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WO2024145615A2 - Compositions and methods for epigenetic regulation of angptl3 expression - Google Patents

Compositions and methods for epigenetic regulation of angptl3 expression Download PDF

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Publication number
WO2024145615A2
WO2024145615A2 PCT/US2023/086491 US2023086491W WO2024145615A2 WO 2024145615 A2 WO2024145615 A2 WO 2024145615A2 US 2023086491 W US2023086491 W US 2023086491W WO 2024145615 A2 WO2024145615 A2 WO 2024145615A2
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domain
sequence
human
peptide linker
seq
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PCT/US2023/086491
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French (fr)
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WO2024145615A3 (en
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Kenneth Kelly
Noorussahar ABUBUCKER
Ricardo Noel RAMIREZ
Morgan Maeder
Frederic Tremblay
Vic MYER
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Chroma Medicine, Inc.
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Publication of WO2024145615A2 publication Critical patent/WO2024145615A2/en
Publication of WO2024145615A3 publication Critical patent/WO2024145615A3/en

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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
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • C07K14/4701—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals not used
    • C07K14/4702—Regulators; Modulating activity
    • 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
    • A61P9/00—Drugs for disorders of the cardiovascular system
    • A61P9/10—Drugs for disorders of the cardiovascular system for treating ischaemic or atherosclerotic diseases, e.g. antianginal drugs, coronary vasodilators, drugs for myocardial infarction, retinopathy, cerebrovascula insufficiency, renal arteriosclerosis
    • 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

  • ANGPTL3 angiopoietin-like 3
  • the human ANGPTL3 gene located on chromosome 1, has CpG islands in the promoter region and has approximately 80% homology with its mouse counterpart.
  • the ANGPTL3 protein is expressed predominantly in the liver.
  • the DNA-binding domain comprises a dead CRISPR Cas (dCas) domain, a ZFP domain, or a TALE domain.
  • the DNA-binding domain may comprise a dCas9 domain, and the system may further comprise (i) one or more guide RNAs (e.g., comprising any one of SEQ ID NOs: 900-988), or (ii) nucleic acid molecules coding for the one or more guide RNAs.
  • the dCas domain comprises a dCas9 sequence, such as a sequence with at least 90% identity to SEQ ID NO: 12 or 13.
  • the DNA-binding domain comprises a ZFP domain that targets a nucleotide sequence selected from SEQ ID NOs: 700-708.
  • the ZFP domain comprises, in order, the F1-F6 amino acid sequences of any one of ZFP810 through ZFP893 as shown in Table 1.
  • the transcriptional repressor domain comprises a sequence with at least 90% identity to a sequence selected from SEQ ID NOs: 33-570.
  • the transcriptional repressor domain is a KRAB domain derived from KOX1, ZIM3, ZFP28, or ZN627.
  • the KRAB domain may comprise, e.g., a sequence with at least 90% identity to a sequence selected from SEQ ID NOs: 89, 116, 245, and 255.
  • the transcriptional repressor domain comprises a fusion of the N- and C- terminal regions of ZIM3 and KOX1 KRAB, and optionally comprises the amino acid sequence of SEQ ID NO: 571 or 572.
  • the fusion protein may comprise, from N-terminus to C-terminus, a first NLS, the DNMT3A domain, the first peptide linker, the DNMT3L domain, the second peptide linker, the DNA-binding domain, the third peptide linker, the transcriptional repressor domain, and a second NLS.
  • the fusion protein may comprise, from N-terminus to C-terminus, first and second NLSs, the DNMT3A domain, the first peptide linker, the DNMT3L domain, the second peptide linker, the DNA- binding domain, the third peptide linker, the transcriptional repressor domain, and third and fourth NLSs.
  • the fusion protein may comprise, from N-terminus to C- terminus, a human DNMT3A domain, a first peptide linker, a human DNMT3L domain, an XTEN80 peptide linker, a first NLS, a dSpCas9 domain, a second NLS, an XTEN 16 peptide linker, and a human KOX1 KRAB domain.
  • the fusion protein comprises SEQ ID NO: 658 or a sequence at least 90% identical thereto.
  • the fusion protein comprises, from N-terminus to C- terminus, first and second NLSs, a human DNMT3A domain, a first peptide linker, a human DNMT3L domain, an XTEN80 peptide linker, a dSpCas9 domain, an XTEN16 peptide linker, a human KOX1 KRAB domain, and third and fourth NLSs.
  • the fusion protein may comprise the amino acid sequence of SEQ ID NO: 660 or a sequence at least 90% identical thereto.
  • the fusion protein comprises, from N-terminus to C- terminus, first and second NLSs, a human DNMT3A domain, a first peptide linker, a human DNMT3L domain, an XTEN80 peptide linker, a ZFP domain, an XTEN16 peptide linker, a human KOX1 KRAB domain, and third and fourth NLSs.
  • the fusion protein comprises, from N-terminus to C- terminus, first and second NLSs, a human DNMT3A domain, a first peptide linker, a human DNMT3L domain, an XTEN80 peptide linker, a ZFP domain, an XTEN16 peptide linker, a human ZFP28 KRAB domain, and third and fourth NLSs.
  • the fusion protein comprises, from N-terminus to C- terminus, first and second NLSs, a human DNMT3A domain, a first peptide linker, a human DNMT3L domain, an XTEN80 peptide linker, a dSpCas9 domain, an XTEN16 peptide linker, a human ZN627 KRAB domain, and third and fourth NLSs.
  • the fusion protein may comprise the amino acid sequence of SEQ ID NO: 662 or a sequence at least 90% identical thereto.
  • the fusion protein comprises, from N-terminus to C- terminus, first and second NLSs, a human DNMT3A domain, a first peptide linker, a human DNMT3L domain, an XTEN80 peptide linker, a ZFP domain, an XTEN16 peptide linker, a human ZIM3 KRAB domain, and third and fourth NLSs.
  • At least one of the NLSs in a fusion protein described herein is an SV40 NLS (e.g., SEQ ID NO: 644).
  • the system comprises: a) a first fusion protein comprising a first DNA-binding domain and comprising or recruiting the DNMT3A domain, a second fusion protein comprising a second DNA-binding domain and comprising or recruiting the DNMT3L domain, and a third fusion protein comprising a third DNA-binding domain and comprising or recruiting the transcriptional repressor domain; or b) one or more nucleic acid molecules encoding the fusion proteins.
  • the present disclosure also provides a human cell comprising a system described herein, or progeny of the cell.
  • the cell is a hepatocyte.
  • the present disclosure also provides use of a system described herein in the manufacture of a medicament for treating a patient in need thereof, e.g., in a method described herein.
  • FIG. 3 is a pair of graphs showing ANGPTL3 silencing after treatment with epigenetic repressors using the top 25 gRNAs at day 7 (left) and day 14 (right).
  • FIG. 7 is a dose response graph showing reduction of ANGPTL3 and PCSK9 secretion at day 7 by a single CRISPR-off repressor in a multiplex format with a single gRNA against each target; “Cutting control” refers to a WT Cas9 control.
  • PLA947 CRISPR-off construct of SEQ ID NO: 1013.
  • PLA1489 CRISPR-off construct of SEQ ID NO: 1009.
  • PLA2628 CRISPR-off construct of SEQ ID NO: 1011.
  • FIG. 8A shows schematic illustrations of fusion protein constructs with variant NLS configurations.
  • FIG. 8B shows schematic illustrations of additional fusion protein constructs with variant KRAB domains.
  • the present disclosure provides epigenetic editors for regulating expression of the ANGPTL3 gene.
  • the systems, compositions and methods described herein may be used for treating conditions such as hypercholesterolemia (e.g., heterozygous familial hypercholesterolemia (HeFH), homozygous familial hypercholesterolemia (HoFH), familial hypercholesterolemia (HF), or established atherosclerotic cardiovascular disease (ASCVD)), or renal insufficiency (RI).
  • hypercholesterolemia e.g., heterozygous familial hypercholesterolemia (HeFH), homozygous familial hypercholesterolemia (HoFH), familial hypercholesterolemia (HF), or established atherosclerotic cardiovascular disease (ASCVD)
  • RI renal insufficiency
  • ANGPTL3 refers herein to human ANGPTL3.
  • a human ANGPTL3 gene sequence can be found at Ensembl Accession No. ENSG00000132855.
  • 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.
  • 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).
  • 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.
  • a linker comprises 7-17 amino acids.
  • the linker may be flexible or rigid.
  • the ZFP domain of the present epigenetic editor binds to a target sequence selected from any one of SEQ ID NOs: 700-708.
  • the ZFP domain comprises, in order, the F1-F6 amino acid sequences of any one of ZFP810- ZFP893 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 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
  • Part 2 the scaffold sequence of a guide polynucleotide as described herein may be, for example, as described in Jinek et al., Science (2012) 337:816-21; U.S. Patent Publication 2016/0208288; or U.S. Patent Publication 2016/0200779. Variants of part 2) are also contemplated by the present disclosure.
  • the tetraloop and stem loop of a gRNA scaffold (tracrRNA) sequence may be modified to include RNA aptamers, which can be bound by specific protein domains.
  • such modified gRNAs can be used to facilitate the recruitment of repressive or activating domains fused to the proteininteracting RNA aptamers.
  • 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.
  • FIG. 1 An exemplary illustration of a Cas9 target site, comprising a 22 nucleotide target domain, and an NGG 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:
  • RNA scaffold [ gRNA scaffold] -3 ' ( RNA) [ target ing domain ( RNA) ] [ binding domain ]
  • FIG. 1 An exemplary illustration of a 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: [ PAM ] [ target domain ( DNA) ]
  • Guide polynucleotides e.g., gRNAs
  • 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 ANGPTL3 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.
  • 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’-0-ethylamine, 2’-O-cyanoethyl, 2’-0-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 ribose stability of
  • 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 human ANGPTL3, as well as the coordinates of the start and end positions of the targeted site on human chromosome 1 (SEQ: SEQ ID NO). The table also shows the distance from the start coordinate to the TSS coordinate of the ANGPTL3 gene.
  • a gRNA described herein has a tracr sequence shown in Table 3 below, or a tracr sequence at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to the tracr sequence shown below (SEQ: SEQ ID NO).
  • 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.
  • 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 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
  • 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.
  • an epigenetic editor described herein comprises a KRAB domain derived from KOX1, ZIM3, ZFP28, or ZN627, and/or an effector domain derived from KAP1, MECP2, HPla, HPlb, CBX8, CDYL2, TOX, TOX3, TOX4, EED, EZH2, RBBP4, RCOR1, or SCML2, optionally wherein the parental protein is a human protein.
  • 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: 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 DNMT-like domain is a mammalian (e.g., human or mouse) DNMT- like domain.
  • the DNMT-like domain is DNMT3L, which may be, for example, human DNMT3L or mouse DNMT3L.
  • 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.
  • an epigenetic editor described herein comprises a DNMT3L domain comprising SEQ ID NO: 580, or a sequence at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 580.
  • an epigenetic editor described herein comprises a DNMT3L domain comprising SEQ ID NO: 581, or a sequence at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 581.
  • 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).
  • 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 above-listed protein.
  • the effector domain herein comprises one or more epigenetic effector domains selected from Table 6, or functional homologs, orthologs, or variants thereof.
  • 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.
  • 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 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: 1029), 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: 666), and (XP)n (SEQ ID NO: 1027)).
  • At least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 105, 110, 115, 120 or more histone tails of the bound histones are altered as compared to the original state of the chromosome or the chromosome in a comparable cell not contacted with the epigenetic editor.
  • an effector domain alters a chemical modification state of a nucleotide or histone tail bound to a nucleotide within 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 1100, 1200, 1300, 1400, 1500, 1600, 1700, 1800, 1900, or 2000 nucleotides flanking the target sequence.
  • “flanking” refers to nucleotide positions 5’ to the 5’ end of and 3’ to the 3’ end of a particular sequence, e.g. a target sequence.
  • the chemical modification may be initiated at less than 2, 3, 5, 10, 20, 30, 40, 50, or 100 nucleotides in the target gene and spread to at least 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 2000, or more nucleotides in the target gene. In some embodiments, the chemical modification spreads to nucleotides in the entire target gene.
  • 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.
  • 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.
  • 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).
  • 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
  • Any AAV serotype e.g., human AAV serotype, can be used for an AAV vector as described herein, including, but not limited to, AAV serotype 1 (AAV1), AAV serotype 2 (AAV2), AAV serotype 3 (AAV3), AAV serotype 4 (AAV4), AAV serotype 5 (AAV5), AAV serotype 6 (AAV6), AAV serotype 7 (AAV7), AAV serotype 8 (AAV8), AAV serotype 9 (AAV9), AAV serotype 10 (AAV10), and AAV serotype 11 (AAV11), as well as variants thereof.
  • 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
  • AAV8 AAV serotype 8
  • 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.
  • 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.
  • the cells may be eukaryotic or prokaryotic.
  • the cells are mammalian (e.g., human) cells.
  • Human cells may include, for example, hepatocytes, biliary epithelial cells (cholangiocytes), stellate cells, Kupffer cells, and liver sinusoidal endothelial cells.
  • 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 ANGPTL3, 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 ) .
  • ‘Treat”, “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).
  • the patient has not received such prior treatment.
  • the patient has failed on a prior treatment for
  • 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 ANGPTL3 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.
  • the additional therapeutic is a PCSK9 antagonist, e.g., a PCSK9 inhibitor.
  • the additional therapeutic is an epigenetic editor targeting PCSK9.
  • the epigenetic editors or components thereof (or nucleic acid molecules encoding the epigenetic editors or components thereof) of the present disclosure may be administered by any method accepted in the art, e.g., subcutaneously, intradermally, intratumorally, intranodally, intramuscularly, intravenously, intralymphatically, or intraperitoneally.
  • a pharmaceutical composition of the present disclosure is administered intravenously to the subject.
  • nucleic acid refers to any oligonucleotide or polynucleotide containing nucleotides (e.g., deoxyribonucleotides or ribonucleotides) in either single- or double-strand form, and includes DNA and RNA.
  • nucleotides contain a sugar deoxyribose (DNA) or ribose (RNA), a base, and a phosphate group, and are linked together through the phosphate groups.
  • 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).
  • 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.
  • the term “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). Such software matches identical or similar sequences by assigning degrees of homology to various substitutions, deletions, and/or other modifications.
  • 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.
  • 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,
  • 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 invention in any manner.
  • a fusion protein comprising dCas9, DNMT3A, DNMT3L, and K0X1 KRAB (“CRISPR-off”) was designed.
  • An exemplary CRISPR-off protein comprises, from N terminus to C terminus, the domains NLS-huDNMT3A-linker-huDNMT3L-XTEN80- dSpCas9- NLS-XTEN16-huKOXl KRAB.
  • the CRISPR-off plasmid construct has been described in Nunez (Nunez et al., Cell (2021) 184(9):2503-19).
  • Another exemplary CRISPR- off protein comprises SEQ ID NO: 658.
  • ZF fusion proteins comprising DNMT3A, 3L, and K0X1 KRAB were also constructed.
  • the constructs have the general structure huDNMT3A-linker-huDNMT3L- XTEN80-NLS-ZFP domain-NLS-XTEN16-KOXlKrab (SEQ ID NO: 659).
  • Example 2 Selection of Target ANGPTL3 Sequences for gRNA Epigenetic Silencing [0239] gRNAs targeting +/- 1 kb from the ANGPTL3 TSS were computationally designed for human (GRCh38), mouse (mmlO), and Macaca fascicularis (5.0) ANGPTL3.
  • gRNAs containing poly-TTTT sequences were first discarded. gRNA off-target analysis was performed using CasOFFinder (Bae et al., Bioinformatics (2014) 30(10): 1473-5). gRNAs were discarded if they matched to multiple locations across the respective genome build for each independent species.
  • a cross -reactivity sequence analysis was performed on human ANGPTL3 gRNAs in order to annotate sequence mismatches with Macaca or mouse gRNA sequences.
  • gRNA sequence alignments were performed to identify the degree of DNA similarity at each nucleotide, including the annotation of guides that contain up to zero, one, or two nucleotide mismatches.
  • a final set of 89 gRNA targeting sequences (Table 7) was selected for the ANGPTL3 primary screen in Huh7 cells.
  • Example 3 Selection of ZF Target Sites and Design of ZF Proteins for Epigenetic Silencing
  • the source of the 2F units was a set of three-finger zinc finger proteins that had been selected to bind specific target sites using a bacterial-2-hybrid (B2H) selection system (Hurt et al., PNAS (2003) 100:12271-6; Maeder et al., Mol Cell (2008) 31(2):294-301).
  • B2H bacterial-2-hybrid
  • a list of targetable DNA sites was created by generating all possible triplet combinations of 6 bp binding sites represented in the library and allowing either 0 or 1 bp between the 6 bp target sites.
  • Design of the six recognition helices used to generate the full proteins was performed by selecting two-finger units and taking into account a number of factors such as known binding preferences of zinc finger proteins, the frequency with which amino acids in positions -1, 2, 3 and 6 had been selected in the B2H selection system to bind the desired target base, avoidance of amino acids in positions -1, 2, 3 and 6 that had been selected to bind multiple different bases in the B2H, and maintaining context dependencies by matching flanking bases where possible.
  • the full ZF sequence is derived from the naturally occurring Zif268 protein, and selected recognition helices were maintained in the sequence context in which they were selected in the B2H (either fingers 1-2 or fingers 2-3 from Zif268).
  • Example 4 CRISPR-off Guide Screening of ANGPTL3 in Huh7 Hepatoma Cell Line
  • the Huh7 hepatoma cell line (Sekisui XenoTech, LLC) is amenable to high- throughput screening and transfection.
  • Guide RNAs (gRNAs) against 89 target sites on the ANGPTL3 locus were tested in Huh7 cells.
