBX8, CDYL2, TOX, TOX3, TOX4, EED, RBBP4, RCOR1, or SCML2 domain).
  • the additional effector domain is a CDYL2, TOX, TOX3, TOX4, or HPla domain.
  • an epigenetic editor described herein may comprise a CDYL2 and/or a TOX domain in combination with a KRAB domain (e.g., a KOX1 KRAB domain).
  • a fusion protein as described herein may comprise one or more linkers that connect components of the epigenetic editor.
  • a linker may be a peptide or non-peptide linker.
  • one or more linkers utilized in an epigenetic editor provided herein is a peptide linker, i.e., a linker comprising a peptide moiety.
  • a peptide linker can be any length applicable to the epigenetic editor fusion proteins described herein.
  • the linker can comprise a peptide between 1 and 250 (e.g., between 1 and 80) amino acids.
  • the linker comprises 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, 1 to 250, 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, 5 to 250, 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, 10 to 250, 20 to 30, 20 to 40, 20 to 50, 20 to 60, 20 to 80, 20 to 100, 20 to 150, 20 to 200, 20 to 250, 30 to 40, 30 to 50, 30 to 60, 30 to 80, 30 to 100, 30 to 150, 30 to 200, 30 to 250, 40 to 50, 40 to 60, 40 to 80, 40 to 100, 40 to 150, 40 to 200, 40 to 250, 50 to 60, 50 to 80, 50 to 100, 50 to 150, 50 to 200, 50 to 250, 60 to 80, 50 to 100, 50 to 150
  • the peptide linker may be any suitable amino acids in length.
  • the peptide linker may be any suitable amino acids in length.
  • the peptide linker may be any suitable amino acids in length.
  • the peptide linker may be a flexible or rigid linker.
  • the peptide linker comprises the amino acid sequence of any one of SEQ ID NOs: 631-637 and 664-665 or a sequence at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical thereto.
  • the peptide linker is an XTEN linker.
  • a linker may comprise part of the XTEN sequence (Schellenberger et al., Nat Biotechnol (2009) 27(1): 1186-90), an unstructured hydrophilic polypeptide consisting only of residues G, S, P, T, E, and A.
  • XTEN refers to a recombinant peptide or polypeptide lacking hydrophobic amino acid residues.
  • XTEN linkers typically are unstructured and comprise a limited set of natural amino acids. Fusion of XTEN to proteins alters its hydrodynamic properties and reduces the rate of clearance and degradation of the fusion protein.
  • the XTEN linker may be, for example, 5, 10, 16, 20, 26, or 80 amino acids in length. In some embodiments, the XTEN linker is 16 amino acids in length. In some embodiments, the XTEN linker is 80 amino acids in length. In certain embodiments, the XTEN linker may be XTEN10, XTEN16, XTEN20, or XTEN80.
  • the XTEN linker may comprise the amino acid sequence of any one of SEQ ID NOs: 638-643 or a sequence at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical thereto.
  • the XTEN linker comprises the amino acid sequence of SEQ ID NO: 638.
  • the XTEN linker comprises the amino acid sequence of SEQ ID NO: 643.
  • one or more linkers utilized in an epigenetic editor provided herein is a non-peptide linker.
  • the linker may be a carbon bond, a disulfide bond, or carbon-heteroatom bond.
  • the linker is a carbon-nitrogen bond of an amide linkage.
  • the linker is a cyclic or acyclic, substituted or unsubstituted, or branched or unbranched aliphatic or heteroaliphatic linker.
  • one or more linkers utilized in an epigenetic editor provided herein is polymeric (e.g., polyethylene, polyethylene glycol, polyamide, polyester, etc.).
  • the linker may comprise, for example, a monomer, dimer, or polymer of aminoalkanoic acid; an aminoalkanoic acid (e.g., glycine, ethanoic acid, alanine, beta- alanine, 3-aminopropanoic acid, 4-aminobutanoic acid, 5-pentanoic acid, etc.); a monomer, dimer, or polymer of aminohexanoic acid (Ahx); or a polyethylene glycol moiety (PEG); or an aryl or heteroaryl moiety.
  • an aminoalkanoic acid e.g., glycine, ethanoic acid, alanine, beta- alanine, 3-aminopropanoic acid, 4-aminobutanoic acid, 5-pentanoic acid,
  • the linker may be based on a carbocyclic moiety (e.g., cyclopentane or cyclohexane) or a phenyl ring.
  • the linker may include functionalized moieties to facilitate attachment of a nucleophile (e.g., thiol, amino) from the peptide to the linker.
  • Any electrophile may 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.
  • linker lengths and flexibilities can be employed between any two components of an epigenetic editor (e.g., between an effector domain (e.g., a repressor domain) and a DNA-binding domain (e.g., a Cas9 domain), between a first effector domain and a second effector domain, etc.).
  • the linkers may range from very flexible linkers, such as glycine/serine-rich linkers, to more rigid linkers, in order to achieve the optimal length for effector domain activity for the specific application.
  • the more flexible linkers are glycine/serine-rich linkers (GS-rich linkers), where more than 45% (e.g., more than 48, 50, 55, 60, 70, 80, or 90%) of the residues are glycine or serine residues.
  • GS-rich linkers are (GGGGS)n (SEQ ID NO: 664), (G)n (SEQ ID NO: 658), and W linker (SEQ ID NO: 637).
  • the more rigid linkers are in the form of the form (EAAAK)n (SEQ ID NO: 665), (SGGS)n (SEQ ID NO: 631), and (XP)n) (SEQ ID NO: 659).
  • n may be 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 comprises a (GGS)n (SEQ ID NO: 660) motif, wherein n is 1, 3, or 7. In some embodiments, the linker comprises a (GGGGS)n motif, wherein n is 4 (SEQ ID NO: 636).
  • a linker in an epigenetic editor described herein comprises a nuclear localization signal, for example, with the amino acid sequence of any one of SEQ ID NOs: 644-649.
  • a linker in an epigenetic editor described herein comprises an expression tag, e.g., a detectable tag such as a green fluorescent protein.
  • a fusion protein described herein may comprise one or more nuclear localization signals, and in certain embodiments, may comprise two or more nuclear localization signals.
  • the fusion protein may comprise 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more nuclear localization signals.
  • a “nuclear localization signal” is an amino acid sequence that directs proteins to the nucleus.
  • the NLS may be an SV40 NLS (e.g., with the amino acid sequence of SEQ ID NO: 644).
  • the fusion protein may comprise two NLSs.
  • the fusion protein may comprise two NLSs at its N-terminus or C-terminus.
  • the fusion protein may comprise one NLS located at its N-terminus and one NLS embedded in the middle of the fusion protein, or one NLS located at its C-terminus and one NLS embedded in the middle of the fusion protein.
  • the fusion protein may comprise two NLSs embedded in the middle of the fusion protein.
  • the fusion protein may comprise four NLSs.
  • the fusion protein may comprise at least two (e.g., two, three, or four) NLSs at its N-terminus or C- terminus.
  • the fusion protein may comprise at least one (e.g., one, two, three, or four) NLSs embedded in the middle of the fusion protein.
  • the fusion protein may comprise two NLSs at its N-terminus and two NLSs at its C-terminus.
  • an epigenetic editor comprising a fusion protein that comprises at least one NLS at the N-terminus and at least one NLS at the C-terminus may increase the efficiency of the epigenetic editor by at least 5%, at least 10%, at least 15%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 100%, at least 200%, at least 300%, at least 400%, at least 500%, at least 600%, at least 700%, at least 800%, at least 900%, at least 1,000%, at least 5,000%, at least 10,000%, at least 50,000%, at least 100,000%, or more as compared to an epigenetic editor with a corresponding fusion protein that does not have at least one NLS at the N-terminus and at least one NLS at the C-terminus.
  • an epigenetic editor comprising a fusion protein that comprises two NLSs at the N-terminus and two NLSs at the C-terminus may increase the efficiency of the epigenetic editor by at least 5%, at least 10%, at least 15%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 100%, at least 200%, at least 300%, at least 400%, at least 500%, at least 600%, at least 700%, at least 800%, at least 900%, at least 1,000%, at least 5,000%, at least 10,000%, at least 50,000%, at least 100,000%, or more as compared to an epigenetic editor with a corresponding fusion protein that does not have two NLSs at the N-terminus and two NLSs at the C-terminus.
  • Epigenetic editors provided herein may comprise one or more additional sequences (“tags”) for tracking, detection, and localization of the editors.
  • the epigenetic editor comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more detectable tags. Each of the detectable tags may be the same or different.
  • an epigenetic editor fusion protein may comprise cytoplasmic localization sequences, export sequences, such as nuclear export sequences, or other localization sequences, as well as sequence tags that are useful for solubilization, purification, or detection of the fusion proteins.
  • a fusion protein of an epigenetic editor described herein may have its components structured in different configurations.
  • the DNA-binding domain may be at the C-terminus, the N-terminus, or in between two or more epigenetic effector domains or additional domains.
  • the DNA-binding domain is at the C-terminus of the epigenetic editor.
  • the DNA-binding domain is at the N-terminus of the epigenetic editor.
  • the DNA-binding domain is linked to one or more nuclear localization signals.
  • the DNA-binding domain is flanked by an epigenetic effector domain and/or an additional domain on both sides.
  • x is an integer between 1 and 10
  • y is an integer between 1 and 4
  • n is an integer between 0 and 1.
  • x is an integer between 1 and 5
  • y is an integer between 1 and 2
  • n is an integer between 0 and 1.
  • x is an integer between 1 and 2
  • y is an integer between 1 and 2
  • n is an integer between 0 and 1.
  • x is an integer between 1 and 2
  • y is 1, and n is an integer between 0 and 1.
  • x is 1, y is 1, and n is 1.
  • x is 2, y is 1, and n is 1. In some embodiments of the configurations above, y is 1. In some embodiments of the configurations above, y is 2. In some embodiments of the configurations above, x is 1. In some embodiments of the configurations above, x is 2.
  • the fusion protein comprises an SDA that binds to DNMT3A.
  • the fusion protein comprises the configuration of: 2XNLS- A 1 -Wlink-C 10- Wlink-DNMT3L-dCas9-2XNLS 2XNLS-G3-Wlink-C10-Wlink-DNMT3L-dCas9-2XNLS 2XNLS-Al-Wlink-C10-Wlink-DNMT3L-dCas9-zim3-2XNLS 2XNLS- A 1 -Wlink-C 10- Wlink-DNMT3L-dCas9-kox lkrab-2XNLS 2XNLS-Al-Wlink-C10-Wlink-DNMT3L-dCas9-ZN627-2XNLS 2XNLS-Al-Wlink-C10-Wlink-DNMT3L(-ADD)-dCas9-zim3-2XNLS 2XNLS-Al-Wlink-C10-Wlink-DNMT3L(-ADD)-dCas9-zim3-2X
  • the fusion protein comprises an SDA that binds to Tet2
  • the present disclosure provides a pharmaceutical composition
  • a pharmaceutical composition comprising as an active ingredient (or as the sole active ingredient) one or more epigenetic editors described herein or component(s) (e.g., fusion proteins and/or guide polynucleotides) thereof, or nucleic acid molecule(s) encoding said epigenetic editors or component(s) thereof.