  • Cells were treated with 400 ng of mRNA encoding the CRISPR-off construct and 50 ng of the respective gRNA.
  • Huh7 cells were seeded at a density of 25,000 cells per well in 96-well plates.
  • Controls included WT Cas9 + ANGPTL3 gRNA (gRNAl 137 or gRNAl 138), and CRISPR-off with no gRNA.
  • mX i.e., mA, mC, mG, or mU
  • rX i.e., rA, rC, rG, or rU
  • * indicates a phosphorothioate linkage. All internucleoside linkages that are not phosphorothioate linkages are phosphate linkages.
  • PXB cells from PhoenixBio are used to test the efficacy of the gRNAs in primary hepatocytes. PXB cultures are maintained according to manufacturer recommendations. Briefly, PXB maintenance media is thawed and made up within 30 minutes of the cells’ arrival. Upon media change, cells are allowed to acclimate for two days in a 37°C, 5% CO2 incubator. On the second day after receipt, LNPs are formulated with CRISPR-off + sgRNA, GFP-mRNA and WT CRISPR Cas9 in various concentrations using the SPARKTM (Precision Nanosystems) and the Genvoy-ILMTM mRNA LNP formulation kit.
  • SPARKTM Precision Nanosystems
  • Negative controls were lipid only and a CRISPR-off construct paired with a gRNA targeted to a different (non-AAGPTL3) genomic locus. Increased potency and durability were observed in dual gRNA treated cells (cells treated with CRISPR-off construct together with paired gRNAs) as compared to single-gRNA mediated CRISPR-off repression (FIG. 5). A number of gRNA pairs were screened, with the screening data for days 7 and 14 shown in Table 11 below.
  • the manufacturer’s instructions are followed to formulate LNPs with CRISPR-off + sgRNA, GFP-mRNA and WT CRISPR Cas9 in various concentrations using the SPARKTM (Precision Nanosystems) and the GenVoy-ILMTM mRNA LNP formulation kit.
  • Liver humanized mice are a special mouse model with functional engraftment of primary human hepatocytes in the host liver (see, e.g., Tateno and Kojima, Lab Anim Res. (2020) 36:2). Such a model has been used to evaluate the efficacy of liver-targeted gene therapies with better predictability than rodent models (see, e.g., Paulk et al., Mol Ther. (2016) 26(l):289-303).
  • 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.
  • 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

This present disclosure relates to compositions and methods comprising epigenetic editors for epigenetic modification of ANGPTL3, as well as nucleic acids and vectors encoding the same. Also disclosed are cells epigenetically modified by the epigenetic editors.

Description

COMPOSITIONS AND METHODS FOR EPIGENETIC REGULATION OF ANGPTL3 EXPRESSION
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application claims the benefit under 35 U.S.C. § 119(e) of U.S. Provisional Application No. 63/478,065, filed December 30, 2022, entitled “COMPOSITIONS AND METHODS FOR EPIGENETIC REGULATION OF ANGPTL3 EXPRESSION,” the entire disclosure of which is hereby incorporated by reference in its entirety.
REFERENCE TO AN ELECTRONIC SEQUENCE LISTING
[0002] The contents of the electronic sequence listing (C1069870013WO00-SEQ- AZW.xml; Size: 1,157,484 bytes; and Date of Creation: December 29, 2023) is herein incorporated by reference in its entirety.
BACKGROUND
[0003] Genome editing has been considered a promising therapeutic approach for the treatment of genetic disease for over a decade. However, manipulation on the DNA level using traditional genetic editors remains risky given the potential for undesired double- strand DNA breaks, heterogenous repair (including large and small insertions and deletions at the intended site), and toxicity. By contrast, targeted epigenetic modification offers the potential to alter gene expression without leading to double-strand break-induced genotoxicity.
SUMMARY
[0004] The angiopoietin-like 3 (ANGPTL3) gene is a promising candidate for epigenetic silencing. ANGPTL3 is a key target in the treatment of heart disease, the leading cause of mortality worldwide (see, e.g., Dewey et al., N Engl J Med. (2017) 377(3):211-21; and Berberich et al., Nature Rev Cardiol. (2019) 16(l):9-20). The human ANGPTL3 gene, located on chromosome 1, has CpG islands in the promoter region and has approximately 80% homology with its mouse counterpart. The ANGPTL3 protein is expressed predominantly in the liver.
[0005] In humans, ANGPTL3 plays a key role in lipid and lipoprotein metabolism as well as angiogenesis. ANGPTL3 regulates plasma levels of triglycerides (TG), triglyceride-rich lipoproteins, high-density lipoprotein cholesterol (HDL-C), low-density lipoprotein cholesterol (LDL-C), and very low-density lipoprotein cholesterol (VLDL-C) by inhibiting the activity of lipoprotein lipase (LPL) and/or endothelial lipase (EL). ANGPTL3 deficiency or pharmacological inhibition is associated with a marked reduction of plasma triglyceride and cholesterol levels, and an increased activity of LPL and EL. Loss-of-function (LOF) mutations in the ANGPTL3 gene have been found to lead to low plasma levels of LDL-C, VLDL-C, HDL-C, and TG, and genome-wide association studies suggest that these mutations are associated with lower risk of cardiovascular disease (CVD).
[0006] In view of the critical role of ANGPTL3 in the pathogenesis of hypercholesterolemia and cardiovascular disease, there is a need for new and improved therapies that target the expression of ANGPTL3.
[0007] 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 a..ANGPTL3, including in host cells and organisms.
[0008] In some aspects, the present disclosure provides a system for repressing transcription of a human ANGPTL3 gene in a human cell, optionally a human hepatocyte, comprising a) one or more fusion proteins that collectively comprise a DNA methyltransferase (DNMT) domain and/or a domain that recruits a DNMT, optionally wherein the DNMT domain and/or the recruiter domain comprise a DNMT3A domain and/or a DNMT3L domain, and optionally wherein the recruited DNMT is DNMT3A, and a transcriptional repressor domain, each domain being linked to a DNA-binding domain that binds to a target region in the human ANGPTL3 gene; or b) one or more nucleic acid molecules encoding the one or more fusion proteins.
[0009] In some embodiments, the DNA-binding domain binds to a target sequence in SEQ ID NO: 1019, 1020, or 1021.
[0010] In certain embodiments, the DNA-binding domain targets the fusion protein(s) to one or more sequences in the ANGPTL3 gene selected from SEQ ID NOs: 700-708 and 811- 899.
[0011] In some embodiments, the DNA-binding domain comprises a dead CRISPR Cas (dCas) domain, a ZFP domain, or a TALE domain. For example, the DNA-binding domain may comprise a dCas9 domain, and the system may further comprise (i) one or more guide RNAs (e.g., comprising any one of SEQ ID NOs: 900-988), or (ii) nucleic acid molecules coding for the one or more guide RNAs. In certain embodiments, the dCas domain comprises a dCas9 sequence, such as a sequence with at least 90% identity to SEQ ID NO: 12 or 13.
[0012] In some embodiments, the DNA-binding domain comprises a ZFP domain that targets a nucleotide sequence selected from SEQ ID NOs: 700-708. In certain embodiments, the ZFP domain comprises, in order, the F1-F6 amino acid sequences of any one of ZFP810 through ZFP893 as shown in Table 1.
[0013] In some embodiments, the DNMT3A domain comprises a sequence with at least 90% identity to SEQ ID NO: 574 or 575.
[0014] The DNMT3L domain may comprise, e.g., a sequence with at least 90% identity to a sequence selected from SEQ ID NOs: 578-581. In some embodiments, the DNMT3L domain comprises a sequence with at least 90% identity to a sequence selected from SEQ ID NOs: 582-603. In some embodiments, the DNMT3L domain comprises a sequence with at least 90% identity to a sequence selected from SEQ ID NOs: 601-603.
[0015] In some embodiments, the transcriptional repressor domain comprises a sequence with at least 90% identity to a sequence selected from SEQ ID NOs: 33-570. In certain embodiments, the transcriptional repressor domain is a KRAB domain derived from KOX1, ZIM3, ZFP28, or ZN627. The KRAB domain may comprise, e.g., a sequence with at least 90% identity to a sequence selected from SEQ ID NOs: 89, 116, 245, and 255. In some embodiments, the transcriptional repressor domain comprises a fusion of the N- and C- terminal regions of ZIM3 and KOX1 KRAB, and optionally comprises the amino acid sequence of SEQ ID NO: 571 or 572. In certain embodiments, the transcriptional repressor domain is derived from KAP1, MECP2, HPla/CBX5, HPlb, CBX8, CDYL2, TOX, TOX3, TOX4, EED, EZH2, RBBP4, RCOR1, or SCML2.
[0016] In some embodiments, the system comprises a) a fusion protein comprising the DNMT3A domain, the DNMT3L domain, the transcriptional repressor domain, and the DNA-binding domain, optionally wherein one or both of the DNMT3A domain and the DNMT3L domain are human, and optionally wherein the DNA-binding domain is a dead CRISPR Cas domain or a ZFP domain; or b) a nucleic acid molecule encoding the fusion protein.
[0017] In certain embodiments, the fusion protein comprises, from N-terminus to C- terminus, the DNMT3A domain, a first peptide linker, the DNMT3L domain, a second peptide linker, the DNA-binding domain, a third peptide linker, and the transcriptional repressor domain. For example, the fusion protein may comprise, from N-terminus to C- terminus, the DNMT3A domain, the first peptide linker, the DNMT3L domain, the second peptide linker, a first nuclear localization signal (NLS), the DNA-binding domain, a second NLS, the third peptide linker, and the transcriptional repressor domain. The fusion protein may comprise, from N-terminus to C-terminus, a first NLS, the DNMT3A domain, the first peptide linker, the DNMT3L domain, the second peptide linker, the DNA-binding domain, the third peptide linker, the transcriptional repressor domain, and a second NLS. The fusion protein may comprise, from N-terminus to C-terminus, first and second NLSs, the DNMT3A domain, the first peptide linker, the DNMT3L domain, the second peptide linker, the DNA- binding domain, the third peptide linker, the transcriptional repressor domain, and third and fourth NLSs. In particular embodiments, the transcriptional repressor domain is a KRAB domain, such as a human K0X1, ZFP28, ZN627, or ZIM3 KRAB domain. In particular embodiments, one or both of the second and third peptide linkers are XTEN linkers, which may be selected from XTEN80 (e.g., SEQ ID NO: 643) and XTEN16 (e.g., SEQ ID NO: 638), e.g., wherein the second peptide linker is XTEN80, and the third peptide linker is XTEN16.
[0018] In some embodiments, the fusion protein may comprise, from N-terminus to C- terminus, a human DNMT3A domain, a first peptide linker, a human DNMT3L domain, an XTEN80 peptide linker, a first NLS, a dSpCas9 domain, a second NLS, an XTEN 16 peptide linker, and a human KOX1 KRAB domain. In certain embodiments, the fusion protein comprises SEQ ID NO: 658 or a sequence at least 90% identical thereto.
[0019] In some embodiments, the fusion protein comprises, from N-terminus to C- terminus, a human DNMT3A domain, a first peptide linker, a human DNMT3L domain, an XTEN80 peptide linker, a first NLS, a ZFP domain, a second NLS, an XTEN 16 linker, and a human KOX1 KRAB domain. In certain embodiments, the fusion protein comprises SEQ ID NO: 659 or a sequence at least 90% identical thereto, optionally wherein the ZFP comprises, in order, the F1-F6 amino acid sequences of any one of ZFP810 through ZFP893 as shown in Table 1.
[0020] In some embodiments, the fusion protein comprises, from N-terminus to C- terminus, first and second NLSs, a human DNMT3A domain, a first peptide linker, a human DNMT3L domain, an XTEN80 peptide linker, a dSpCas9 domain, an XTEN16 peptide linker, a human KOX1 KRAB domain, and third and fourth NLSs. In particular embodiments, the fusion protein may comprise the amino acid sequence of SEQ ID NO: 660 or a sequence at least 90% identical thereto.
[0021] In some embodiments, the fusion protein comprises, from N-terminus to C- terminus, first and second NLSs, a human DNMT3A domain, a first peptide linker, a human DNMT3L domain, an XTEN80 peptide linker, a ZFP domain, an XTEN16 peptide linker, a human KOX1 KRAB domain, and third and fourth NLSs.
[0022] In some embodiments, the fusion protein comprises, from N-terminus to C- terminus, first and second NLSs, a human DNMT3A domain, a first peptide linker, a human DNMT3L domain, an XTEN80 peptide linker, a dSpCas9 domain, an XTEN16 peptide linker, a human ZFP28 KRAB domain, and third and fourth NLSs. In particular embodiments, the fusion protein may comprise the amino acid sequence of SEQ ID NO: 661 or a sequence at least 90% identical thereto.
[0023] In some embodiments, the fusion protein comprises, from N-terminus to C- terminus, first and second NLSs, a human DNMT3A domain, a first peptide linker, a human DNMT3L domain, an XTEN80 peptide linker, a ZFP domain, an XTEN16 peptide linker, a human ZFP28 KRAB domain, and third and fourth NLSs.
[0024] In some embodiments, the fusion protein comprises, from N-terminus to C- terminus, first and second NLSs, a human DNMT3A domain, a first peptide linker, a human DNMT3L domain, an XTEN80 peptide linker, a dSpCas9 domain, an XTEN16 peptide linker, a human ZN627 KRAB domain, and third and fourth NLSs. In particular embodiments, the fusion protein may comprise the amino acid sequence of SEQ ID NO: 662 or a sequence at least 90% identical thereto.
[0025] In some embodiments, the fusion protein comprises, from N-terminus to C- terminus, first and second NLSs, a human DNMT3A domain, a first peptide linker, a human DNMT3L domain, an XTEN80 peptide linker, a ZFP domain, an XTEN16 peptide linker, a human ZN627 KRAB domain, and third and fourth NLSs.
[0026] In some embodiments, the fusion protein comprises, from N-terminus to C- terminus, first and second NLSs, a human DNMT3A domain, a first peptide linker, a human DNMT3L domain, an XTEN80 peptide linker, a dSpCas9 domain, an XTEN16 peptide linker, a human ZIM3 KRAB domain, and third and fourth NLSs. In particular embodiments, the fusion protein may comprise the amino acid sequence of SEQ ID NO: 663 or a sequence at least 90% identical thereto.
[0027] In some embodiments, the fusion protein comprises, from N-terminus to C- terminus, first and second NLSs, a human DNMT3A domain, a first peptide linker, a human DNMT3L domain, an XTEN80 peptide linker, a ZFP domain, an XTEN16 peptide linker, a human ZIM3 KRAB domain, and third and fourth NLSs.
[0028] In some embodiments, at least one of the NLSs in a fusion protein described herein is an SV40 NLS (e.g., SEQ ID NO: 644).
[0029] In some embodiments, the system comprises: a) a first fusion protein comprising a first DNA-binding domain and comprising or recruiting the DNMT3A domain, a second fusion protein comprising a second DNA-binding domain and comprising or recruiting the DNMT3L domain, and a third fusion protein comprising a third DNA-binding domain and comprising or recruiting the transcriptional repressor domain; or b) one or more nucleic acid molecules encoding the fusion proteins.
[0030] The present disclosure also provides a human cell comprising a system described herein, or progeny of the cell. In some embodiments, the cell is a hepatocyte.
[0031] The present disclosure also provides a pharmaceutical composition comprising a system described herein and a pharmaceutically acceptable excipient. In some embodiments, the composition comprises lipid nanoparticles (LNPs) comprising the system, and/or the DNA-binding domain is a dCas domain and the LNPs further comprise one or more gRNAs. [0032] The present disclosure also provides a method of treating a patient in need thereof, comprising administering a system or pharmaceutical composition described herein to the patient (e.g., intravenously). In some embodiments, the patient 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 (e.g., heterozygous familial hypercholesterolemia (HeFH), or homozygous familial hypercholesterolemia (HoFH)).
[0033] The present disclosure also provides a system or pharmaceutical composition described herein for use in treating a patient in need thereof, e.g., in a method described herein.
[0034] The present disclosure also provides use of a system described herein in the manufacture of a medicament for treating a patient in need thereof, e.g., in a method described herein.
[0035] The present disclosure also provides articles and kits comprising the systems described herein. [0036] Other features, objectives, and advantages of the invention are apparent in the detailed description that follows. It should be understood, however, that the detailed description, while indicating embodiments and embodiments of the invention, is given by way of illustration only, not limitation. Various changes and modifications within the scope of the invention will become apparent to those skilled in the art from the detailed description.
BRIEF DESCRIPTION OF THE DRAWINGS
[0037] FIG. 1A is a diagram summarizing results of ANGPTL3 silencing after treatment with epigenetic repressors (DNMT3A-DNMT3L-dCas9-KRAB) using different gRNAs in a primary ANGPTL3 gRNA screen. FIG. IB is a pair of scatter plots showing agreement of ANGPTL3 silencing results between different replicates. Rectangular boxes indicate the area comprising the no_gRNA replicates. Triangles indicate the area comprising WTCas replicates, indicating replicates using WT Cas9 and an ANGPTL3 gRNA, as described in Example 4.
[0038] FIG. 2 is a set of graphs showing relative ANGPTL3 expression (y-axis) at days 7 and 14 in cells treated with CRISPR-off (DNMT3A-DNMT3L-dCas9-KRAB). The genomic distance from the gRNA target site to the ANGPTL3 transcription start site (TSS) is shown on the x-axis and in the table (right).
[0039] FIG. 3 is a pair of graphs showing ANGPTL3 silencing after treatment with epigenetic repressors using the top 25 gRNAs at day 7 (left) and day 14 (right).