  • a pharmaceutical composition may comprise nucleic acid molecule(s) encoding the fusion protein(s) (and guide polynucleotides, where applicable) of an epigenetic editor described herein.
  • separate pharmaceutical compositions comprise the fusion protein(s) and the guide polynucleotide(s).
  • a pharmaceutical composition may also comprise cells that have undergone epigenetic modification(s) mediated or induced by an epigenetic editor provided herein.
  • the epigenetic editors described herein or component(s) thereof, or nucleic acid molecule(s) encoding said epigenetic editors or component(s) thereof, of the present disclosure are suitable to be administered as a formulation in association with one or more pharmaceutically acceptable excipient(s), e.g., as described below.
  • excipient is used herein to describe any ingredient other than the compound(s) of the present disclosure.
  • the choice of excipient(s) will to a large extent depend on factors such as the particular mode of administration, the effect of the excipient on solubility and stability, and the nature of the dosage form.
  • pharmaceutically acceptable excipient includes any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like that are physiologically compatible.
  • Some examples of pharmaceutically acceptable excipients are water, saline, phosphate buffered saline, dextrose, glycerol, ethanol and the like, as well as combinations thereof.
  • isotonic agents for example, sugars, polyalcohols such as mannitol, sorbitol, or sodium chloride in the composition.
  • additional examples of pharmaceutically acceptable substances are wetting agents or minor amounts of auxiliary substances such as wetting or emulsifying agents, preservatives, or buffers, which enhance the shelf life or effectiveness of the antibody.
  • Formulations of a pharmaceutical composition suitable for parenteral administration typically comprise the active ingredient combined with a pharmaceutically acceptable carrier, such as sterile water or sterile isotonic saline. Such formulations may be prepared, packaged, or sold in a form suitable for bolus administration or for continuous administration.
  • a pharmaceutically acceptable carrier such as sterile water or sterile isotonic saline.
  • the epigenetic editor or its component(s) are introduced to target cells in the form of nucleic acid molecule(s) encoding the epigenetic editor or its component(s); accordingly, the pharmaceutical compositions herein comprise the nucleic acid molecule(s).
  • nucleic acid molecule(s) may be, for example, DNA, RNA or mRNA, and/or modified nucleic acid sequence(s) (e.g., with chemical modifications, a 5’ cap, or one or more 3’ modifications).
  • the nucleic acid molecule(s) may be delivered as naked DNA or RNA, for instance by means of transfection or electroporation, or can be conjugated to molecules (e.g., N-acetylgalactosamine) promoting uptake by target cells.
  • the nucleic acid molecule(s) may be in nucleic acid expression vector(s), which may include expression control sequences such as promoters, enhancers, transcription signal sequences, transcription termination sequences, introns, polyadenylation signals, Kozak consensus sequences, internal ribosome entry sites (IRES), etc. Such expression control sequences are well known in the art.
  • a vector may also comprise a sequence encoding a signal peptide (e.g., for nuclear localization, nucleolar localization, or mitochondrial localization), associated with (e.g., inserted into or fused to) a sequence coding for a protein.
  • a signal peptide e.g., for nuclear localization, nucleolar localization, or mitochondrial localization
  • vectors include, but are not limited to, plasmid vectors; viral vectors based on vaccinia virus, poliovirus, adenovirus, adeno-associated virus, SV40, herpes simplex virus, human immunodeficiency virus, retrovirus (e.g., Murine Leukemia Virus, or spleen necrosis virus, vectors derived from retroviruses such as Rous Sarcoma Virus, Harvey Sarcoma Virus, avian leukosis virus, a lentivirus, human immunodeficiency virus, myeloproliferative sarcoma virus, and mammary tumor virus); and other recombinant vectors.
  • retrovirus e.g., Murine Leukemia Virus, or spleen necrosis virus, vectors derived from retroviruses such as Rous Sarcoma Virus, Harvey Sarcoma Virus, avian leukosis virus, a lentivirus, human immunodeficiency virus, mye
  • the vector is a plasmid or a viral vector.
  • Viral particles or virus-like particles may also be used to deliver nucleic acid molecule(s) encoding epigenetic editors or component(s) thereof as described herein.
  • empty viral particles can be assembled to contain any suitable cargo.
  • Viral vectors and viral particles may also be engineered to incorporate targeting ligands to alter target tissue specificity.
  • an epigenetic editor as described herein or component(s) thereof are encoded by nucleic acid sequence(s) present in one or more viral vectors, or a suitable capsid protein of any viral vector.
  • viral vectors include adeno- associated viral vectors (e.g., derived from AAV3, AAV3b, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAVrh8, AAV10, and/or variants thereof); retroviral vectors (e.g., Maloney murine leukemia virus, MML-V), adenoviral vectors (e.g., AD100), lentiviral vectors (e.g., HIV and FIV-based vectors), and herpesvirus vectors (e.g., HSV-2).
  • adeno- associated viral vectors e.g., derived from AAV3, AAV3b, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAVrh8, AAV10,
  • delivery involves an adeno-associated virus (AAV) vector.
  • AAV vector delivery may be particularly useful where the DNA-binding domain of an epigenetic editor fusion protein is a zinc finger array.
  • the smaller size of zinc finger arrays compared to larger DNA-binding domains such as Cas protein domains may allow such a fusion protein to be conveniently packed in viral vectors such as an AAV vector.
  • AAV serotype e.g., human AAV serotype
  • AAV serotype 1 AAV1
  • AAV2 AAV serotype 2
  • AAV3 AAV 3
  • 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
  • AAV11 AAV serotype 11
  • an AAV variant has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% amino acid sequence identity to a wildtype AAV.
  • the AAV variant may be engineered such that its capsid proteins have reduced immunogenicity or enhanced transduction ability in humans.
  • one or more regions of at least two different AAV serotype viruses are shuffled and reassembled to generate a chimeric variant.
  • a chimeric AAV may comprise inverted terminal repeats (ITRs) that are of a heterologous serotype compared to the serotype of the capsid.
  • a chimeric variant of an AAV includes amino acid sequences from 2, 3, 4, 5, or more different AAV serotypes.
  • Non-viral systems are also contemplated for delivery as described herein.
  • Non-viral systems include, but are not limited to, nucleic acid transfection methods including electroporation, sonoporation, calcium phosphate transfection, microinjection, DNA biolistics, lipid-mediated transfection, transfection through heat shock, compacted DNA- mediated transfection, lipofection, cationic agent-mediated transfection, and transfection with liposomes, immunoliposomes, exosomes, or cationic facial amphiphiles (CFAs).
  • nucleic acid transfection methods including electroporation, sonoporation, calcium phosphate transfection, microinjection, DNA biolistics, lipid-mediated transfection, transfection through heat shock, compacted DNA- mediated transfection, lipofection, cationic agent-mediated transfection, and transfection with liposomes, immunoliposomes, exosomes, or cationic facial amphiphiles (CFAs).
  • one or more mRNAs encoding epigenetic editor fusion proteins as described herein may be co-electroporated with one or more guide polynucleotides (e.g., gRNAs) as described herein.
  • guide polynucleotides e.g., gRNAs
  • One important category of non-viral nucleic acid vectors is nanoparticles, which can be organic (e.g., lipid) or inorganic (e.g., gold).
  • organic (e.g. lipid and/or polymer) nanoparticles can be suitable for use as delivery vehicles in certain embodiments of this disclosure.
  • LNP compositions are typically sized on the order of micrometers or smaller and may include a lipid bilayer.
  • a LNP refers to any particle that has a diameter of less than 1000 nm, 500 nm, 250 nm, 200 nm, 150 nm, 100 nm, 75 nm, 50 nm, or 25 nm.
  • a nanoparticle may range in size from 1-1000 nm, 1-500 nm, 1-250 nm, 25- 200 nm, 25-100 nm, 35-75 nm, or 25-60 nm.
  • Nanoparticle compositions encompass lipid nanoparticles (LNPs), liposomes (e.g., lipid vesicles), and lipoplexes.
  • an LNP as described herein may be made from cationic, anionic, or neutral lipids.
  • an LNP may comprise neutral lipids, such as the fusogenic phospholipid l,2-Dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE) or the membrane component cholesterol, as helper lipids to enhance transfection activity and nanoparticle stability.
  • DOPE fusogenic phospholipid l,2-Dioleoyl-sn-glycero-3-phosphoethanolamine
  • an LNP may comprise hydrophobic lipids, hydrophilic lipids, or both hydrophobic and hydrophilic lipids. Any lipid or combination of lipids that are known in the art can be used to produce an LNP. The lipids may be combined in any molar ratios to produce the LNP.
  • the LNP is a liver-targeting (e.g., preferentially or specifically targeting the liver) LNP.
  • LNP formulations and methods of LNP delivery that can be used will be apparent to those skilled in the art from the present disclosure and the state of the art.
  • Non-limiting exemplary compositions and methods can be found in Shah, R., Eldridge, D., Palombo, E., and Harding, I., Lipid Nanoparticles: Production, Characterization and Stability, Springer, 2015, ISBN- 13 978-3319107103; Mitchell, M.J., Billingsley, M.M., Haley, R.M. et al. Engineering precision nanoparticles for drug delivery, Nat Rev Drug Discov 20, 101-124 (2021); Hou, X., Zaks, T., Langer, R. et al. Lipid nanoparticles for mRNA delivery.
  • an epigenetic editor described herein, or component(s) thereof are delivered to a host cell for transient expression, e.g., via a transient expression vector.
  • Transient expression of the epigenetic editor or its component(s) may result in prolonged or permanent epigenetic modification of the target gene.
  • the epigenetic modification may be stable for at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10. 11, or 12 weeks or more; or 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 months or more, after introduction of the epigenetic editor into the host cell.
  • the epigenetic modification may be maintained after one or more mitotic and/or meiotic events of the host cell. In particular embodiments, the epigenetic modification is maintained across generations in offspring generated or derived from the host cell.
  • the present disclosure also provides methods for treating or preventing a condition in a subject, comprising administering to the subject an epigenetic editor or pharmaceutical composition as described herein.
  • the epigenetic editor may effect an epigenetic modification of a target polynucleotide sequence in a target gene associated with a disease, condition, or disorder in the subject, thereby modulating expression of the target gene to treat or prevent the disease, condition, or disorder.
  • the epigenetic editor reduces the expression of the target gene to an extent sufficient to achieve a desired effect, e.g., a therapeutically relevant effect such as the prevention or treatment of the disease, condition, or disorder.
  • a subject is administered a system for modulating (e.g., repressing) expression of PCSK9, wherein the system comprises (1) the fusion protein(s) and, where relevant, guide polynucleotide(s) of an epigenetic editor as described herein, or (2) nucleic acid molecules encoding said fusion protein(s) and, where relevant, guide poly nucleotide(s ) .
  • the system comprises (1) the fusion protein(s) and, where relevant, guide polynucleotide(s) of an epigenetic editor as described herein, or (2) nucleic acid molecules encoding said fusion protein(s) and, where relevant, guide poly nucleotide(s ) .
  • Treating”, “treating” and “treatment” refer to a method of alleviating or abrogating a biological disorder and/or at least one of its attendant symptoms.
  • to “alleviate” a disease, disorder or condition means reducing the severity and/or occurrence frequency of the symptoms of the disease, disorder, or condition.