[0040] FIG. 4 is a set of graphs showing ANGPTL3 silencing (y-axis) at days 7 and 14 in cells treated with ANGPTL3 targeting zinc-finger based epigenetic repressors (DNMT3A- DNMT3L-ZF-KRAB, also sometimes referred to herein as “ZF-off”). Repression levels of treatment with WT Cas9 using /WGPT -targcting guide RNAs (positive controls) and Cas9 without gRNA (negative control) are shown for reference. The genomic distance from the gRNA target site to the ANGPTL3 TSS is shown on the x-axis.
[0041] FIG. 5 is a graph showing ANGPTL3 silencing by the top 19 gRNAs (single and dual) targeting the ANGPTL3 locus in an unbiased multiplex screen.
[0042] FIG. 6 is a series of line graphs showing increased silencing potency and durability out to 14 days in Huh7 cells from top hits of a dual gRNA screen against the ANGPTL3 and PCSK9 loci. PLA2628: CRISPR-off construct of SEQ ID NO: 1011. PLA2075: CRISPRi fusion protein (dCas9-KRAB).
[0043] FIG. 7 is a dose response graph showing reduction of ANGPTL3 and PCSK9 secretion at day 7 by a single CRISPR-off repressor in a multiplex format with a single gRNA against each target; “Cutting control” refers to a WT Cas9 control. PLA947: CRISPR-off construct of SEQ ID NO: 1013. PLA1489: CRISPR-off construct of SEQ ID NO: 1009. PLA2628: CRISPR-off construct of SEQ ID NO: 1011.
[0044] FIG. 8A shows schematic illustrations of fusion protein constructs with variant NLS configurations. FIG. 8B shows schematic illustrations of additional fusion protein constructs with variant KRAB domains.
[0045] FIGs. 9A-9D are a set of graphs showing alternative KRAB domains tested for epi-silencing activity against CRISPR-off when using 0.5 ng effector DNA using CLTA-GFP as a marker (FIG. 9A), 3 ng effector DNA using GFP as a marker (FIG. 9B), and 0.5 ng effector DNA using GFP as a marker (FIG. 9C). FIG 9D shows results after 30 days using varying nanogram amounts of effector DNA.
DETAILED DESCRIPTION
[0046] The present disclosure provides epigenetic editors for regulating expression of the ANGPTL3 gene. By altering expression of ANGPTL3, the systems, compositions and methods described herein may be used for treating conditions such as hypercholesterolemia (e.g., heterozygous familial hypercholesterolemia (HeFH), homozygous familial hypercholesterolemia (HoFH), familial hypercholesterolemia (HF), or established atherosclerotic cardiovascular disease (ASCVD)), or renal insufficiency (RI). Unless otherwise stated, ‘ANGPTL3” refers herein to human ANGPTL3. A human ANGPTL3 gene sequence can be found at Ensembl Accession No. ENSG00000132855. The present epigenetic editors have several advantages compared to other genome engineering methods, including reversibility, decreased risk of translocation, and durable, inheritable silencing.
[0047] In some embodiments, the region of the human ANGPTL3 gene targeted for epigenetic regulation is about 2 kb long, and is approximately +/- 1 kb of the ANGPTL3 TSS. In certain embodiments, the region has the nucleotide sequence of SEQ ID NO: 1020. In some embodiments, the targeted ANGPTL3 region is about 1000 bps long, and stretches from approximately 750 bps upstream of the ANGPTL3 transcription start site (TSS) to approximately 250 bps downstream of the TSS. In certain embodiments, the region targeted has the nucleotide sequence of SEQ ID NO: 1021. The TSS of ANGPTL3 is at #chrl:62597514 of Genome GRCh38.
[0048] In some embodiments, an epigenetic editor as described herein may comprise one or more fusion proteins, wherein each fusion protein comprises a DNA-binding domain linked to one or more effector domains for epigenetic modification. In certain embodiments, where the DNA-binding domain is a polynucleotide guided DNA-binding domain, the epigenetic editor may further comprise one or more guide polynucleotides. DNA-binding domains, effector domains, and guide polynucleotides of an epigenetic editor as described herein may be selected, e.g., from those described below, in any functional combination. [0049] The epigenetic editors described herein may be expressed in a host cell transiently, or may be integrated in a genome of the host cell; such cells and their progeny are also contemplated by the present disclosure. Both transiently expressed and integrated epigenetic editors or components thereof can effect stable epigenetic modifications. For example, after introducing to a host cell an epigenetic editor described herein, the target gene in the host cell may be stably or permanently repressed or silenced. In some embodiments, expression of the target gene is reduced or silenced for at least 1 week, at least 2 weeks, at least 3 weeks, at least 4 weeks, at least 5 weeks, at least 6 weeks, at least 7 weeks, at least 2 months, at least 3 months, at least 4 months, at least 5 months, at least 6 months, at least 1 year, at least 2 years, or for the entire lifetime of the cell or the subject carrying the cell, as compared to the level of expression in the absence of the epigenetic editor. The epigenetic modification may be inherited by the progeny of the host cells into which the epigenetic editor was introduced.
[0050] The present epigenetic editors may be introduced to a patient in need thereof (e.g., a human patient), e.g., into the patient’s hepatocytes, biliary epithelial cells (cholangiocytes), stellate cells, Kupffer cells, and liver sinusoidal endothelial cells.
I. DNA-Binding Domains
[0051] 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 the ANGPTL3 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.
[0052] In some embodiments, 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. A. Polynucleotide Guided DNA-Binding Domains
[0053] In some embodiments, 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 the ANGPTL3 gene locus. In certain embodiments, the protein domain may be derived from a CRISPR-associated nuclease, such as a Class I or II CRIS PR-associated nuclease. In some embodiments, 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. In certain embodiments, 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, Csf2, CsO, Csf4, and homologues and modified versions thereof. “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).
[0054] In some embodiments, 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. In some embodiments, 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). In some embodiments, said wildtype polypeptide is Cas9 from Staphylococcus aureus (SEQ ID NO: 3). In some embodiments, 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 Franscisella novicida (UniProt Ref. No. U2UMQ6 or SEQ ID NO: 4). In certain embodiments, 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.
[0055] 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 dassonvillei, Streptomyces pristinaespiralis, Streptomyces viridochromo genes, Streptosporangium roseum, Alicyclobacillus acidocaldarius, Bacillus pseudomycoides, Bacillus selenitireducens, Exiguobacterium sibiricum, Lactobacillus delbrueckii, Lactobacillus salivarius, Lactobacillus buchneri, Treponema denticola, Microscilla marina, Burkholderiales bacterium, Polar omonas naphthalenivorans, Polar omonas sp., Crocosphaera watsonii, Cyanothece sp., Microcystis aeruginosa, Synechococcus sp. , Acetohalobium arabaticum, Ammonifex degensii, Caldicelulosiruptor becscii, Candidatus desulforudis, Clostridium botulinum, Clostridium difficile, Finegoldia magna, Natranaerobius thermophilus, Pelotomaculum thermopropionium, Acidithiobacillus caldus, Acidithiobacillus ferrooxidans, Allochromatium vinosum, Marinobacter sp., Nitrosococcus halophilus, Nitrosococcus watsoni, Pseudoalteromonas haloplanktis, Ktedonobacter racemifer, Methanohalobium evestigatum, Anabaena variabilis, Nodularia spumigena, Nostoc sp., Arthrospira maxima, Arthrospira platensis, Arthrospira sp., Lyngbya sp., Microcoleus chthonoplastes, Oscillator ia sp., Petrotoga mobilis, Thermosipho africanus, Streptococcus pasteurianus, Neisseria cinerea, Campylobacter lari, Parvibaculum lavamentivorans, Coryne bacterium diphtheria, and Acaryochloris marina. Cas9 sequences also include those from the organisms and loci disclosed in Chylinski et al., RNA Biol. (2013) 10(5):726-37.
[0056] In some embodiments, the Cas9 domain is from Streptococcus pyogenes. In some embodiments, the Cas9 domain is from Staphylococcus aureus.
[0057] Other 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).
[0058] For epigenetic editing, the nuclease-derived protein domain (e.g., a Cas9 or Cpfl 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. For example, 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. In some embodiments, a CRIS PR-associated protein domain described herein is catalytically inactive (“dead”). Examples of such domains include, for example, dCas9 (“dead” Cas9), dCpfl, ddCpfl, dCasPhi, ddCasl2a, dLbCpfl, and dFnCpfl. A dCas9 protein domain, for example, 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. For example, 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. In some embodiments, the dCas9 comprises at least one mutation in the HNH subdomain and/or the RuvCl subdomain that reduces or abrogates nuclease activity. In some embodiments, 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.
[0059] In some embodiments, a dCas9 protein herein comprises a mutation at position(s) corresponding to position D10 (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). In particular embodiments, the dCas9 comprises the amino acid sequence of dSpCas9 (D10A and H840A) (SEQ ID NO: 12).
[0060] In some embodiments, 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). In particular embodiments, the dCas9 comprises the amino acid sequence of dSaCas9 (D10A and N580A) (SEQ ID NO.: 13).
[0061] 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). [0062] 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. In some embodiments, 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). In certain embodiments, 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. In some embodiments, the dCpfl comprises a D917A mutation. In particular embodiments, the dCpfl comprises the amino acid sequence of dFnCpfl (SEQ ID NO: 14).
[0063] Further nuclease inactive CRISPR-associated protein domains contemplated herein include those from, for example, dNmeCas9 (e.g., SEQ ID NO: 15), dCjCas9 (e.g., SEQ ID NO: 16), dStlCas9 (e.g., SEQ ID NO: 17), dSt3Cas9 (e.g., SEQ ID NO: 18), dLbCpfl (e.g., SEQ ID NO: 19), dAsCpfl (e.g., SEQ ID NO: 20), denAsCpfl (e.g., SEQ ID NO: 21), dHFAsCpfl (e.g., SEQ ID NO: 22), dRVRAsCpfl (e.g., SEQ ID NO: 23), dRRAsCpfl (e.g., SEQ ID NO: 24), dCasX (e.g., SEQ ID NO: 25), and dCasPhi (e.g., SEQ ID NO: 26).
[0064] In some embodiments, 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. In certain embodiments, the high fidelity Cas9 domain may be nuclease inactive as described herein.
[0065] 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. For example, 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. Nuclease inactive versions of these Cas9 domains are also contemplated, such as nuclease inactive VRER SpCas9 (e.g., SEQ ID NO: 27), nuclease inactive EQR SpCas9 (e.g., SEQ ID NO: 28), nuclease inactive VQR SpCas9 (e.g., SEQ ID NO: 29), nuclease inactive SpG Cas9 (e.g., SEQ ID NO: 30), nuclease inactive SpRY Cas9 (e.g., SEQ ID NO: 31), and nuclease inactive KKH SaCas9 (e.g., SEQ ID NO: 32). Another example is the Cas9 of Francisella novicida engineered to recognize 5’-YG-3’ (where “Y” is a pyrimidine).
[0066] Additional suitable CRIS PR-associated proteins, orthologs, and variants, including nuclease inactive variants and sequences, will be apparent to those of skill in the art based on this disclosure.
[0067] Guide RNAs that can be used in conjunction with the CRIS PR-associated protein domains herein are further described in Section II below.
B. Zinc Finger Protein Domains
[0068] In some embodiments, 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.
[0069] 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 (C2H2 class) 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. In some embodiments, a ZFP domain herein may comprise a ZF array comprising sequential C2H2-ZFs each contacting three or more sequential nucleotides. [0070] 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. In some embodiments, a ZFP domain comprising at least three ZFs recognizes a target DNA sequence of 9 or 10 nucleotides. In some embodiments, a ZFP domain comprising at least four ZFs recognizes a target DNA sequence of 12 to 14 nucleotides. In some embodiments, a ZFP domain comprising at least six ZFs recognizes a target DNA sequence of 18 to 21 nucleotides.
[0071] In some embodiments, 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. In some embodiments, a linker comprises
5 or more amino acids. In some embodiments, a linker comprises 7-17 amino acids. The linker may be flexible or rigid.
[0072] In some embodiments a zinc finger array may have the sequence:
SRPGERPFQCRICMRNFSXXXXXXXHXXTHTGEKPFQCRICMRNFSXXXXXXXHXXT H [linker] FQCRICMRNFSXXXXXXXHXXTHTGEKPFQCRICMRNFSXXXXXXXHX XTH [linker] PFQCRICMRNFSXXXXXXXHXXTHTGEKPFQCRICMRNFSXXXXXX XHXXTHLRGS ( SEQ ID NO : 650 ) , or a sequence at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical thereto, where “XXXXXXX” represents the amino acids of the ZF recognition helix, which confers DNA-binding specificity upon the zinc finger; each X may be independently chosen. In the above sequence, “XX” in italics may be TR, LR or LK, and “[linker]” represents a linker sequence. In some embodiments, the linker sequence is TGSQKP (SEQ ID NO: 651); this linker may be used when sub-sites targeted by the ZFs are adjacent. In some embodiments, 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.
[0073] 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. In some embodiments, directly adjacent fingers bind to contiguous nucleic acid sequences, i.e., to adjacent trinucleotides/triplets. In some embodiments, 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. In some embodiments, distant ZFs within the ZFP domain may recognize (or bind to) non-contiguous nucleotide sequences.
[0074] The amino acid sequences of the ZF DNA-recognition helices of exemplary ZFP domains herein, and their ANGPTL3 target sequences, are shown below in Table 1, where numbers within the parentheses denote SEQ ID NOs:
Table 1. ZF Sequences of Exemplary ZFP Domains Targeting ANGPTL3
Figure imgf000018_0001
Figure imgf000019_0001
Figure imgf000020_0001
Figure imgf000021_0001
Figure imgf000022_0001
Figure imgf000023_0001
[0075] In some embodiments, the ZFP domain of the present epigenetic editor binds to a target sequence selected from any one of SEQ ID NOs: 700-708. In further embodiments, the ZFP domain comprises, in order, the F1-F6 amino acid sequences of any one of ZFP810- ZFP893 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.
C. TALES
[0076] In some embodiments, 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. TALEs 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. (2007) 25(7):778-85; Christian et al., Genetics (2008) 186(2):757-61; Li et al., Nucl Acids Res. (2010) 39(l):359-72; and Moscou et al., Science (2009) 326(5959): 1501.
D. Other DNA-Binding Domains
[0077] Other DNA-binding domains are contemplated for the epigenetic editors described herein. In some embodiments, 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. In contrast to Cas9, the NgAgo-gDNA system does not require a protospacer- adjacent motif (PAM). Thus, using a nuclease inactive NgAgo (dNgAgo) can greatly expand the bases that may be targeted. 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.
[0078] In some embodiments, the DNA-binding domain comprises an inactivated nuclease, for example, an inactivated meganuclease. Additional non-limiting examples of 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.
II. Guide Polynucleotides
[0079] 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. For example, where the polynucleotide guided DNA-binding domain is a CRISPR-associated protein domain, the guide polynucleotide may be a guide RNA (gRNA). 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. Methods of using guide polynucleotide sequences with programmable DNA-binding proteins (e.g., CRISPR-associated protein domains) for site-specific DNA targeting (e.g., to modify a genome) are known in the art.
[0080] A guide polynucleotide sequence (e.g., a gRNA 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. In some embodiments, a guide polynucleotide comprises parts 1) and 2) connected by a linker. In some embodiments, a guide polynucleotide comprises parts 1) and 2) connected by a non-nucleic acid linker, for example, a peptide linker or a chemical linker.
[0081] Part 2 (the scaffold sequence) of a guide polynucleotide as described herein may be, for example, as described in Jinek et al., Science (2012) 337:816-21; U.S. Patent Publication 2016/0208288; or U.S. Patent Publication 2016/0200779. Variants of part 2) are also contemplated by the present disclosure. For example, the tetraloop and stem loop of a gRNA scaffold (tracrRNA) sequence may be modified to include RNA aptamers, which can be bound by specific protein domains. In some embodiments, such modified gRNAs can be used to facilitate the recruitment of repressive or activating domains fused to the proteininteracting RNA aptamers.
[0082] A gRNA as provided herein typically comprises a targeting domain and a binding domain. The targeting domain (also termed “targeting sequence”) may comprise a nucleic acid sequence that binds to a target site, e.g., to a genomic nucleic acid molecule within a cell. The target site may be a double-stranded DNA sequence comprising a PAM sequence as well as the target sequence, which is located on the same strand as, and directly adjacent to, the PAM sequence. The targeting domain of the gRNA may comprise an RNA sequence that corresponds to the target sequence, i.e., it resembles the sequence of the target domain, sometimes with one or more mismatches, but typically comprising an RNA sequence instead of a DNA sequence. 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. For an illustration of the location of the PAM and the mechanism of gRNA binding to a target site, see, e.g., Figure 1 of Vanegas et al., Fungal Biol Biotechnol. (2019) 6:6, which is incorporated by reference herein. For additional illustration and description of the mechanism of gRNA targeting of an RNA-guided nuclease to a target site, see Fu et al., Nat Biotechnol (2014) 32(3):279-84 and Sternberg et al., Nature (2014) 507(7490):62-7, each incorporated herein by reference. [0083] In some embodiments, 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.