  • references herein to “treatment” include references to curative, palliative and prophylactic treatment.
  • alleviating a symptom may involve reduction of the symptom by at least 3%, 5%, 10%, 20%, 40%, 50%, 60%, 80%, 90%, 95%, 98%, 99%, 99.5%, 99.9%, or 100% as measured by any standard technique.
  • the subject may be a mammal, e.g., a human.
  • the subject is selected from a non-human primate such as chimpanzee, cynomolgus monkey, or macaque, and other ape and monkey species.
  • the human patient has a condition selected from hypercholesterolemia (e.g., familial hypercholesterolemia such as heterozygous familial hypercholesterolemia (HeFH) or homozygous familial hypercholesterolemia (HoFH), or established atherosclerotic cardiovascular disease (ASCVD)) or renal insufficiency (RI).
  • hypercholesterolemia e.g., familial hypercholesterolemia such as heterozygous familial hypercholesterolemia (HeFH) or homozygous familial hypercholesterolemia (HoFH), or established atherosclerotic cardiovascular disease (ASCVD)
  • RI renal insufficiency
  • a patient to be treated with an epigenetic editor of the present disclosure has received prior treatment for the condition to be treated (e.g., hypercholesterolemia (such as HeFH, HoFH, HF, or established ASCVD) or RI).
  • prior treatment for the condition to be treated e.g., hypercholesterolemia (such as HeFH, HoFH, HF, or established ASCVD) or RI.
  • the patient has not received such prior treatment.
  • the patient has failed on a prior treatment for the condition (e.g., a prior hypercholesterolemia treatment).
  • An epigenetic editor of the present disclosure may be administered in a therapeutically effective amount to a patient with a condition described herein.
  • “Therapeutically effective amount,” as used herein, refers to an amount of the therapeutic agent being administered that will relieve to some extent one or more of the symptoms of the disorder being treated, and/or result in clinical endpoint(s) desired by healthcare professionals.
  • An effective amount for therapy may be measured by its ability to stabilize disease progression and/or ameliorate symptoms in a patient, and preferably to reverse disease progression.
  • the ability of an epigenetic editor of the present disclosure to reduce or silence PCSK9 expression may be evaluated by in vitro assays, e.g., as described herein, as well as in suitable animal models that are predictive of the efficacy in humans. Suitable dosage regimens will be selected in order to provide an optimum therapeutic response in each particular situation, for example, administered as a single bolus or as a continuous infusion, and with possible adjustment of the dosage as indicated by the exigencies of each case.
  • An epigenetic editor of the present disclosure may be administered without additional therapeutic treatments, i.e., as a stand-alone therapy (monotherapy).
  • treatment with an epigenetic editor of the present disclosure may include at least one additional therapeutic treatment (combination therapy).
  • the additional therapeutic agent is any known in the art to treat hypercholesterolemia or RI.
  • Therapeutic agents include, but are not limited to, statins, fibrates, HMG-CoA reductase inhibitors, niacin, bile acid modulators or sequestrants, cholesterol absorption inhibitors or modulators, CETP inhibitors, MTTP inhibitors, and PPAR agonists.
  • Bases include purines and pyrimidines, which include natural compounds such as adenine, thymine, guanine, cytosine, uracil, inosine, and natural analogs; as well as synthetic derivatives of purines and pyrimidines, which include, but are not limited to, modified versions which place new reactive groups such as amines, alcohols, thiols, carboxylates, alkylhalides, etc.
  • Nucleic acids may contain known nucleotide analogs and/or modified backbone residues or linkages, which may be synthetic, naturally occurring, and non-naturally occurring. Such nucleotide analogs, modified residues, and modified linkages are well known in the art, and may provide a nucleic acid molecule with enhanced cellular uptake, reduced immunogenicity, and/or increased stability in the presence of nucleases.
  • an “isolated” or “purified” nucleic acid molecule is a nucleic acid molecule that exists apart from its native environment.
  • an “isolated” or “purified” nucleic acid molecule (1) has been separated away from the nucleic acids of the genomic DNA or cellular RNA of its source of origin; and/or (2) does not occur in nature.
  • an “isolated” or “purified” nucleic acid molecule is a recombinant nucleic acid molecule.
  • variants, derivatives, homologs, and fragments thereof may have the specific sequence of residues (whether amino acid or nucleic acid residues) modified in such a manner that the polypeptide or polynucleotide in question substantially retains at least one of its endogenous functions.
  • a variant sequence can be obtained by addition, deletion, substitution, modification, replacement and/or variation of at least one residue present in the naturally occurring sequence (in some embodiments, no more than 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, or 20 residues).
  • the present disclosure also contemplates any of the protein’s naturally occurring forms, or variants or homologs that retain at least one of its endogenous functions (e.g., at least 50%, 60%, 70%, 80%, 90%, 85%, 96%, 97%, 98%, or 99% of its function as compared to the specific protein described).
  • fusion proteins embraced by the present disclosure are provided herein. It will be appreciated by the skilled artisan, that these exemplary proteins are nonlimiting examples and that additional proteins are within the scope of the present disclosure. For example, where fusion exemplary proteins comprising a specific domain, e.g., a mammalian DNMT3L and/or KRAB domain, such as a human or mouse DNMT3L and/or KRAB domain, are provided, the skilled artisan will be able to ascertain that, in some embodiments, fusion proteins with the same configuration, but with one or more of the mammalian domains substituted for a homologous domain from another mammal, e.g., one or more mouse domains substituted for one or more human domains, are also embraced by the present disclosure. For example, where an exemplary fusion protein is provided that comprises a mouse DNMT3L domain, a fusion protein of the same architecture but with the mouse DNMT3L substituted for a human DNMT3L domain is also
  • a homologue of any polypeptide or nucleic acid sequence contemplated herein includes sequences having a certain homology with the wildtype amino acid and nucleic sequence.