[0084] An exemplary illustration of a Cas9 target site, comprising a 22 nucleotide target domain, and an NGG 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:
[ target domain ( DNA) ] [ PAM ]
5 ' -N-N-N-N-N-N-N-N-N-N-N-N-N-N-N-N-N-N-N-N-N-N-N-G-G-3 ' ( DNA) 3 ' -N-N-N-N-N-N-N-N-N-N-N-N-N-N-N-N-N-N-N-N-N-N-N-C-C-5 ' ( DNA)
I I I I I I I I I I I I I I I I I I I I I I
5 ' -N-N-N-N-N-N-N-N-N-N-N-N-N-N-N-N-N-N-N-N-N-N- [ gRNA scaffold] -3 ' ( RNA) [ target ing domain ( RNA) ] [ binding domain ]
[0085] An exemplary illustration of a 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: [ PAM ] [ target domain ( DNA) ]
5 ' -T-T-N-N-N-N-N-N-N-N-N-N-N-N-N-N-N-N-N-N-N-N-N-N-N-3 ' ( DNA)
3 ' -A-A-N-N-N-N-N-N-N-N-N-N-N-N-N-N-N-N-N-N-N-N-N-N-N-5 ' ( DNA)
I I I I I I I I I I I I I I I I I I I I I I
5 ' - [ gRNA scaffold] -N-N-N-N-N-N-N-N-N-N-N-N-N-N-N-N-N-N-N-N-N-N-3 ' ( RNA)
[ binding domain ] [ target ing domain ( RNA) ]
[0086] While not wishing to be bound by theory, at least in some embodiments, it is believed that 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. In some embodiments, 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. In some embodiments, the targeting domain is 17 nucleotides in length. In some embodiments, the targeting domain is 18 nucleotides in length. In some embodiments, the targeting domain is 19 nucleotides in length. In some embodiments, the targeting domain is 20 nucleotides in length. In some embodiments, the targeting domain is 21 nucleotides in length. In some embodiments, the targeting domain is 22 nucleotides in length. In some embodiments, the targeting domain is 23 nucleotides in length. In some embodiments, the targeting domain is 24 nucleotides in length. In some embodiments, the targeting domain is 25 nucleotides in length. In certain embodiments, the targeting domain fully corresponds, without mismatch, to a target sequence provided herein, or a part thereof. In some embodiments, 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.
[0087] Methods for designing, selecting, and validating gRNAs are described herein and known in the art. Software tools can be used to optimize the gRNAs corresponding to a target DNA sequence, e.g., to minimize total off-target activity across the genome. For example, DNA sequence searching algorithms can be used to identify a target sequence in crRNAs of a gRNA for use with Cas9. Exemplary gRNA design tools include the ones described in Bae et al., Bioinformatics (2014) 30:1473-5.
[0088] Guide polynucleotides (e.g., gRNAs) described herein may be of various lengths. In some embodiments, the length of the spacer or targeting sequence depends on the CRISPR-associated protein component of the epigenetic editor system used. For example, Cas proteins from different bacterial species have varying optimal targeting sequence lengths. Accordingly, 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. In some embodiments, the spacer comprises 10-24, 11-20, 11-16, 18-24, 19-21, or 20 nucleotides in length. In some embodiments, a guide polynucleotide (e.g., 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. In some embodiments, a guide polynucleotide described herein may be truncated, e.g., by 1, 2, 3, 4, 5, 10, 15, 20, 25, 30, 40, 50 or more nucleotides.
[0089] In certain embodiments, the 3’ end of the ANGPTL3 target sequence is immediately adjacent to 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%. In particular embodiments, the targeting and the target sequence may be 100% complementary. In other embodiments, the targeting sequence and the target sequence may contain, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 mismatches.
[0090] A guide polynucleotide (e.g., gRNA) may be modified with, for example, chemical alterations and synthetic modifications. A modified gRNA, for instance, 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.
[0091] In some embodiments, one or more ribose groups of the gRNA may be modified. Examples of 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’-0-ethylamine, 2’-O-cyanoethyl, 2’-0-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 oligonucleotides.
[0092] In some embodiments, one or more phosphate groups of the gRNA may be chemically modified. Examples of 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. In some embodiments, 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.
[0093] In some embodiments, the gRNA herein comprises a mixture of ribonucleotides and deoxyribonucleotides and/or one or more PS linkages.
[0094] In some embodiments, one or more nucleobases of the gRNA may be chemically modified. Examples of 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.
[0095] Table 2 below lists exemplary gRNA target sequences for epigenetic modification of human ANGPTL3, as well as the coordinates of the start and end positions of the targeted site on human chromosome 1 (SEQ: SEQ ID NO). The table also shows the distance from the start coordinate to the TSS coordinate of the ANGPTL3 gene.
Table 2. Exemplary Target Sequences of gRNAs Targeting ANGPTL3
Figure imgf000029_0001
Figure imgf000030_0001
Figure imgf000031_0001
[0096] Any tracr sequence known in the art is contemplated for a gRNA described herein. In some embodiments, a gRNA described herein has a tracr sequence shown in Table 3 below, or a tracr sequence at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to the tracr sequence shown below (SEQ: SEQ ID NO).
Table 3. Exemplary TRACR Sequences
Figure imgf000031_0002
Figure imgf000032_0001
[0097] In some embodiments, a gRNA described herein is used alone or in combination with one or more gRNAs targeting the same sequence or one or more different sequences in the same gene. In some embodiments, a gRNA described herein is used in combination with one or more gRNAs targeting different genes. In some embodiments, an ANGPTL3 targeting gRNA described herein (e.g., comprising any one of SEQ ID NOs: 900-988) is used in combination with another ANGPTL3 targeting gRNA described herein (e.g., comprising any one of SEQ ID NOs: 900-988). For example, a combination of ANGPTL3 targeting gRNAs may be any combination shown in Table 11. In some embodiments, an ANGPTL3 targeting gRNA described herein (e.g., comprising any one of SEQ ID NOs: 900-988) is used in combination with one or more PCSK9 targeting gRNAs. For example, the PCSK9 targeting gRNA may be gRNA41 (SEQ ID NO: 1024). In certain embodiments, the ANGPTL3 targeting gRNA is RNA1094 (SEQ ID NO: 940) or RNA1096 (SEQ ID NO: 962) and the PCSK9 targeting gRNA is gRNA41 (SEQ ID NO: 1024). In some embodiments, the gRNA herein is provided to the cell directly (e.g., through an RNP complex together with the CRISPR-associated protein domain). In some embodiments, 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. Methods of introducing gRNAs and expression vectors into cells are well known in the art.
III. Effector Domains
[0098] 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. In some embodiments, an effector domain described herein may provide repression or silencing of expression of a target gene such as ANGPTL3, e.g., by repressing transcription or by modifying or remodeling chromatin. Such effector domains are also referred to herein as “repression domains,” “repressor domains,” or “epigenetic repressor domains.” Non-limiting examples of chemical modifications that may be mediated by effector domains include methylation, demethylation, acetylation, deacetylation, phosphorylation, SUMOylation and/or ubiquitination of DNA or histone residues. [0099] In some embodiments, 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.
[0100] In some embodiments, 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.
[0101] In some embodiments, 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.
[0102] 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. For example, 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.
[0103] 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. In particular embodiments, 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. [0104] In some embodiments, 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. In certain embodiments, an effector domain comprises a fusion of a truncated form of an effector domain and a second effector domain. In certain embodiments, 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). [0105] In some embodiments, 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. In certain embodiments, 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. In some embodiments, 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.
[0106] In certain embodiments, an effector domain described herein (e.g., DNMT3A and/or DNMT3L) is encoded by a nucleotide sequence as found in the native genome (e.g., human or murine) for that effector domain. In other embodiments, an effector domain described herein is encoded by a nucleotide sequence that has been codon-optimized for optimal expression in human cells.
[0107] Effector domains described herein may include, for example, transcriptional repressors, DNA methyltransferases, and/or histone modifiers, as further detailed below.
A. Transcriptional Repressors
[0108] In some embodiments, 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 domain, or any combination thereof. The effector domain may recruit one or more protein domains that repress expression of the target gene, e.g., through a scaffold protein. In some embodiments, 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.
[0109] In some embodiments, 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.
[0110] In certain embodiments, the effector domain comprises a repressor domain (e.g., KRAB) derived from KOX1/ZNF10, KOX8/ZNF708, ZNF43, ZNF184, ZNF91, HPF4, HTF10, or HTF34. In some embodiments, 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, ZNF764, ZNF785, or any combination thereof. For example, the repressor domain may be a KRAB domain derived from KOX1, ZIM3, ZFP28, or ZN627. In particular embodiments, the repressor domain is a ZIM3 KRAB domain. In further embodiments, the effector domain is derived from a human protein, e.g., a human ZIM3, a human KOX1, a human ZFP28, or a human ZN627.
[0111] Sequences of exemplary effector domains that may reduce or silence target gene expression, or protein sequences that contain them, are provided in Table 4 below (SEQ: SEQ ID NO). Further examples of repressors and transcriptional repressor domains can be found, e.g., in PCT Patent Publication WO 2021/226077 and Tycko et al., Cell (2020) 183(7):2020-35, each of which is incorporated herein by reference in its entirety.
Table 4. Exemplary Effector Domains That May Reduce or Silence Gene Expression
Figure imgf000035_0001
Figure imgf000035_0002
Figure imgf000036_0001
Figure imgf000036_0002
Figure imgf000037_0001
Figure imgf000037_0002
Figure imgf000038_0001
Figure imgf000038_0002
Figure imgf000039_0001
Figure imgf000039_0002
Figure imgf000040_0001
Figure imgf000040_0002
Figure imgf000041_0001
Figure imgf000041_0002
Figure imgf000042_0001
Figure imgf000042_0002
Figure imgf000043_0002
Figure imgf000043_0001
[0112] 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. For example, 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. In some embodiments, 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. [0113] In certain embodiments, an epigenetic editor described herein comprises a KRAB domain derived from KOX1, ZIM3, ZFP28, or ZN627, and/or an effector domain derived from KAP1, MECP2, HPla, HPlb, CBX8, CDYL2, TOX, TOX3, TOX4, EED, EZH2, RBBP4, RCOR1, or SCML2, optionally wherein the parental protein is a human protein. In particular embodiments, an epigenetic editor described herein comprises a domain derived from KOX1, ZIM3, ZFP28, and/or ZN627, optionally wherein the parental protein is a human protein. In certain embodiments, the epigenetic editor may comprise a KRAB domain derived from KOX1 (ZNF10), e.g., a human KOX1. In certain embodiments, the epigenetic editor may comprise a KRAB domain derived from ZIM3 (ZNF657 or ZNF264), e.g., a human ZIM3. In certain embodiments, the epigenetic editor may comprise a KRAB domain derived from ZFP28, e.g., a human ZFP28. In certain embodiments, the epigenetic editor may comprise a KRAB domain derived from ZN627, e.g., a human ZN627. In certain embodiments, 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).
[0114] In certain embodiments, an epigenetic effector described herein comprises a repressor domain derived from KOX1/ZNF10 (SEQ ID NO: 89). For example, the repressor domain may comprise the sequence of SEQ ID NO: 89, or a sequence at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 89.
[0115] In certain embodiments, an epigenetic effector described herein comprises a repressor domain derived from KOX1/ZNF10, as shown in Table 5 below: Table 5. Exemplary Effector Domains Derived from KOX1/ZNF10
Figure imgf000044_0001
[0116] In particular embodiments, 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.
[0117] In particular embodiments, 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.
[0118] In particular embodiments, 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.
[0119] In particular embodiments, 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.
[0120] In particular embodiments, the repressor domain may comprise the amino acid sequence of SEQ ID NO: 569, or a sequence at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 569.
[0121] In particular embodiments, 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.
[0122] In particular embodiments, 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.
[0123] In particular embodiments, 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. B. DNA Methyltransferases
[0124] In some embodiments, an effector domain of an epigenetic editor described herein alters target gene expression through DNA modification, such as methylation. Highly methylated areas of DNA tend to be less transcriptionally active than less methylated areas. DNA methylation occurs primarily at CpG sites (shorthand for “C-phosphate-G-” or “cytosine-phosphate-guanine” sites). Many mammalian genes have promoter regions near or including CpG islands (nucleic acid regions with a high frequency of CpG dinucleotides). [0125] 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., DNMT1, DNMT3A, DNMT3B, and DNMT3C). This term also encompasses non-canonical family members that do not catalyze methylation themselves but that recruit (including activate) catalytically active DNMTs; a non-limiting examples of such a DNMT is DNMT3L. See, e.g., Lyko, Nat Review (2018) 19:81-92. Unless otherwise indicated, a DNMT domain may refer to a polypeptide domain derived from a catalytically active DNMT (e.g., DNMT1, DNMT3A, and DNMT3B) or from a catalytically inactive DNMT (e.g., DNMT3L). A DNMT may repress expression of the target gene through the recruitment of repressive regulatory proteins. In some embodiments, the methylation is at a CG (or CpG) dinucleotide sequence. In some embodiments, the methylation is at a CHG or CHH sequence, where H is any one of A, T, or C.
[0126] In some embodiments, 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. In certain embodiments, the bacterial species is Mycoplasmatales bacterium, Mycoplasma marinum, or Spiroplasma chinense. In certain embodiments, the bacterial species is not M. penetrans, S. monbiae, H. parainfluenzae, A. luteus, H. aegyptius, H. haemolyticus, Moraxella, E. coli, T. aquaticus, C. crescentus, or C. difficile. In certain embodiments, an epigenetic editor described herein comprises a DNMT domain comprising SEQ ID NO: 601, or a sequence at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 601. In certain embodiments, an epigenetic editor described herein comprises a DNMT domain comprising SEQ ID NO: 602, or a sequence at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 602. In certain embodiments, an epigenetic editor described herein comprises a DNMT domain comprising SEQ ID NO: 603, or a sequence at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 603.
[0127] In certain embodiments, DNMTs in the epigenetic editors described herein may include, e.g., DNMT1, DNMT3A, DNMT3B, and/or DNMT3C. In some embodiments, the DNMT is a mammalian (e.g., human or murine) DNMT. In particular embodiments, the DNMT is DNMT3A (e.g., human DNMT3A). In certain embodiments, an epigenetic editor described herein comprises 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. In certain embodiments, an epigenetic editor described herein comprises 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. In some embodiments, the DNMT3A domain may have, e.g., a mutation at position H739 (such as H739A or H739E), R771 (such as R771L) and/or R836 (such as R836A or R836Q), or any combination thereof (numbering according to SEQ ID NO: 574). [0128] In some embodiments, an effector domain described herein may be a DNMT-like domain. As used herein a “DNMT-like domain” is a regulatory factor of DNMT that may activate or recruit other DNMT domains, but does not itself possess methylation activity. In some embodiments, the DNMT-like domain is a mammalian (e.g., human or mouse) DNMT- like domain. In certain embodiments, the DNMT-like domain is DNMT3L, which may be, for example, human DNMT3L or mouse DNMT3L. In certain embodiments, 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. In certain embodiments, 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. In certain embodiments, an epigenetic editor described herein comprises a DNMT3L domain comprising SEQ ID NO: 580, or a sequence at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 580. In certain embodiments, an epigenetic editor described herein comprises a DNMT3L domain comprising SEQ ID NO: 581, or a sequence at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 581. In some embodiments, 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).
[0129] In certain embodiments, an epigenetic editor herein may comprise comprising both DNMT and DNMT-like effector domains. For example, the epigenetic editor may comprise a DNMT3A-3L domain, wherein DNMT3A and DNMT3L may be covalently linked. In other embodiments, an epigenetic editor described herein may comprise an effector domain that comprises only a DNMT3A domain (e.g., human DNMT3A), or only a DNMT-like domain (e.g., DNMT3L, which may be human or mouse DNMT3L).
[0130] Table 6 below provides exemplary DNMTs that may be part of an epigenetic effector domain described herein, or from which an effector domain of an epigenetic editor described herein may be derived.
Table 6. Exemplary DNMT Sequences
Figure imgf000047_0001
Figure imgf000048_0001
Figure imgf000049_0001
[0131] 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. For example, 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. In some embodiments, 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. In some embodiments, the effector domain herein comprises only the functional domain (or functional analog thereof), e.g., the catalytic domain or recruiting domain, of an above-listed protein. In some embodiments, the effector domain herein comprises one or more epigenetic effector domains selected from Table 6, or functional homologs, orthologs, or variants thereof.
[0132] As used herein, a DNMT domain (e.g., a DNMT3A domain or a DNMT3L domain) refers to a protein domain that is identical to the parental protein (e.g., a human or murine DNMT3A or DNMT3L) or a functional analog thereof (e.g., having a functional fragment, such as a catalytic fragment or recruiting fragment, of the parental protein; and/or having mutations that improve the activity of the DNMT protein).
[0133] 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. For example, 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%. As used herein, an observed-to-expected CpG ratio is determined by Number of CpG * (sequence length) / (Number of C * Number of G). In some embodiments, the CpG island has an observed-to-expected CpG ratio of at least 60%, 70%, 80%, 90% or more. A CpG island may be a sequence or region of, e.g., at least 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, or 800 nucleotides. In some embodiments, only 1, or less than 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 30, 40, or 50 CpG dinucleotides are methylated by the epigenetic editor.
[0134] In some embodiments, 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.
[0135] In some embodiments, an epigenetic editor described herein induces methylation at a hypermethylated nucleic acid sequence.
[0136] In some embodiments, methylation may be introduced by the epigenetic editor at a site other than a CpG dinucleotide. For example, the target gene sequence may be methylated at the C nucleotide of CpA, CpT, or CpC sequences. In some embodiments, an epigenetic editor comprises a DNMT3A domain and effects methylation at CpG, CpA, CpT, CpC sequences, or any combination thereof. In some embodiments, an epigenetic editor comprises a DNMT3A domain that lacks a regulatory subdomain and only maintains a catalytic domain. In some embodiments, the epigenetic editor comprising a DNMT3A catalytic domain effects methylation exclusively at CpG sequences. In some embodiments, 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.
C. Histone Modifiers
[0137] In some embodiments, 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. Recent studies have revealed that 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.
[0138] In particular, 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. For example, histone acetyltransferases (HATs) utilize acetyl-CoA as a cofactor and catalyze the transfer of an acetyl group to the epsilon amino group of the lysine side chains. 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. On the other hand, histone methylation of lysine 9 of histone H3 may be associated with heterochromatin, or transcriptionally silent chromatin.