  • a homologous sequence may include a sequence, e.g. an amino acid sequence which may be at least 50%, 55%, 65%, 75%, 85%, 90%, 91%, 92% ⁇ 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the subject sequence.
  • percent identical in the context of amino acid or nucleotide sequences refers to the percent of residues in two sequences that are the same when aligned for maximum correspondence.
  • the length of a reference sequence aligned for comparison purposes is at least 30%, (e.g., at least 40, 50, 60, 70, 80, or 90%, or 100%) of the reference sequence.
  • Sequence identity may be measured using sequence analysis software (for example, Sequence Analysis Software Package of the Genetics Computer Group, University of Wisconsin Biotechnology Center, 1710 University Avenue, Madison, Wis. 53705, BLAST, BESTFIT, GAP, or PILEUP/PRETTYBOX programs).
  • sequence analysis software for example, Sequence Analysis Software Package of the Genetics Computer Group, University of Wisconsin Biotechnology Center, 1710 University Avenue, Madison, Wis. 53705, BLAST, BESTFIT, GAP, or PILEUP/PRETTYBOX programs.
  • Such software matches identical or similar sequences by assigning degrees of homology to various substitutions, deletions, and/or other modifications.
  • a BLAST program may be used, with a probability score between e-3 and e-100 indicating a closely related sequence.
  • the percent identity of two nucleotide or polypeptide sequences is determined by, e.g., BLAST® using default parameters (available at the U.S. National Library of Medicine’s National Center for Biotechnology Information website).
  • the length of a reference sequence aligned for comparison purposes is at least 30%, (e.g., at least 40, 50, 60, 70, 80, or 90%) of the reference sequence.
  • an epigenetic editor as described herein may modulate the activity of a promoter sequence by binding to a motif within the promoter, thereby inducing, enhancing, or suppressing transcription of a gene operatively linked to the promoter sequence.
  • an epigenetic editor as described herein may block RNA polymerase from transcribing a gene, or may inhibit translation of an mRNA transcript.
  • inhibitor when used in reference to an epigenetic editor or a component thereof as described herein, refers to decreasing or preventing the activity (e.g., transcription) of a nucleic acid sequence (e.g., a target gene) or protein relative to the activity of the nucleic acid sequence or protein in the absence of the epigenetic editor or component thereof.
  • the term may include partially or totally blocking activity, or preventing or delaying activity.
  • the inhibited activity may be, e.g., 10%, 20%, 30%, 40%, 50%, 60%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% less than that of a control, or may be, e.g., at least 1.5-fold, 2-fold, 3-fold, 4- fold, 5-fold, or 10-fold less than that of a control.
  • Ranges provided herein are understood to be shorthand for all of the values within the range.
  • 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 intervening decimal values between the aforementioned integers such as, for example, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, and 1.9.
  • a nested sub-range of an exemplary range of 1 to 50 may comprise 1 to 10, 1 to 20, 1 to 30, and 1 to 40 in one direction, or 50 to 40, 50 to 30, 50 to 20, and 50 to 10 in the other direction.
  • scientific and technical terms used in connection with the present disclosure shall have the meanings that are commonly understood by those of ordinary skill in the art. Exemplary methods and materials are described below, although methods and materials similar or equivalent to those described herein can also be used in the practice or testing of the present disclosure. In case of conflict, the present specification, including definitions, will control.
  • back-references in the dependent claims are meant as short-hand writing for a direct and unambiguous disclosure of each and every combination of claims that is indicated by the back-reference.
  • headers herein are created for ease of organization and are not intended to limit the scope of the claimed methods and compositions in any manner.
  • Some protein sequences e.g., so me fusion protein sequences, provided herein include a peptide tag, e.g., a His6 tag, or a DYKDDDDK (SEQ ID NO: 1528) tag, which are useful for detection and/or purification of tagged proteins, but do not affect protein function.
  • a peptide tag e.g., a His6 tag, or a DYKDDDDK (SEQ ID NO: 1528) tag, which are useful for detection and/or purification of tagged proteins, but do not affect protein function.
  • tags can be substituted for other suitable peptide tags, and that fusion proteins of the same or highly similar sequence, but not including such peptide tags, e.g., from which the peptide tag has been cleaved or which are created without a peptide tag, are suitable for carrying out embodiments of the present disclosure as well.
  • fusion proteins of the same or highly similar sequence but not including such peptide tags, e.g., from which the peptide tag has been cleaved or which are created without a peptide tag, are suitable for carrying out embodiments of the present disclosure as well.
  • the following examples are set forth. These examples are for purposes of illustration only and are not to be construed as limiting the scope of the present disclosure in any manner.
  • a VHH single domain antibody library was derived from 16 alpacas, 10 camels, and 31 llamas. Animals were immunized with full length DNMT3A (DNMT3A-FL) (SEQ ID NO: 574) and the DNMT3A catalytic domain (DNMT3A-CD (SEQ ID NO: 575). 1.5xl0 A 10 library members were isolated.
  • Phage display variants of interest were validated for DNMT3A-binding using standard ELISA techniques.
  • Candidate single domain antibodies were tested for binding to DMNT3A-FL and DNMT3A-CD. Results of the ELISA experiments are shown in FIG. 3.
  • Fusion proteins comprising dCas9, a single domain antibody that binds DNMT3A, DNMT3L, and KOX1 KRAB (“CRISPR-off”) were designed and constructed. From N terminus to C terminus, the proteins have the structure SDA-SDA-3L-DBD-ED and SDA-SDA-3L-DBD, as described in this disclosure. Non-limiting examples of fusion proteins are provided in FIG.5A and FIG. 6.