[0139] In certain embodiments, an effector domain of an epigenetic editor described herein comprises a histone methyltransferase domain. The effector domain may comprise, for example, a DOT1L domain, a SET domain, a SUV39H1 domain, a G9a/EHMT2 protein domain, an EZH1 domain, an EZH2 domain, a SETDB1 domain, or any combination thereof. In particular embodiments, the effector domain comprises a histone-lysine-N- methyltransferase SETDB1 domain.
[0140] In some embodiments, the effector domain comprises a histone deacetylase protein domain. In certain embodiments, the effector domain comprises a HD AC family protein domain, for example, a HDAC1, HDAC3, HDAC5, HDAC7, or HDAC9 protein domain. In particular embodiments, the effector domain comprises a nucleosome remodeling and deacetylase complex (NURD), which removes acetyl groups from histones.
D. Other Effector Domains
[0141] In some embodiments, the effector domain comprises a tripartite motif containing protein (TRIM28, TIFl-beta, or KAP1). In certain embodiments, 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. For example, 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. In some embodiments, KAP1 interacts with or recruits a histone deacetylase protein, a histone-lysine methyltransferase protein, a chromatin remodeling protein, and/or a heterochromatin protein. For example, 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. In some embodiments, 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.
[0142] In some embodiments, the effector domain comprises a protein domain that interacts with or is recruited by one or more DNA epigenetic marks. For example, 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). An MECP2 protein domain in an epigenetic editor described herein may induce condensed chromatin structure, thereby reducing or silencing expression of the target gene. In some embodiments, 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. In some embodiments, 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. [0143] Also contemplated as 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 Yaf2/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-binding domain, a homeodomain, a MYM-type zinc finger with FCS sequence domain (ZF-FCS), an interferon regulatory factor 2-binding protein zinc finger domain (IRF-2BP1_2), an SSX repressor domain (SSXRD), a B-box-type zinc finger domain (ZF-B_box), a CXXC zinc finger domain (ZF-CXXC), a regulator of chromosome condensation 1 domain (RCC1), an SRC homology 3 domain (SH3_9), a sterile alpha motif domain (SAM_1), a sterile alpha motif domain (SAM_2), a sterile alpha motif/Pointed domain (SAM_PNT), a Vestigial/Tondu family domain (Vg_Tdu), a LIM domain, an RNA recognition motif domain (RRM_1), a paired amphipathic helix domain (PAH), a proteasomal ATPase OB C-terminal domain (Prot_ATP_ID_OB), a nervy homology 2 domain (NHR2), a hinge domain of cleavage stimulation factor subunit 2 (CSTF2_hinge), a PPAR gamma N-terminal region domain (PPARgamma_N), a CDC48 N- terminal domain (CDC48_2), a WD40 repeat domain (WD40), a Fipl motif domain (Fipl), a PDZ domain (PDZ_6), a Von Willebrand factor type C domain (VWC), a NAB conserved region 1 domain (NCD1), an SI RNA-binding domain (SI), an HNF3 C-terminal domain (HNF_C), a Tudor domain (Tudor_2), a histone-like transcription factor (CBF/NF-Y) and archaeal histone domain (CBFD_NFYB_HMF), a zinc finger protein domain (DUF3669), an EGF-like domain (cEGF), a GATA zinc finger domain (GATA), a TEA/ATTS domain (TEA), a phorbol esters/diacylglycerol binding domain (Cl-1), polycomb-like MTF2 factor 2 domain (Mtf2_C), a transactivation domain of FOXO protein family (FOXO-TAD), a homeobox KN domain (Homeobox_KN), a BED zinc finger domain (ZF-BED), a zinc finger of C3HC4-type RING domain (ZF-C3HC4_4), a RAD51 interacting motif domain (RAD5 l_interact), a p55-binding region of a nethyl-CpG-binding domain protein MBD (MBDa), a Notch domain, a Raf-like Ras-binding domain (RBD), a Spin/Ssty family domain (Spin-Ssty), a PHD finger domain (PHD_3), a Low-density lipoprotein receptor domain class A (Ldl_recept_a), a CS domain, a DM DNA-binding domain, and a QLQ domain.
[0144] In some embodiments, the effector domain is a protein domain comprising a YAF2_RYBP domain or homeodomain or any combination thereof. In certain embodiments, the homeodomain of the YAF2_RYBP domain is a PRD domain, an NKL domain, a HOXL domain, or a LIM domain. In particular embodiments, the YAF2_RYBP domain may comprise a 32 amino acid Yaf2/RYBP C-terminal binding motif domain (32 aa RYBP).
[0145] In some embodiments, 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).
[0146] In some embodiments, the effector domain comprises an HNF3 C-terminal domain (HNF_C). The HNF_C domain may be from FOXA1 or FOXA2. In certain embodiments, the HNF_C domain comprises an EH1 (engrailed homology 1) motif. [0147] In some embodiments, 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.
IV. Epigenetic Editors
[0148] Provided herein are epigenetic editors (i.e., epigenetic editing systems) that direct epigenetic modification(s) to a target sequence in a gene of interest, e.g., using one or more DNA-binding domains as described herein and one or more effector domains (e.g., epigenetic repressor domains) as described herein, in any combination. 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 the target sequence, resulting in gene repression or silencing that may be durable and inheritable across cell generations. In some aspects, the epigenetic editors described herein can repress or silence genes reversibly or irreversibly in cells.
[0149] In particular embodiments, an epigenetic editor described herein comprises one or more fusion proteins, each comprising (1) DNA-binding domain(s) and (2) effector domain(s). The effector domains may be on one or more fusion proteins comprised by the epigenetic editor. For example, a single fusion protein may comprise all of the effector domains with a DNA-binding domain. Alternatively, the effector domains or subsets thereof may be on separate fusion proteins, each with a DNA-binding domain (which may be the same or different). A fusion protein described herein may further comprise one or more linkers (e.g., peptide linkers), detectable tags, nuclear localization signals (NESs), or any combination thereof. As used herein, a “fusion protein” refers to a chimeric protein in which two or more coding sequences (e.g., for DNA-binding domain(s) and/or effector domain(s)) are covalently or non-covalently joined, directly or indirectly.
[0150] In some embodiments, 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. In certain embodiments, 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. For example, 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). Such an epigenetic editor may comprise, for instance, a KRAB domain, a DNMT3A domain, and a DNMT3L domain. In some embodiments, 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). In some embodiments, the additional effector domain is a CDYL2, TOX, TOX3, TOX4, or HPla domain. For example, 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).
A. Linkers
[0151] 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.
[0152] In some embodiments, 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. In some embodiments, the linker can comprise a peptide between 1 and 200 (e.g., between 1 and 80) amino acids. In some embodiments, 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, 5 to 10, 5 to 20, 5 to 30, 5 to 40, 5 to 60, 5 to 80, 5 to 100, 5 to 150, 5 to 200, 10 to 20, 10 to 30, 10 to 40, 10 to 50, 10 to 60, 10 to 80, 10 to 100, 10 to 150, 10 to 200, 20 to 30, 20 to 40, 20 to 50, 20 to 60, 20 to 80, 20 to 100, 20 to 150, 20 to 200, 30 to 40, 30 to 50, 30 to 60, 30 to 80, 30 to 100, 30 to 150, 30 to 200, 40 to 50, 40 to 60, 40 to 80, 40 to 100, 40 to 150, 40 to 200, 50 to 60 50 to 80, 50 to 100, 50 to 150, 50 to 200, 60 to 80, 60 to 100, 60 to 150, 60 to 200, 80 to 100, 80 to 150, 80 to 200, 100 to 150, 100 to 200, or 150 to 200 amino acids in length. Longer or shorter linkers are also contemplated. In some embodiments, the peptide linker is 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 25, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or 100 amino acids in length. For example, the peptide linker may be 4, 5, 16, 20, 24, 27, 32, 40, 64, 92, or 104 amino acids in length. The peptide linker may be a flexible or rigid linker. In particular embodiments, the peptide linker comprises the amino acid sequence of any one of SEQ ID NOs: 631-637 and 664-666 or a sequence at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical thereto.
[0153] In certain embodiments, the peptide linker is an XTEN linker. Such 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. The term “XTEN” as used herein 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. These XTEN fusion proteins are produced using recombinant technology, without the need for chemical modifications, and degraded by natural pathways. 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. In certain embodiments, 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. In particular embodiments, the XTEN linker comprises the amino acid sequence of SEQ ID NO: 638. In particular embodiments, the XTEN linker comprises the amino acid sequence of SEQ ID NO: 643.
[0154] In some embodiments, one or more linkers utilized in an epigenetic editor provided herein is a non-peptide linker. For example, the linker may be a carbon bond, a disulfide bond, or carbon-heteroatom bond. In certain embodiments, the linker is a carbonnitrogen bond of an amide linkage. In certain embodiments, the linker is a cyclic or acyclic, substituted or unsubstituted, or branched or unbranched aliphatic or heteroaliphatic linker. [0155] In some embodiments, 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, betaalanine, 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. In certain embodiments, 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. [0156] Various 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. In some embodiments, 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. Nonlimiting examples of the GS-rich linkers are (GGGGS)n (SEQ ID NO: 664), (G)n (SEQ ID NO: 1029), and W linker (SEQ ID NO: 637). In some embodiments, the more rigid linkers are in the form of the form (EAAAK)n (SEQ ID NO: 665), (SGGS)n (SEQ ID NO: 666), and (XP)n (SEQ ID NO: 1027)). In the aforementioned formulae of flexible and rigid linkers, 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: 1028) 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).
[0157] In some embodiments, 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. In some embodiments, a linker in an epigenetic editor described herein comprises an expression tag, e.g., a detectable tag such as a green fluorescent protein.
B. Nuclear Localization Signals
[0158] 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. For example, the fusion protein may comprise 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more nuclear localization signals. As used herein, a “nuclear localization signal” (NLS) is an amino acid sequence that directs proteins to the nucleus. In certain embodiments, the NLS may be an SV40 NLS (e.g., with the amino acid sequence of SEQ ID NO: 644). The fusion protein may comprise an NLS at its N-terminus, C-terminus, or both, and/or an NLS may be embedded in the middle of the fusion protein (e.g., at the N- or C- terminus of a DNA-binding domain or an effector domain).
[0159] In some embodiments, 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.
[0160] In some embodiments, 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. In particular embodiments, the fusion protein may comprise two NLSs at its N-terminus and two NLSs at its C-terminus.
[0161] An NLS described herein may be an endogenous NLS sequence. In certain embodiments, an NLS described herein comprises the amino acid sequence of any one of SEQ ID NOs: 644-649, or a sequence at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the selected sequence. In particular embodiments, the NLS comprises the amino acid sequence of SEQ ID NO: 644. Additional NLSs are known in the art.
[0162] In some embodiments, 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.
[0163] In some embodiments, 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.
C. Tags
[0164] Epigenetic editors provided herein may comprise one or more additional sequences (“tags”) for tracking, detection, and localization of the editors. In some embodiments, 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.
[0165] For example, 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. Suitable protein tags provided herein include, but are not limited to, biotin carboxylase carrier protein (BCCP) tags, myc-tags, calmodulin-tags, FEAG- tags, hemagglutinin (HA)-tags, poly-histidine tags (also referred to as histidine tags or His- tags), maltose binding protein (MBP)-tags, nus-tags, glutathione-S-transferase (GST)-tags, green fluorescent protein (GFP)-tags, thioredoxin-tags, S-tags, Softags (e.g., Softag 1 or Softag 3), strep-tags, biotin ligase tags, FlAsH tags, V5 tags, and SBP-tags. Additional suitable sequences will be apparent to those of skill in the art.
D. Fusion Protein Configurations
[0166] A fusion protein of an epigenetic editor described herein may have its components structured in different configurations. For example, 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. In some embodiments, the DNA-binding domain is at the C-terminus of the epigenetic editor. In some embodiments, the DNA-binding domain is at the N-terminus of the epigenetic editor. In some embodiments, the DNA-binding domain is linked to one or more nuclear localization signals. In some embodiments, the DNA-binding domain is flanked by an epigenetic effector domain and/or an additional domain on both sides. In some embodiments, where “DBD” indicates DNA-binding domain and “ED” indicates effector domain, the epigenetic editor comprises the configuration of:
- N’]-[ED1]-[DBD]-[ED2]-[C’
- N’]-[ED1]-[DBD]-[ED2]-[ED3]-[C’
- N’]-[ED1]-[ED2]-[DBD]-[ED3]-[C’ or
- N’]-[ED1]-[ED2]-DBD]-[ED3]-[ED4]-[C’.
[0167] In some embodiments, an epigenetic editor comprises a DNA-binding domain (DBD), a DNA methyltransferase (DNMT) domain, and a transcriptional repressor (“repressor”) domain that represses or silences expression of a target gene. The DBD, DNMT, and transcriptional repressor domains may be any as described herein, in any combination. The DBD, DNMT domain, and repressor domain may be in any configuration, e.g., with any of said domains at the N-terminus, at the C-terminus, or in the middle of the fusion protein. In some embodiments, the epigenetic editor comprises a fusion protein with the configuration of:
N’]-[DNMT domain]-[DBD]-[repressor domain]-[C’ N’]-[repressor domain]-[DBD]-[DNMT domain]-[C’ N’]-[DNMT domain] -[repressor domain]- [DBD]-[C’ or
N’]-[repressor domain]-[DNMT domain]- [DBD]-[C’.
[0168] In some embodiments, a connecting structure “]-[“in any one of the epigenetic editor structures is a linker, e.g., a peptide linker; a detectable tag; a peptide bond; a nuclear localization signal; and/or a promoter or regulatory sequence. In an epigenetic editor structure, the multiple connecting structures “]-[“ may be the same or may each be a different linker, tag, NLS, or peptide bond. In some embodiments, the DNMT domain may comprise any one of the domains in Table 6, or any combinations or homologs thereof. In particular embodiments, the DNMT domain comprises DNMT3A or a truncated version thereof, DNMT3L or a truncated version thereof, or both. In particular embodiments, the DBD is a catalytically inactive polynucleotide guided DNA-binding domain (e.g., a dCas9) or a ZFP domain. In certain embodiments, the repressor domain comprises any one of the domains shown in Table 4 or 5, or any combinations or homologs thereof. For example, the repressor domain may be a KRAB domain. In certain embodiments, the repressor domain is a ZFP28, ZN627, KAP1, MeCP2, HPlb, CBX8, CDYL2, TOX, Tox3, Tox4, EED, RBBP4, RCOR1, or SCML2 domain, or a fusion of two of said domains (e.g., a fusion of the N- and C-terminal regions of ZIM3 and KOX1 KRAB). In particular embodiments, the repressor domain is a KRAB domain from ZFP28, ZN627, ZIM3, or KOX1.
[0169] In some embodiments, the epigenetic editor comprises a configuration selected from
N’ ] -[DNMT3 A-DNMT3L] -[DBD] - [repressor]- [C’ N’ ] - [repressor] - [DBD] - [DNMT3 A-DNMT3L] - [C ’ N’ ] - [repressor] - [DBD] - [DNMT3 A] - [C ’ N’ ] - [DNMT3 A] - [DBD] - [repressor] - [C ’ N’ ] - [repressor] - [DBD] - [DNMT3 A] - [DNMT3L] - [C ’ N’ ] - [DNMT3 A] - [DNMT3L] - [DBD] - [repressor] - [C ’ N’]-[DNMT3A]-[DBD]-[C’ N’]-[DBD]-[DNMT3A]-[C’ N’ ] - [DNMT3L] - [DBD] - [C’ N’ ] -[DBD]- [DNMT3L] - [C’ wherein [DNMT3A-DNMT3L] indicates that the DNMT3A and DNMT3L domains are directly fused via a peptide bond, and wherein the connecting structure ]-[ is any one of the linkers as described herein, a detectable tag, an affinity domain, a peptide bond, a nuclear localization signal, a promoter, and/or a regulatory sequence. The DBD, repressor, DNMT3A, and DNMT3L domains may be any as described herein, in any combination. For example, the DNMT3A and DNMT3L domains may be selected from those in Table 6. In particular embodiments, the DBD is a CRIS PR-associated protein domain (e.g., dCas9) or a ZFP domain; the repressor domain is a KRAB domain derived from K0X1, ZIM3, ZFP28, or ZN627; the DNMT3A domain is a human DNMT3A domain; and the DNMT3L domain is a human or mouse DNMT3L domain; any combination of these components is also contemplated by the present disclosure.
[0170] In some embodiments, the epigenetic editor comprises a configuration selected from
N’ ] - [DNMT3 A] - [DBD] - [SETDB 1 ] - [C ’
N’]-[DNMT3A]- [DNMT3L]- [DBD]-[SETDB1]-[C’ N’]- [DNMT3A-DNMT3L]- [DBD]- [SETDB 1]- [C’ N’ ] - [SETDB 1 ] - [DBD] - [DNMT3 A] - [DNMT3L] - [C ’ N’ ] - [SETDB 1 ] - [DBD] - [DNMT3 A] - [C ’ wherein [DNMT3A-DNMT3L] indicates that the DNMT3A and DNMT3L domains are directly fused via a peptide bond, and wherein the connecting structure ]-[ is any one of the linkers as described herein, a detectable tag, an affinity domain, a peptide bond, a nuclear localization signal, a promoter, and/or a regulatory sequence. The DBD, SETDB 1, DNMT3A, and DNMT3L domains may be any as described herein, in any combination. In particular embodiments, the DBD is a CRIS PR-associated protein domain (e.g., dCas9) or a ZFP domain; the SETDB 1 domain is derived from human SETDB 1, ZIM3, ZFP28, or ZN627; the DNMT3A domain is a human DNMT3A domain; and the DNMT3L domain is a human or mouse DNMT3L domain; any combination of these components is also contemplated by the present disclosure.
[0171] Particular constructs contemplated herein include: DNMT3A-DNMT3L-XTEN80-NLS-dCas9-NLS-XTENl 6-KOX 1 KRAB (Configuration 1),
DNMT3A-DNMT3L-XTEN80-NLS-ZFP domain-NLS-XTEN16-KOXl KRAB (Configuration 2),
NLS-DNMT3 A-DNMT3L-XTEN80-dCas9-XTEN 16-KOX 1 KRAB -NLS (Configuration 3),
NLS-DNMT3A-DNMT3L-XTEN80-ZFP domain-XTENl 6-KOX 1 KRAB-NLS (Configuration 4),
NLS-NLS-DNMT3A-DNMT3L-XTEN80-dCas9-XTENl 6-KOX 1 KRAB-NLS-NLS (Configuration 5), and
NLS-NLS-DNMT3A-DNMT3L-XTEN80-ZFP domain-XTENl 6-KOX 1 KRAB- NLS-NLS (Configuration 6).
The DNMT3L and DNMT3A may be derived from human parental proteins, mouse parental proteins, or any combination thereof. In certain embodiments, the DNMT3L and DNMT3A are derived from mouse and human parental proteins, respectively (mDNMT3L and hDNMT3A). In certain embodiments, the DNMT3L and DNMT3A are both derived from human parental proteins (hDNMT3L and hDNMT3A). In some embodiments, the dCas9 is dSpCas9. In some embodiments, the K0X1 is human K0X1. Also contemplated is any of Configurations 1-6 wherein the K0X1 KRAB domain is replaced by a ZFP28, ZN627, or ZIM3 KRAB domain. In some embodiments, the ZFP28, ZN627, and ZIM3 are human ZFP28, ZN627, and ZIM3, respectively. In particular embodiments, the fusion construct may have the configuration:
NLS-NLS-hDNMT3A-hDNMT3L-XTEN80-dCas9-XTENl 6-KOX 1 KRAB-NLS- NLS (Configuration 7),
NLS-NLS-DNMT3A-DNMT3L-XTEN80-ZFP domain-XTENl 6-KOX 1 KRAB- NLS-NLS (Configuration 8), NLS-NLS-hDNMT3A-hDNMT3L-XTEN80-dCas9-XTEN16-ZFP28 KRAB-NLS- NLS (Configuration 9),
NLS-NLS-DNMT3A-DNMT3L-XTEN80-ZFP domain-XTENl 6-ZFP28 KRAB- NLS-NLS (Configuration 10), NLS-NLS-hDNMT3A-hDNMT3L-XTEN80-dCas9-XTEN16-ZN627 KRAB-NLS- NLS (Configuration 11),
NLS-NLS-DNMT3A-DNMT3L-XTEN80-ZFP domain-XTENl 6-ZN627 KRAB- NLS-NLS (Configuration 12), NLS-NLS-hDNMT3A-hDNMT3L-XTEN80-dCas9-XTEN16-ZIM3 KRAB-NLS-
NLS (Configuration 13), or
NLS-NLS-DNMT3A-DNMT3L-XTEN80-ZFP domain-XTEN16-ZIM3 KRAB-
NLS-NLS (Configuration 14).
[0172] In particular embodiments, a fusion construct described herein may have Configuration 1 and comprise SEQ ID NO: 658, or a sequence at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical thereto. In SEQ ID NO: 658 below, the XTEN linkers are underlined, the W linker is bolded, underlined, and italicized, the NLS sequences are bolded, the DNMT3A sequence is italicized, the DNMT3L sequence is underlined and italicized, the dCas9 domain is bolded and italicized, and the KOX1 KRAB domain is underlined and bolded:
MNHDQEFDPPKVYPPVPAEKRKPIRVLSLFDGIATGLLVLKDLGIQVDRYIASEVCE DSITVGMVRHQGKIMYVGDVRSVTQKHIQEWGPFDLVIGGSPCNDLSIVNPARKGLY EGTGRLFFEFYRLLHDARPKEGDDRPFFWLFENWAMGVSDKRDISRFLESNPVMID AKEVSAAHRAR YFWGNLPGMNRPLASTVNDKLELQECLEHGRIAKFSKVR TI TTRSN SIKQGKDQHFPVFMNEKEDILWCTEMERVFGFPVHYTDVSNMSRLARQRLLGRSWSV PVIRHLFAPLKEYFACVSSGNSNANSRGPSFSSGLVPLSLRGSH IGPMEIYKTVSAW KRQPVRVLSLFRNIDKVLKSLGFLESGSGSGGGTLKYVEDVTNWRRDVEKWGPFDL VYGSTQPLGSSCDRCPGWYMFQFHRILQYALPRQESQRPFFWIFMDNLLLTEDDQET TTRFLQTEAVTLQDVRGRDYQNAMRVWSNIPGLKSKHAPLTPKEEEYLQAQVRSRSK LDAPKVDLLVKNCLLPLREYFKYFSQNSLPLGGPSSGAPPPSGGSPAGSPYSYPPGY SESATPESGPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEPKK KRKV MDKKYSIGLAIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKNLIGALL FDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEESFLVEE DKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIKFRG HFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRLE NLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLA QIGDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKA LVRQQLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLN REDLLRKQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYY VGPLARGNSRFAWMTRKSEETITPWNFEEWDKGASAQSFIERMTNFDKNLPNEKVL PKHSLLYEYFTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLK EDYFKKIECFDSVEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLT LTLFEDREMIEERLKTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTI LDFLKSDGFANRNFMQLIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKG ILQTVKWDELVKVMGRHKPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGS QILKEHPVENTQLQNEKLYLYYLQNGRDMYVDQELDINRLSDYDVDAIVPQSFLKDD SIDNKVLTRSDKNRGKSDNVPSEEWKKMKNYWRQLLNAKLITQRKFDNLTKAERGG LSELDKAGFIKRQLVETRQITKHVAQILDSRMNTKYDENDKLIREVKVITLKSKLVS DFRKDFQFYKVREINNYHHAHDAYLNAWGTALIKKYPKLESEFVYGDYKVYDVRKM IAKSEQEIGKATAKYFFYSNIMNFFKTEITLANGEIRKRPLIETNGETGEIVWDKGR DFATVRKVLSMPQVNIVKKTEVQTGGFSKESILPKRNSDKLIARKKDWDPKKYGGFD SPTVAYSVLWAKVEKGKSKKLKSVKELLGITIMERSSFEKNPIDFLEAKGYKEVKK DLIIKLPKYSLFELENGRKRMLASAGELQKGNELALPSKYVNFLYLASHYEKLKGSP EDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILADANLDKVLSAYNKHRDKPIREQA ENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVLDATLIHQSITGLYETRIDLS QLGGDPKKKRKVSGSETPGTSESATPESTGRTLVTFKDVFVDFTREEWKLLDTAQQI VYRNVMLENYKNLVSLGYQLTKPDVILRLEKGEEP ( SEQ ID NO : 658 )
[0173] In particular embodiments, a fusion construct described herein may have
Configuration 2 and comprise SEQ ID NO: 659, or a sequence at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical thereto. In SEQ ID NO: 659 below, the XTEN linkers are underlined, the W linker is bolded, underlined, and italicized, the NLS sequences are bolded and underlined, the DNMT3A sequence is italicized, the DNMT3L sequence is underlined and italicized, the ZFP domain is bolded, and the KOX1 KRAB domain is underlined and bolded. Variable amino acids represented by Xs are the amino acids of the DNA-recognition helix of the zinc finger and XX in italics may be either TR, LR or LK.
MNHDQEFDPPKVYPPVPAEKRKPIRVLSLFDGIATGLLVLKDLGIQVDRYIASEVCE DSITVGMVRHQGKIMYVGDVRSVTQKHIQEWGPFDLVIGGSPCNDLSIVNPARKGLY EGTGRLFFEFYRLLHDARPKEGDDRPFFWLFENWAMGVSDKRDISRFLESNPVMID AKEVSAAHRAR YFWGNLPGMNRPLASTVNDKLELQECLEHGRIAKFSKVR TI TTRSN SIKQGKDQHFPVFMNEKEDILWCTEMERVFGFPVHYTDVSNMSRLARQRLLGRSWSV PVIRHLFAPLKEYFACVSSGNSNANSRGPSFSSGLVPLSLRGSH IGPMEIYKTVSAW KRQPVRVLSLFRNIDKVLKSLGFLESGSGSGGGTLKYVEDVTNWRRDVEKWGPFDL VYGSTQPLGSSCDRCPGWYMFQFHRILQYALPRQESQRPFFWIFMDNLLLTEDDQET TTRFLQTEAVTLQDVRGRDYQNAMRVWSNIPGLKSKHAPLTPKEEEYLQAQVRSRSK LDAPKVDLLVKNCLLPLREYFKYFSQNSLPLGGPSSGAPPPSGGSPAGSPYSYPPGY SESATPESGPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEPKK KRKVYSRPGERPFQCRICMRNFSXXXXXXXHXXTHTGEKPFQCRICMRNFSXXXXXX XHXXTH [linker] PFQCRICMRNFSXXXXXXXHXXTHTGEKPFQCRICMRNFSXXX XXXXHXXTH [linker] PFQCRICMRNFSXXXXXXXHXXTHTGEKPFQCRICMRNFS XXXXXXXHXXTHLRGSPKKKRKVS GSETPGTSESATPEST GRTLVTFKDVFVDFTRE EWKLLDTAQQIVYRNVMLENYKNLVSLGYQLTKPDVILRLEKGEEP ( SEQ ID NO : 659 )
In certain embodiments, the six “XXXXXXX” regions in SEQ ID NO: 659 comprise, in order, the F1-F6 amino acid sequences shown in Table 1 for any one of ZF810-ZF893. [linker] represents a linker sequence. In some embodiments, one or both linker sequences may be TGSQKP (SEQ ID NO: 651). In some embodiments, one or both linker sequences may be TGGGGSQKP (SEQ ID NO: 652). In some embodiments, one linker sequence may have the amino acid sequence of SEQ ID NO: 651 and the other linker sequence may have the amino acid sequence of SEQ ID NO: 652. [0174] In particular embodiments, a fusion construct described herein may have Configuration 7 and comprise SEQ ID NO: 660, or a sequence at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical thereto.
[0175] In particular embodiments, a fusion construct described herein may have Configuration 9 and comprise SEQ ID NO: 661, or a sequence at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical thereto.
[0176] In particular embodiments, a fusion construct described herein may have Configuration 11 and comprise SEQ ID NO: 662, or a sequence at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical thereto.
[0177] In particular embodiments, a fusion construct described herein may have Configuration 13 and comprise SEQ ID NO: 663, or a sequence at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical thereto.
[0178] In some embodiments, a fusion construct described herein (e.g., the fusion construct of any one of Configurations 1-14) is within an expression construct that comprises a WPRE sequence, a polyadenylation site, or both. In certain embodiments, the WPRE sequence is in a 3’ noncoding region. In certain embodiments, the WPRE sequence is upstream from a poly-adenylation site. In particular embodiments, the expression construct comprises the fusion construct (e.g., of any one of Configurations 1-14) and a WPRE sequence in a 3’ noncoding region upstream from a polyadenylation site.
[0179] In some embodiments, a fusion construct described herein may have the sequence of any one of Fusion Proteins 1-15 as described in Example 13.
[0180] Multiple fusion proteins may be used to effect activation or repression of a target gene or multiple target genes. For example, an epigenetic editor fusion protein comprising a DNA-binding domain (e.g., a dCas9 domain) and an effector domain may be co-delivered with two or more guide polynucleotides (e.g., gRNAs), each targeting a different target DNA sequence. The target sites for two of the DNA-binding domains may be the same or in the vicinity of each other, or separated by, for example, about 100 base pairs, about 200 base pairs, about 300 base pairs, about 400 base pairs, about 500 base pairs, or about 600 or more base pairs. In addition, when targeting double-strand DNA, such as an endogenous gene locus, the guide polynucleotides may target the same or different strands (one or more to the positive strand and/or one or more to the negative strand). V. Target Sequences
[0181] An epigenetic editor herein may be directed to a target sequence in ANGPTL3 to effect epigenetic modification of the ANGPTL3 gene. As used herein, a “target sequence,” a “target site,” or a “target region” is a nucleic acid sequence present in a gene of interest; in some instances, the target sequence may be outside but in the vicinity of the gene of interest wherein methylation or binding by a repressor of the target sequence represses expression of the gene. In some embodiments, the target sequence may be a hypomethylated or hypermethylated nucleic acid sequence.
[0182] The target sequence may be in any part of a target gene. In some embodiments, the target sequence is part of or near a noncoding sequence of the gene. In some embodiments, the target sequence is part of an exon of the gene. In some embodiments, the target sequence is part of or near a transcriptional regulatory sequence of the gene, such as a promoter or an enhancer. In some embodiments, the target sequence is adjacent to, overlaps with, or encompasses a CpG island. In certain embodiments, the target sequence is within about 3000, 2900, 2800, 2700, 2600, 2500, 2400, 2300, 2200, 2100, 2000, 1900, 1800, 1700, 1600, 1500, 1400, 1300, 1200, 1100, 1000, 900, 800, 700, 600, 500, 400, 300, 200, or 100 base pairs (bp) flanking a ANGPTL3 TSS. In certain embodiments, the target sequence is within 500 bp flanking the ANGPTL3 TSS. In certain embodiments, the target sequence is within 1000 bp flanking the ANGPTL3 TSS.
[0183] In some embodiments, the target sequence may hybridize to a guide polynucleotide sequence (e.g., gRNA) complexed with a fusion protein comprising a polynucleotide guided DNA-binding domain (e.g., a CRISPR protein such as dCas9) and effector domain(s). The guide polynucleotide sequence may be designed to have complementarity to the target sequence, or identity to the opposing strand of the target sequence. In some embodiments, the guide polynucleotide comprises a spacer sequence that is about 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to a protospacer sequence in the target sequence. In particular embodiments, the guide polynucleotide comprises a spacer sequence that is 100% identical to a protospacer sequence in the target sequence.
[0184] In some embodiments, where the DNA-binding domain of an epigenetic editor described herein is a zinc finger array, the target sequence may be recognized by said zinc finger array.
[0185] In some embodiments, where the DNA-binding domain of an epigenetic editor described herein is a TALE, the target sequence may be recognized by said TALE. [0186] A target sequence described herein may be specific to one copy of a target gene, or may be specific to one allele of a target gene. Accordingly, the epigenetic modification and modulation of expression thereof may be specific to one copy or one allele of the target gene. For example, an epigenetic editor may repress expression of a specific copy harboring a target sequence recognized by the DNA-binding domain (e.g., a copy associated with a disease or condition, or that harbors a mutation associated with a disease or condition).