  • FIG. 5B shows a diagram of one example of a fusion protein as described herein interacting with DNA. Constructs comprising various combinations of single domain antibodies (SDA) were generated.
  • SDA single domain antibodies
  • ZF-off comprising a single domain antibody that binds DNMT3A, 3L, and KOX1 KRAB are also constructed.
  • Fusion proteins comprising dCas9, a single domain antibody that binds DNMT3A, DNMT3L, and KOX1 KRAB are designed and constructed. From N terminus to C terminus, the proteins have the structure SDA-3L-DBD-ED and SDA-3L-DBD, as described in this disclosure.
  • the CRISPR-off plasmid construct has been described in Nunez (Nunez et al., Cell (2021) 184(9):2503-19) and was ordered from Twist Biosciences.
  • ZF fusion proteins comprising a single domain antibody that binds DNMT3A, 3L, and KOX1 KRAB are also constructed.
  • Single domain antibodies of interest were generated and selected as described in Examples 1 and 2. Constructs comprising one or more single domain antibody described herein were generated to test in HeLa PCSK9-TdTomato and HEK293T GripTite (CLTA- GFP) cells. Results from GripTite CLTA-GFP cells are shown in FIGs. 2A and 2B. Results from HeLa PCSK9-TdTomato cells showing epigenetic silencing by a wider range of singledomain antibody-containing constructs are shown in FIGs. 1A-1B.
  • RNAseq RNAseq
  • methylation array RNAseq
  • whole genome bisulfite sequencing assays Genome- wide expression and methylation changes after epigenetic editing compared to negative controls will be profiled.
  • Specificity of 5 gRNAs is tested in PXB cells. Fresh human hepatocytes are isolated from PXB mouse model mice. Long-term stability and functionality of hepatocytes as well as robust PCSK9 secretion is confirmed. Cells are treated with the epigenetic editor or controls.
  • PCSK9 secretion is measured and plotted as a percent of secreted PCSK9 produced by a negative control (epigenetic editor with a non-PCSK9-targeting gRNA (“off target”). Specificity of constructs is tested by RNAseq at day 14 post-delivery for all gRNAs.
  • single domain antibody epigenetic effector constructs of the present disclosure to mediate epigenetic silencing of endogenous PCSK9 in vivo is tested.
  • Constructs are delivered using a single IV administration of mRNA (and, for CRISPR-off silencing, gRNA) formulated into an LNP.
  • Silencing is tested in wildtype mice over a period of two to six months.
  • the readout is serum PCSK9 levels and serum cholesterol levels.
  • a subset of each cohort is selected for liver hematoxylin and eosin (H&E) stain RNAseq and analysis. For several constructs, robust, stable, and inheritable PCSK9 silencing is observed.
  • H&E liver hematoxylin and eosin
  • hPCSK9-Tg mPCSK9+/-
  • hPCSK9-Tg mPCSK9+/+
  • homozygous mouse hPCSK9+/+
  • hPCSK9-Tg mPCSK9-/- mouse.
  • the hPCSK9-Tg (mPCSK9-/-) mouse line available for use is C57BL/6J-Pcsk9-/- Tg(RPl l-55M23-AbsI), which expresses human PCSK9 under the control of its own promoter. See, e.g., Weider et al., J Biol Chem (2016) 291(32):16659-71.
  • Constructs are delivered via single IV administration of epigenetic silencers formulated into LNPs.
  • Durability is tested over six to twelve months. Readouts are serum PCSK9 levels and serum cholesterol levels. A subset of the cohort is selected for liver H&E and RNAseq analysis.
  • Example 9 Fusion proteins with Variant NLS Configurations Several improved fusion protein constructs are developed using variant nuclear localization sequence (NLS) configurations to have significantly higher epi-silencing activity.
  • NLS nuclear localization sequence
  • HeLa ATCC-CRM-CCL-2
  • Hepal-6 PCSK9-IRES-TdTomato
  • Huh7 Sekisui XenoTech, LLC
  • HEK293T GripTite CTA-GEP
  • HeLa cells are reverse transfected using TransIT-X2 transfection reagent from Minis (Cat# MIR6003).
  • Huh7 cells are reverse transfected using MessengerMAX reagent from Invitrogen (Cat# LMRNA003).
  • HEK293T Griptite cells with GPP knocked into the CLTA locus as an in-frame CLTA fusion are co-transfected with plasmids encoding effector construct and human CLTA guide RNA using TransIT-X2 transfection reagent from Minis (Cat# MIR6003).
  • GPP is measured by LACS for GPP expression as a surrogate for CLTA expression.
  • Hepal-6 cells are co-transfected with plasmids encoding effector construct and mouse PCSK9 guide RNA using SP Cell Line 96-well Nucleofector Kit (Cat # V4SC-2096, program code: CM- 138) in Amaxa 4D nucleofector device from Lonza. At the indicated timepoint, cells are LACS analyzed for TdTomato expression as a surrogate for PCSK9 levels.
  • 1 pg of linearized effector template is used to set up in-vitro transcription reactions using T7 mScriptTM Standard mRNA Production System from CellScript (Cat# C- MSC 100625) according to manufacturer’s instructions to obtain RNA that had a Cap 1 structure on the 5’ end and was 3’polyadenylated.
  • End- modified sgRNA that have three 2’0- methyl modified nucleotides with phosphorothioate linkages on both 5’ and 3’ ends are obtained from Integrated DNA Technologies.
  • Genomic DNA is extracted from each well of a 96-well culture plate using a DNAdvance DNA Extraction from Tissue Kit (Beckman Coulter). After quantification of genomic DNA via High-Sensitivity DNA IX kit (Quant-IT), each genomic DNA sample is bisulfite converted using an EZ-96 DNA Methylation- Gold MagPrep kit (Zymo Research) according to manufacturer’s instructions.