[0187] In some embodiments, the target ANGPTL3 genomic region may fall within the sequence shown below:
CTCTTAACCACTATGGAATACTGCCTCTACTGTAACTATTATACCCAAAACCCTTAATCCTAAGTCATC AAAAGGAAGAGC CTCTATTTTACACAAT GAAGAGGC AT T T C T AAGAAT AGAAAT T T AGGGAC GAGC AC A GTGGCTTACTCCTTTAATATCAGCACTTTGAGAGGCTGATATGGGAGGTTCACTTGAAGTCAGGAGTTC AAGGTCAGCTTGGGCAACATAGTGAAACACAGTCTCTACAAAATATTTAAAAATTAGCTGGGTGTGGTG GCATGCATCTATAGTCTCAGCTACTT GGGAGAC AGAGGGAGGAGGC T T GC T C GAGC C C AGGAGT T C GT G GC T AT AGT GAGC T AT GAT C AT GC C AC T GC AC T C C AGC C T GGAC AAC AGAGC AAGAC C C T GT C T C T AAAA AAGAAAAGAAATTTGGAAATGGTTTATTTTGTATTAACAATTTATAATTTACACTGAAATTTATTATGA TAAAACTTTTCCCTGTGTTAAAAAGCTATTAACTTTATGAAAAATTTCTTTTAGGTAAGGTTGATTATA TATACCCACACACATACACAGGTTAAAAGTTAGTTTCATGTGACATAATAACTAGCATTTTGAGCACTA CCTGTTTGCCCAGCACTGTTCTAAGTGCTCTACATGTATTATTGTTAAATTATCATAACACTATGAATT ATGTACTATAATTACCCCAGCTTTACAGATGAGGAGACTAATCCATGGGGAGGTTAAGTAACTTGTCCA AGGC C AGAC AGC T AGAGC CGGCTTTT GGAC C C AC AC C AC AGT C T GAC TCCAGCACCCATATTCT T AAC A ATTTCACCATATTAATATGTCAAGATTAAGCAGTTTTAAAGGATGCTATTTTCTCACAAATTTCTTAAT AT GAAC AC T CAAT AAGAAT AAT C AC T AAT AT AAGC AT TTAGTATTTTTT T AAC AC T AAGT T GGAAGC AT AGTGGAACATTTATTTTTAGAAATATTATTAATTGGCTGGGCTCACGCTTGTAATCGGCTGGGCTCATG CCTGTAAATTTTGGGAGGCCAAGGTAAAAGAATTGCTTGAGCCCAGTATTTCCAGACCAGCATGGGCAA TACATTAAGACATCATCTTTAAAAAAAAAATGTTATTAATCTCCTCTTTTTGTTAAATGTATATTATCA AAATTGTTACTAAGCTAACAAACTTCAGAAAAACTTATGATGGGCAAGCTGCTTGTGACATTGAAGGTA TTTAAGATTCAATTCTAGTTTGGTCCTAGATGACCACATATCCATTGTTCCTTCAACGAGCACATGGTA AAGAGC C T AGAAC AC AGAGAC AC AGAAC AC AGT GGAGAAAAGGGAGT GAAAT GT C T T T AAT GAC AC T T A CTATATATGGGATTTTGTGACAATATACAAGGATGGTTAAGACATATAAGGTGATGCAAAAAAACATAT T AAC AAT T AT AGT GAC AAAAAAT GAGGAGCAT AT AAT TAT AC ATT GATT T AT AC AGAGT AC C AGAGGAA C AC AGC AT T GAGAGC C GT AAC AC C AC C T GAGGGAGT GGAGAAAGGC T T C AGAGAGAAAGT GT T T T T T GG AATGGATCACTGTTTCCAAAAGAACTAAAGTACAGTTTGAGAAATGCATACTTAATTCATTACTTTTTT C C C C T C AAC T T T AAT AAT AAAT T T AC C C AAC AAAAAAGT T T AT T T T T GAC T T GT AAAT C T C T T AAAAT C ATAAAAAAGTAAAATTAGCTTTTAAAAACAGGTAGTCACCATAGCATTGAATGTGTAGTTTATAATACA GCAAAGTTAAATACAATTTCAAATTACCTATTAAGTTAGTTGCTCATTTCTTTGATTTCATTTAGCATT GATCTAACTCAATGTGGAAGAAGGTTACATTCGTGCAAGTTAACACGGCTTAATGATTAACTATGTTCA CCTACCAACCTTACCTTTTCTGGGCAAATATTGGTATATATAGAGTTAAGAAGTCTAGGTCTGCTTCCA GAAGAAAAC AGT T C C AC GT T GC T T GAAAT T GAAAAT C AAGAT AAAAAT GT T C AC AAT T AAGC T C C T T C T TTTTATTGTTCCTCTAGTTATTTCCTCCAGAATTGATCAAGACAATTCATCATTTGATTCTCTATCTCC AGAGCCAAAATCAAGATTTGCTATGTTAGACGATGTAAAAATTTTAGCCAATGGCCTCCTTCAGTTGGG ACATGGTCTTAAAGACTTTGTCCATAAGACGAAGGGCCAAATTAATGACATATTTCAAAAACTCAACAT ATTTGATCAGTCTTTTTATGATCTATCGCT GC AAAC C AGT GAAAT C AAAGAAGAAGAAAAGGAAC T GAG AAGAAC T AC AT AT AAAC T AC AAGT C AAAAAT GAAGAGGT AAAGAAT AT GT C AC T T GAAC T C AAC T C AAA AC T T GAAAGC C T C C T AGAAGAAAAAAT T C T AC T T C AAC AAAAAGT GAAAT AT T T AGAAGAGC AAC T AAC TAACTTAATTCAAAATCAACCTGAAACTCCAGAACACCCAGAAGTAACTTCACTTAAA ( SEQ ID NO : 1020 )
[0188] In some embodiments, the target ANGPTL3 genomic region may fall within the sequence shown below (chrl:62596520-62598520):
GGCTGGGCTCACGCTTGTAATCGGCTGGGCTCATGCCTGTAAATTTTGGGAGGCCAAGGTAAAAGAATT GC T T GAGC CCAGTATTTC C AGAC CAGCATGGGCAATACAT T AAGAC AT C AT C T T T AAAAAAAAAAT GT T ATTAATCTCCTCTTTTTGTTAAATGTATATTATCAAAATTGTTACTAAGCTAACAAACTTCAGAAAAAC TTATGATGGGCAAGCTGCTTGTGACATTGAAGGTATTTAAGATTCAATTCTAGTTTGGTCCTAGATGAC CACATATCCATTGTTCCTT C AAC GAGC AC AT GGT AAAGAGC C T AGAAC AC AGAGAC AC AGAAC AC AGT G GAGAAAAGGGAGT GAAAT GT C T T T AAT GAC AC TTACTATATATGGGATTT T GT GAC AAT AT AC AAGGAT GGTTAAGACATATAAGGTGATGCAAAAAAACATATTAACAATTATAGTGACAAAAAATGAGGAGCATAT AATTATACATTGATT T AT AC AGAGT AC C AGAGGAAC AC AGC AT T GAGAGC C GT AAC AC C AC C T GAGGGA GTGGAGAAAGGCTTCAGAGAGAAAGTGTTTTTTGGAATGGATCACTGTTTCCAAAAGAACTAAAGTACA GTTTGAGAAATGCATACTTAATTCATTACTTTTTTCCCCTCAACTTTAATAATAAATTTACCCAACAAA AAAGT T T AT T T T T GAC T T GT AAAT C T C T T AAAAT C AT AAAAAAGT AAAAT T AGC T T T T AAAAAC AGGT A GTCACCATAGCATTGAATGTGTAGTTTATAATACAGCAAAGTTAAATACAATTTCAAATTACCTATTAA GTTAGTTGCTCATTTCTTTGATTTCATTTAGCATTGATCTAACTCAATGTGGAAGAAGGTTACATTCGT GCAAGTTAACACGGCTTAATGATTAACTATGTTCACCTACCAACCTTACCTTTTCTGGGCAAATATTGG TATATATAGAGTTAAGAAGTCTAGGTCTGCTTCCAGAAGAAAACAGTTCCACGTTGCTTGAAATTGAAA ATCAAGATAAAAATGTTCACAATTAAGCTCCTTCTTTTTATTGTTCCTCTAGTTATTTCCTCCAGAATT GAT C AAGAC AAT TCATCATTTGATTCTCTATCTC C AGAGC C AAAAT C AAGAT T T GC TAT GT T AGAC GAT GTAAAAATTTTAGCCAATGGCCTCCTTCAGTTGGGACATGGTCTTAAAGACTTTGTCCATAAGACGAAG GGCCAAATTAATGACATATTTCAAAAACTCAACATATTTGATCAGTCTTTTTATGATCTATCGCTGCAA AC C AGT GAAAT C AAAGAAGAAGAAAAGGAAC T GAGAAGAAC T AC AT AT AAAC T AC AAGT C AAAAAT GAA GAGGT AAAGAAT AT GT C AC T T GAAC T C AAC T C AAAAC T T GAAAGC C T C C T AGAAGAAAAAAT T C T AC T T C AAC AAAAAGT GAAAT AT T T AGAAGAGC AAC T AAC T AAC T T AAT T C AAAAT C AAC C T GAAAC T C C AGAA CACCCAGAAGTAACTTCACTTAAAGTAAGTAGAAAATAAAGAGGGTTCATGTTTATGTTTTCAATGTGG ATCTTTTAAAAAAAATATTTCTAAGGCATGCCATTTGAAATACTTTGTTGCATTGTTGAAATACTTTTT T T T C CAAGAAAAAT AAT C T C CAGAAAAT AAAAT T T C C TAT TAT AAT T T C AAGT T AGT T T T T T GT T T C C C TAATGTTATATATGAAAACACTGAAAATTTGCATTTTATATGAAAATTACAAATCGGTTAAATTATACA ATCTAGAACACTATGTCATTACACTATTGTAAATTACTGAAGGTAAGTAAAAAGTTAAAAAAAATTTAA AAC TATTCTCCAGTGTT T AAAAC AGAT T AAAT AAT AC AGT AAAT GGAAAAGAT TTATTCATAT GAAAAT ATGCTGGGCTTTTTCTTTTAATTGAAGTTCAGAAAATCAAATTTTAGAGATAGTACAATTTAAATAAA ( SEQ ID NO : 1019 ) [0189] In some embodiments, the target ANGPTL3 genomic region may fall within the sequence shown below:
GGCTGGGCTCACGCTTGTAATCGGCTGGGCTCATGCCTGTAAATTTTGGGAGGCCAAGGTAAAAGAATT GC T T GAGC CCAGTATTTC C AGAC CAGCATGGGCAATACAT T AAGAC AT C AT C T T T AAAAAAAAAAT GT T ATTAATCTCCTCTTTTTGTTAAATGTATATTATCAAAATTGTTACTAAGCTAACAAACTTCAGAAAAAC TTATGATGGGCAAGCTGCTTGTGACATTGAAGGTATTTAAGATTCAATTCTAGTTTGGTCCTAGATGAC CACATATCCATTGTTCCTT C AAC GAGC AC AT GGT AAAGAGC C T AGAAC AC AGAGAC AC AGAAC AC AGT G GAGAAAAGGGAGT GAAAT GT C T T T AAT GAC AC TTACTATATATGGGATTT T GT GAC AAT AT AC AAGGAT GGTTAAGACATATAAGGTGATGCAAAAAAACATATTAACAATTATAGTGACAAAAAATGAGGAGCATAT AATTATACATTGATT T AT AC AGAGT AC C AGAGGAAC AC AGC AT T GAGAGC C GT AAC AC C AC C T GAGGGA GTGGAGAAAGGCTTCAGAGAGAAAGTGTTTTTTGGAATGGATCACTGTTTCCAAAAGAACTAAAGTACA GTTTGAGAAATGCATACTTAATTCATTACTTTTTTCCCCTCAACTTTAATAATAAATTTACCCAACAAA AAAGT T T AT T T T T GAC T T GT AAAT C T C T T AAAAT C AT AAAAAAGT AAAAT T AGC T T T T AAAAAC AGGT A GTCACCATAGCATTGAATGTGTAGTTTATAATACAGCAAAGTTAAATACAATTTCAAATTACCTATTAA GTTAGTTGCTCATTTCTTTGATTTCATTTAGCATTGATCTAACTCAATGTGGAAGAAGGTTACATTCGT GCAAGTTAACACGGCTTAATGATTAACTATGTTCACCTACCAACCTTACCTTTTCTGGGCAAATATTGG TATATATAGAGTTAAGAAGTCTAGGTCTGCTTCC ( SEQ ID NO : 1021 )
VI. Epigenetic Modifications
[0190] An epigenetic editor described herein may perform sequence- specific epigenetic modification(s) (e.g., alteration of chemical modification(s)) of a target gene that harbors the target sequence. Such epigenetic modulation may be safer and more easily reversible than modulation due to gene editing, e.g., with generation of DNA double-strand breaks. In some embodiments, the epigenetic modulation may reduce or silence the target gene. In some embodiments, the modification is at a specific site of the target sequence. In some embodiments, the modification is at a specific allele of the target gene. Accordingly, the epigenetic modification may result in modulated (e.g., reduced) expression of one copy of a target gene harboring a specific allele, and not the other copy of the target gene. In some embodiments, the specific allele is associated with a disease, condition, or disorder.
[0191] In some embodiments, the epigenetic modification reduces or abolishes transcription of the target gene harboring the target sequence. In some embodiments, the epigenetic modification reduces or abolishes transcription of a copy of the target gene harboring a specific allele recognized by the epigenetic editor. In some embodiments, the epigenetic editor reduces the level of or eliminates expression of a protein encoded by the target gene. In some embodiments, the epigenetic editor reduces the level of or eliminates expression of a protein encoded by a copy of the target gene harboring a specific allele recognized by the epigenetic editor. The target ANGPTL3 gene may be epigenetically modified in vitro, ex vivo, or in vivo.
[0192] The effector domain of an epigenetic editor described herein may alter (e.g., deposit or remove) a chemical modification at a nucleotide of the target gene or at a histone associated with the target gene. The chemical modification may be altered at a single nucleotide or a single histone, or may be altered at 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, 1000, 1500, 2000, 2500, 3000 or more nucleotides.
[0193] In some embodiments, an effector domain of an epigenetic editor described herein may alter a CpG dinucleotide within the target gene. In some embodiments, all CpG dinucleotides within 2000, 1500, 1000, 500, or 200 bps flanking a target sequence (e.g., in an alteration site as described herein) are altered according to a modification type described herein, as compared to the original state of the gene or the gene in a comparable cell not contacted with the epigenetic editor. In some embodiments, at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, 50, 60, 70, 80, 90, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700 or more of the CpG dinucleotides are altered as compared to the original state of the gene or the gene in a comparable cell not contacted with the epigenetic editor. In some embodiments, at least 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% of the CpG dinucleotides are altered as compared to the original state of the gene or the gene in a comparable cell not contacted with the epigenetic editor. In some embodiments, one single CpG dinucleotide is altered, as compared to the original state of the gene or the gene in a comparable cell not contacted with the epigenetic editor.
[0194] An effector domain of an epigenetic editor described herein may alter a histone modification state of a histone associated with or bound to the target gene. For example, an effector domain may deposit a modification on one or more lysine residues of histone tails of histones associated with the target gene. In some embodiments, the effector domain may result in deacetylation of one or more histone tails of histones associated with the target gene, thereby reducing or silencing expression of the target gene. In some embodiments, the histone modification state is a methylation state. For example, the effector domain may result in a H3K9, H3K27 or H4K20 methylation (e.g. one or more of a H3K9me2, H3K9me3, H3K27me2, H3K27me3, and H4K20me3 methylation) at one or more histone tails associated with the target gene, thereby reducing or silencing expression of the target gene. [0195] In some embodiments, all histone tails of histones bound to DNA nucleotides within 2000, 1500, 1000, 500, or 200 bps flanking the target sequence are altered according to a modification type as described herein, as compared to the original state of the chromosome or the chromosome in a comparable cell not contacted with the epigenetic editor. In some embodiments, at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 105, 110, 115, 120 or more histone tails of the bound histones are altered as compared to the original state of the chromosome or the chromosome in a comparable cell not contacted with the epigenetic editor. In some embodiments, at least 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% of histone tails of the bound histones are altered as compared to the original state of the chromosome or the chromosome in a comparable cell not contacted with the epigenetic editor. For example, one single histone tail of the bound histones may be altered as compared to the original state of the chromosome or the chromosome in a comparable cell not contacted with the epigenetic editor. As another example, one single bound histone octamer may be altered as compared to the original state of the chromosome or the chromosome in a comparable cell not contacted with the epigenetic editor.
[0196] The chemical modification deposited at target gene DNA nucleotides or histone residues may be at or in close proximity to a target sequence in the target gene. In some embodiments, an effector domain of an epigenetic editor described herein alters a chemical modification state of a nucleotide or histone tail bound to a nucleotide 100-200, 200-300, 300-400, 400-55, 500-600, 600-700, or 700-800 nucleotides 5’ or 3’ to the target sequence in the target gene. In some embodiments, an effector domain alters a chemical modification state of a nucleotide or histone tail bound to a nucleotide within 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 1100, 1200, 1300, 1400, 1500, 1600, 1700, 1800, 1900, or 2000 nucleotides flanking the target sequence. As used herein, “flanking” refers to nucleotide positions 5’ to the 5’ end of and 3’ to the 3’ end of a particular sequence, e.g. a target sequence.
[0197] In some embodiments, an effector domain mediates or induces a chemical modification change of a nucleotide or a histone tail bound to a nucleotide distant from a target sequence. Such modification may be initiated near the target sequence, and may subsequently spread to one or more nucleotides in the target gene distant from the target sequence. For example, an effector domain may initiate alteration of a chemical modification state of one or more nucleotides or one or more histone residues bound to one or more nucleotides within 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500 nucleotides flanking the target sequence, and the chemical modification state alteration may spread to one or more nucleotides at least 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 1100, 1200, 1300, 1400, 1500, 1600, 1700, 1800, 1900, 2000, 2500, 3000, or more nucleotides from the target sequence in the target gene, either upstream or downstream of the target sequence. In certain embodiments, the chemical modification may be initiated at less than 2, 3, 5, 10, 20, 30, 40, 50, or 100 nucleotides in the target gene and spread to at least 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 2000, or more nucleotides in the target gene. In some embodiments, the chemical modification spreads to nucleotides in the entire target gene.
Additional proteins or transcription factors, for example, transcription repressors, methyltransferases, or transcription regulation scaffold proteins, may be involved in the spreading of the chemical modification. Alternatively, the epigenetic editor alone may be involved.
[0198] In some embodiments, an epigenetic editor described herein reduces expression of a target gene by at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 95%, at least about 99%, or more, as measured by transcription of the target gene in a cell, a tissue, or a subject as compared to a control cell, control tissue, or a control subject (e.g., in the absence of the epigenetic editor). In some embodiments, the epigenetic editors described herein reduces expression of a copy of target gene by at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 95%, at least about 99%, or more, as measured by transcription of the copy of the target gene in a cell, a tissue, or a subject as compared to a control cell, control tissue, or a control subject. In certain embodiments, the copy of the target gene harbors a specific sequence or allele recognized by the epigenetic editor. In particular embodiments, the epigenetically modified copy encodes a functional protein, and accordingly an epigenetic editor disclosed herein may reduce or abolish expression and/or function of the protein. For example, an epigenetic editor described herein may reduce expression and/or function of a protein encoded by the target gene by at least 3- fold, at least 4-fold, at least 5-fold, at least 6-fold, at least 7-fold, at least 8-fold, at least 9- fold, at least 10-fold, at least 11-fold, at least 12-fold, at least 13-fold, at least 14-fold, at least 15-fold, at least 20-fold, at least 25-fold, at least 30-fold, at least 35-fold, at least 40-fold, at least 45-fold, at least 50-fold, at least 60-fold, at least 70-fold, at least 80-fold, at least 90- fold, or at least 100 fold in a cell, a tissue, or a subject as compared to a control cell, control tissue, or a control subject.
[0199] Modulation of target gene expression can be assayed by determining any parameter that is indirectly or directly affected by the expression of the target gene. Such parameters include, e.g., changes in RNA or protein levels; changes in protein activity; changes in product levels; changes in downstream gene expression; changes in transcription or activity of reporter genes such as, for example, luciferase, CAT, beta-galactosidase, or GFP; changes in signal transduction; changes in phosphorylation and dephosphorylation; changes in receptor- ligand interactions; changes in concentrations of second messengers such as, for example, cGMP, cAMP, IP3, and Ca2+; changes in cell growth; changes in neovascularization; and/or changes in any functional effect of gene expression.
Measurements can be made in vitro, in vivo, and/or ex vivo, and can be made by conventional methods, e.g., measurement of RNA or protein levels, measurement of RNA stability, and/or identification of downstream or reporter gene expression. Readout can be by way of, for example, chemiluminescence, fluorescence, colorimetric reactions, antibody binding, inducible markers, ligand binding assays, changes in intracellular second messengers such as cGMP and inositol triphosphate (IP3), changes in intracellular calcium levels; cytokine release, and the like.