  • DNA libraries are prepared using the xGenTM Methyl-Seq DNA Library Prep Kit (IDT) and hybrid capture is conducted using the xGenTM Hybridization Capture of DNA libraries kit (IDT).
  • DNA libraries are prepared using the xGenTM Methyl- Seq DNA Library Prep Kit (IDT) and hybrid capture was conducted using the xGenTM Hybridization Capture of DNA libraries (IDT).
  • IDT xGenTM Methyl- Seq DNA Library Prep Kit
  • IDT Hybridization Capture of DNA libraries
  • Bisulfite-converted DNA from each sample is used to seed PCR corresponding to each of the two VIM amplicons using a Platinum Taq kit (Invitrogen). Pooled products are cleaned using the AMPure XP kit (Beckman Coulter) and fragment size assessment via D1000 screentape on a Tapestation 4200 (Agilent) prior to sequencing by commercial service (Azenta).
  • fusion proteins are constructed with alternative KRAB domains and show improved activity as compared to CRISPR-off when tested using the experimental procedures described above.
  • ZIM3 and KOX1KRAB are KRAB family proteins with extensive homology.
  • sequences are designed which represent halfway points between ZIM3 and KOX1KRAB.
  • KOX1KRAB and ZIM3 constructs encode a small region of KOX1KRAB and ZIM3 focused around the zinc finger domain of the protein. While the regions used of KOX1KRAB and ZIM3 are very similar within the first ⁇ 75bp of their sequence, ZIM3 also possesses a small alpha-helical region at the C-terminus, not present in KOX1KRAB.
  • the KOX1KRAB-FL sequence includes the KOX1KRAB sequence equivalent of this extra piece, while the ZIM3 truncation has this extra piece removed from the ZIM3 sequence.
  • the ZIM3/KOX1KRAB chimeras are fusions of the N- and C-terminal pieces of the two proteins.
  • the ZIM3-like KOX1KRAB variants were both assembled by first, BLAST of ZIM3 or KOX1KRAB proteins from nonhuman species to assemble the closest 100 homologs (‘families’) of each gene; second, identifying the 3 members of the KOX1KRAB family that most closely resemble ZIM3 and the 3 members of the ZIM3 family that most closely resemble KOX1KRAB; and third, rationally modifying the KOX1KRAB-FL sequence to resemble each set of three (Table 11).
  • Varying dosage amounts of epigenetic effector constructs of the present disclosure are tested in mice expressing human PCSK9. Constructs which have shown durable silencing of PCSK9 at 3 mg/kg are chosen for further study. Constructs are delivered as described in previous examples. Each construct of interest is tested at 0.2 mg/kg, 0.375 mg/kg, 0.75 mg/kg, and 3 mg/kg. Baseline hPCSK9 levels are determined in the two weeks before administration of constructs. Efficacy of PCSK9 silencing is measured over 28 days. Post- IV human PCSK9 serum levels are measured by ELISA. Durable silencing of hPCSK9, to levels of about 10% or less of baseline, is observed with each construct at several of the tested dosages.
  • Partial hepatectomy is an established method of inducing liver regeneration in mice and has been used for testing durability of genetic and epigenetic effects in mice.
  • Mice are treated with an epigenetic editor targeting PCSK9 formulated into lipid nanoparticles using a single IV administration. Groups of mice each are given (1) a sham administration (vehicle only - negative control), (2) 1.5 mg/kg of the epigenetic editor, or (3) 3 mg/kg of the epigenetic editor. Mice are then split into Cohort A and Cohort B. After 3 months, mice in Cohort B have 70 percent of their livers surgically resected, while mice in Cohort A do not undergo surgery. After 2 months, the livers of mice in Cohort B have regenerated. Serum levels of PCSK9 are measured periodically after injection in both Cohorts. Silencing of mouse PCSK9 is expected to be durable through 140 days post-injection in both Cohort A and Cohort B.
  • Example 14 Stable PCSK9 Silencing via Epigenetic Editing in a human subject
  • Constructs are delivered via single IV administration of epigenetic silencers formulated into LNPs.
  • SEQ ID NOs (SEQ) of nucleotide (nt) and amino acid (aa) sequences described in the present disclosure are listed below.

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Abstract

La présente invention concerne des anticorps à domaine unique se liant à des DNMT (par exemple, DNMT3A) ou des protéines Tet (par exemple, Tet1 ou Tet2), et des compositions d'éditeur épigénétique et des procédés de modification épigénétique de gènes cibles les utilisant. La présente divulgation concerne également des acides nucléiques et des vecteurs codant pour les anticorps à domaine unique anti-DNMT3A, ou des anticorps à domaine unique anti-Tet, et des éditeurs épigénétiques. L'invention concerne en outre cellules épigénétiquement modifiées par les éditeurs épigénétiques.
PCT/US2024/040780 2023-08-03 2024-08-02 Procédés et compositions comprenant des anticorps se liant à dnmt3a Pending WO2025030130A1 (fr)

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WO2022031871A1 (fr) * 2020-08-05 2022-02-10 Synthekine, Inc. Compositions et méthodes se rapportant à la liaison du récepteur il27
WO2022140577A2 (fr) * 2020-12-22 2022-06-30 Chroma Medicine, Inc. Compositions et méthodes pour l'édition épigénétique

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* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2022031871A1 (fr) * 2020-08-05 2022-02-10 Synthekine, Inc. Compositions et méthodes se rapportant à la liaison du récepteur il27
WO2022140577A2 (fr) * 2020-12-22 2022-06-30 Chroma Medicine, Inc. Compositions et méthodes pour l'édition épigénétique

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