[0200] Methods for determining the expression level of a gene, for example the target of an epigenetic editor, may include, e.g., determining the transcript level of a gene by reverse transcription PCR, quantitative RT-PCR, droplet digital PCR (ddPCR), Northern blot, RNA sequencing, DNA sequencing (e.g., sequencing of complementary deoxyribonucleic acid (cDNA) obtained from RNA); next generation (Next-Gen) sequencing, nanopore sequencing, pyrosequencing, or Nanostring sequencing. Levels of protein expressed from a gene may be determined, e.g., by Western blotting, enzyme linked immuno-absorbance assays, mass- spectrometry, immunohistochemistry, or flow cytometry analysis. Gene expression product levels may be normalized to an internal standard such as total messenger ribonucleic acid (mRNA) or the expression level of a particular gene, e.g., a housekeeping gene.
[0201] In some embodiments, the effect of an epigenetic editor in modulating target gene expression may be examined using a reporter system. For example, an epigenetic editor may be designed to target a reporter gene encoding a reporter protein, such as a fluorescent protein. Expression of the reporter gene in such a model system may be monitored by, e.g., flow cytometry, fluorescence-activated cell sorting (FACS), or fluorescence microscopy. In some embodiments, a population of cells may be transfected with a vector that harbors a reporter gene. The vector may be constructed such that the reporter gene is expressed when the vector transfects a cell. Suitable reporter genes include genes encoding fluorescent proteins, for example green, yellow, cherry, cyan or orange fluorescent proteins. The population of cells carrying the reporter system may be transfected with DNA, mRNA, or vectors encoding the epigenetic editor targeting the reporter gene.
VII. Pharmaceutical Compositions
[0202] In one aspect, the present disclosure provides 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. For example, 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. In some embodiments, 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.
[0203] Generally, 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.
[0204] The term “excipient” is used herein to describe any ingredient other than the compound(s) of the present disclosure. The choice of excipient(s) will to a large extent depend on factors such as the particular mode of administration, the effect of the excipient on solubility and stability, and the nature of the dosage form. As used herein, “pharmaceutically acceptable excipient” includes any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like that are physiologically compatible. Some examples of pharmaceutically acceptable excipients are water, saline, phosphate buffered saline, dextrose, glycerol, ethanol and the like, as well as combinations thereof. In many cases, it will be preferable to include isotonic agents, for example, sugars, polyalcohols such as mannitol, sorbitol, or sodium chloride in the composition. Additional examples of pharmaceutically acceptable substances are wetting agents or minor amounts of auxiliary substances such as wetting or emulsifying agents, preservatives, or buffers, which enhance the shelf life or effectiveness of the antibody. [0205] 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.
VIII. Delivery Methods
[0206] In some embodiments, the epigenetic editor or its component(s) are introduced to target cells in the form of nucleic acid molecule(s) encoding the epigenetic editor or its component(s); accordingly, the pharmaceutical compositions herein comprise the nucleic acid molecule(s). Such nucleic acid molecule(s) may be, for example, DNA, RNA or mRNA, and/or modified nucleic acid sequence(s) (e.g., with chemical modifications, a 5’ cap, or one or more 3’ modifications). In some embodiments, the nucleic acid molecule(s) may be delivered as naked DNA or RNA, for instance by means of transfection or electroporation, or can be conjugated to molecules (e.g., N-acetylgalactosamine) promoting uptake by target cells. In some embodiments, the nucleic acid molecule(s) may be in nucleic acid expression vector(s), which may include expression control sequences such as promoters, enhancers, transcription signal sequences, transcription termination sequences, introns, polyadenylation signals, Kozak consensus sequences, internal ribosome entry sites (IRES), etc. Such expression control sequences are well known in the art. A vector may also comprise a sequence encoding a signal peptide (e.g., for nuclear localization, nucleolar localization, or mitochondrial localization), associated with (e.g., inserted into or fused to) a sequence coding for a protein.
[0207] Examples of 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. In certain embodiments, the vector is a plasmid or a viral vector. Viral particles or virus-like particles (VLPs) may also be used to deliver nucleic acid molecule(s) encoding epigenetic editors or component(s) thereof as described herein. For example, “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. [0208] In certain embodiments, 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. Examples of viral vectors include adeno- associated viral vectors (e.g., derived from AAV3, AAV3b, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAVrh8, AAV10, and/or variants thereof); retroviral vectors (e.g., 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).
[0209] In some embodiments, 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. Without wishing to be bound by any theory, 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.
[0210] Any AAV serotype, e.g., human AAV serotype, can be used for an AAV vector as described herein, including, but not limited to, AAV serotype 1 (AAV1), AAV serotype 2 (AAV2), AAV serotype 3 (AAV3), AAV serotype 4 (AAV4), AAV serotype 5 (AAV5), AAV serotype 6 (AAV6), AAV serotype 7 (AAV7), AAV serotype 8 (AAV8), AAV serotype 9 (AAV9), AAV serotype 10 (AAV10), and AAV serotype 11 (AAV11), as well as variants thereof. In some embodiments, an AAV variant has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% amino acid sequence identity to a wildtype AAV. In certain embodiments, the AAV variant may be engineered such that its capsid proteins have reduced immunogenicity or enhanced transduction ability in humans. In some instances, one or more regions of at least two different AAV serotype viruses are shuffled and reassembled to generate a chimeric variant. For example, a chimeric AAV may comprise inverted terminal repeats (ITRs) that are of a heterologous serotype compared to the serotype of the capsid. The resulting chimeric AAV can have a different antigenic reactivity or recognition compared to its parental serotypes. In some embodiments, a chimeric variant of an AAV includes amino acid sequences from 2, 3, 4, 5, or more different AAV serotypes.
[0211] 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). In certain embodiments, 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. One important category of non-viral nucleic acid vectors is nanoparticles, which can be organic (e.g., lipid) or inorganic (e.g., gold). For instance, organic (e.g. lipid and/or polymer) nanoparticles can be suitable for use as delivery vehicles in certain embodiments of this disclosure.
[0212] In some embodiments, delivery is accomplished using a lipid nanoparticle (LNP). LNP compositions are typically sized on the order of micrometers or smaller and may include a lipid bilayer. In some embodiments, a LNP refers to any particle that has a diameter of less than 1000 nm, 500 nm, 250 nm, 200 nm, 150 nm, 100 nm, 75 nm, 50 nm, or 25 nm. In some embodiments, 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.
[0213] An LNP as described herein may be made from cationic, anionic, or neutral lipids. In some embodiments, an LNP may comprise neutral lipids, such as the fusogenic phospholipid l,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE) or the membrane component cholesterol, as helper lipids to enhance transfection activity and nanoparticle stability. In some embodiments, an LNP may comprise hydrophobic lipids, hydrophilic lipids, or both hydrophobic and hydrophilic lipids. Any lipid or combination of lipids that are known in the art can be used to produce an LNP. The lipids may be combined in any molar ratios to produce the LNP. In some embodiments, the LNP is a liver-targeting (e.g., preferentially or specifically targeting the liver) LNP.
[0214] Any type of cell may be targeted for delivery of an epigenetic editor or component(s) thereof as described herein. For example, the cells may be eukaryotic or prokaryotic. In some embodiments, the cells are mammalian (e.g., human) cells. Human cells may include, for example, hepatocytes, biliary epithelial cells (cholangiocytes), stellate cells, Kupffer cells, and liver sinusoidal endothelial cells.
[0215] In some embodiments, 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. For example, the epigenetic modification may be stable for at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10. 11, or 12 weeks or more; or 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 months or more, after introduction of the epigenetic editor into the host cell. The epigenetic modification may be maintained after one or more mitotic and/or meiotic events of the host cell. In particular embodiments, the epigenetic modification is maintained across generations in offspring generated or derived from the host cell.
IX. Therapeutic Uses of Epigenetic Editors
[0216] 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. In some embodiments, 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.
[0217] In some embodiments, a subject is administered a system for modulating (e.g., repressing) expression of ANGPTL3, 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 ) .
[0218] ‘Treat”, “treating” and “treatment” refer to a method of alleviating or abrogating a biological disorder and/or at least one of its attendant symptoms. As used herein, to “alleviate” a disease, disorder or condition means reducing the severity and/or occurrence frequency of the symptoms of the disease, disorder, or condition. Further, references herein to “treatment” include references to curative, palliative and prophylactic treatment. In some embodiments, as compared with an equivalent untreated control, 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.
[0219] In some embodiments, the subject may be a mammal, e.g., a human. In some embodiments, the subject is selected from a non-human primate such as chimpanzee, cynomolgus monkey, or macaque, and other ape and monkey species.
[0220] In some embodiments, 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). [0221] In some embodiments, 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). In other embodiments, the patient has not received such prior treatment. In some embodiments, the patient has failed on a prior treatment for the condition (e.g., a prior hypercholesterolemia treatment).
[0222] 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 ANGPTL3 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.
[0223] An epigenetic editor of the present disclosure may be administered without additional therapeutic treatments, i.e., as a stand-alone therapy (monotherapy). Alternatively, treatment with an epigenetic editor of the present disclosure may include at least one additional therapeutic treatment (combination therapy). In some embodiments, 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. In some embodiments, the additional therapeutic is a PCSK9 antagonist, e.g., a PCSK9 inhibitor. In some embodiments, the additional therapeutic is an epigenetic editor targeting PCSK9.
[0224] The epigenetic editors or components thereof (or nucleic acid molecules encoding the epigenetic editors or components thereof) of the present disclosure may be administered by any method accepted in the art, e.g., subcutaneously, intradermally, intratumorally, intranodally, intramuscularly, intravenously, intralymphatically, or intraperitoneally. In particular embodiments, a pharmaceutical composition of the present disclosure is administered intravenously to the subject.
X. Definitions
[0225] The term “nucleic acid” as used herein refers to any oligonucleotide or polynucleotide containing nucleotides (e.g., deoxyribonucleotides or ribonucleotides) in either single- or double-strand form, and includes DNA and RNA. “Nucleotides” contain a sugar deoxyribose (DNA) or ribose (RNA), a base, and a phosphate group, and are linked together through the phosphate groups. “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.
[0226] As used herein, an “isolated” or “purified” nucleic acid molecule is a nucleic acid molecule that exists apart from its native environment. For example, 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. In some embodiments, an “isolated” or “purified” nucleic acid molecule is a recombinant nucleic acid molecule.
[0227] It will be understood that in addition to the specific proteins and nucleic acid molecules mentioned herein, the present disclosure also contemplates the use of variants, derivatives, homologs, and fragments thereof. A variant of any given sequence 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). For specific proteins described herein (e.g., KRAB, dCas9, DNMT3A, and DNMT3L proteins described herein), 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).
[0228] As used herein, 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. The term “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. In some embodiments, 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). Such software matches identical or similar sequences by assigning degrees of homology to various substitutions, deletions, and/or other modifications. In an exemplary approach to determining the degree of identity, a BLAST program may be used, with a probability score between e-3 and e-100 indicating a closely related sequence.
[0229] 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). In some embodiments, 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.
[0230] It will be understood that the numbering of the specific positions or residues in polypeptide sequences depends on the particular protein and numbering scheme used. Numbering might be different, e.g., in precursors of a mature protein and the mature protein itself, and differences in sequences from species to species may affect numbering. One of skill in the art will be able to identify the respective residue in any homologous protein and in the respective encoding nucleic acid by methods well known in the art, e.g., by sequence alignment and determination of homologous residues.
[0231] The term “modulate” or “alter” refers to a change in the quantity, degree, or extent of a function. For example, 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. As other examples, an epigenetic editor as described herein may block RNA polymerase from transcribing a gene, or may inhibit translation of an mRNA transcript. The terms “inhibit,” “repress,” “suppress,” “silence” and the like, 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.
[0232] The term “about” or “approximately” means within an acceptable error range for the particular value as determined by one of ordinary skill in the art, which will depend in part on how the value is measured or determined, e.g., the limitations of the measurement system. For example, “about” can mean within one or more than one standard deviation, per the practice in the given value. Where particular values are described in the application and claims, unless otherwise stated, the term “about” should be assumed to mean an acceptable error range for the particular value.
[0233] Ranges provided herein are understood to be shorthand for all of the values within the range. For example, a range of 1 to 50 is understood to include any number, combination of numbers, or sub-range from the group consisting of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38,
39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50, as well as all 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. With respect to sub-ranges, “nested sub-ranges” that extend from either end point of the range are specifically contemplated. For example, 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.
[0234] Unless otherwise defined herein, 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. Further, unless otherwise required by context, singular terms shall include pluralities and plural terms shall include the singular. Throughout this specification and embodiments, the words “have” and “comprise,” or variations such as “has,” “having,” “comprises,” or “comprising,” will be understood to imply the inclusion of a stated integer or group of integers but not the exclusion of any other integer or group of integers. Unless otherwise indicated, the recitation of a listing of elements herein includes any of the elements singly or in any combination. The recitation of an embodiment herein includes that embodiment as a single embodiment, or in combination with any other embodiment(s) herein. All publications, patents, patent applications, and other references mentioned herein are incorporated by reference in their entirety. To the extent that references incorporated by reference contradict the disclosure contained in the specification, the specification is intended to supersede and/or take precedence over any such contradictory material. Although a number of documents are cited herein, this citation does not constitute an admission that any of these documents forms part of the common general knowledge in the art.
[0235] According to the present disclosure, 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. Further, headers herein are created for ease of organization and are not intended to limit the scope of the claimed invention in any manner.
[0236] In order that the present disclosure may be better understood, the following examples are set forth. These examples are for purposes of illustration only and are not to be construed as limiting the scope of the present disclosure in any manner.
EXAMPLES
Example 1: Fusion Protein Design and Synthesis
[0237] A fusion protein comprising dCas9, DNMT3A, DNMT3L, and K0X1 KRAB (“CRISPR-off”) was designed. An exemplary CRISPR-off protein comprises, from N terminus to C terminus, the domains NLS-huDNMT3A-linker-huDNMT3L-XTEN80- dSpCas9- NLS-XTEN16-huKOXl KRAB. The CRISPR-off plasmid construct has been described in Nunez (Nunez et al., Cell (2021) 184(9):2503-19). Another exemplary CRISPR- off protein comprises SEQ ID NO: 658. [0238] ZF fusion proteins (“ZF-off”) comprising DNMT3A, 3L, and K0X1 KRAB were also constructed. The constructs have the general structure huDNMT3A-linker-huDNMT3L- XTEN80-NLS-ZFP domain-NLS-XTEN16-KOXlKrab (SEQ ID NO: 659). Example 2: Selection of Target ANGPTL3 Sequences for gRNA Epigenetic Silencing [0239] gRNAs targeting +/- 1 kb from the ANGPTL3 TSS were computationally designed for human (GRCh38), mouse (mmlO), and Macaca fascicularis (5.0) ANGPTL3. gRNAs containing poly-TTTT sequences were first discarded. gRNA off-target analysis was performed using CasOFFinder (Bae et al., Bioinformatics (2014) 30(10): 1473-5). gRNAs were discarded if they matched to multiple locations across the respective genome build for each independent species.
[0240] A cross -reactivity sequence analysis was performed on human ANGPTL3 gRNAs in order to annotate sequence mismatches with Macaca or mouse gRNA sequences. In particular, gRNA sequence alignments were performed to identify the degree of DNA similarity at each nucleotide, including the annotation of guides that contain up to zero, one, or two nucleotide mismatches. A final set of 89 gRNA targeting sequences (Table 7) was selected for the ANGPTL3 primary screen in Huh7 cells.
Table 7. Exemplary gRNA Sequences Targeting ANGPTL3
Figure imgf000084_0001
Figure imgf000085_0001
Figure imgf000086_0001
Example 3: Selection of ZF Target Sites and Design of ZF Proteins for Epigenetic Silencing [0241] A library of two-finger ZFPs (2F units), each recognizing 6 bp DNA sites, was used to design larger six-finger ZFP arrays targeting 18 bp DNA binding sites. The source of the 2F units was a set of three-finger zinc finger proteins that had been selected to bind specific target sites using a bacterial-2-hybrid (B2H) selection system (Hurt et al., PNAS (2003) 100:12271-6; Maeder et al., Mol Cell (2008) 31(2):294-301). A list of targetable DNA sites was created by generating all possible triplet combinations of 6 bp binding sites represented in the library and allowing either 0 or 1 bp between the 6 bp target sites. To identify zinc finger target sites within A NGPTL3, the sequence +/- Ikb from TSS (human (GRCh38)) was interrogated against this list. For each identified ZF target site, multiple ZF proteins could be designed. Design of the six recognition helices used to generate the full proteins was performed by selecting two-finger units and taking into account a number of factors such as known binding preferences of zinc finger proteins, the frequency with which amino acids in positions -1, 2, 3 and 6 had been selected in the B2H selection system to bind the desired target base, avoidance of amino acids in positions -1, 2, 3 and 6 that had been selected to bind multiple different bases in the B2H, and maintaining context dependencies by matching flanking bases where possible. The full ZF sequence is derived from the naturally occurring Zif268 protein, and selected recognition helices were maintained in the sequence context in which they were selected in the B2H (either fingers 1-2 or fingers 2-3 from Zif268). Two-finger units were joined by the linker TGSQKP (SEQ ID NO: 651) where 6 bp binding sites were contiguous and joined by the linker TGGGGSQKP (SEQ ID NO: 652) where 1 bp separated the 6 bp binding sites. A final set of 84 ZFPs targeted to 9 distinct binding sites (Table 1) were selected for the ANGPTL3 primary screen in Huh7 cells.
Example 4: CRISPR-off Guide Screening of ANGPTL3 in Huh7 Hepatoma Cell Line [0242] The Huh7 hepatoma cell line (Sekisui XenoTech, LLC) is amenable to high- throughput screening and transfection. Guide RNAs (gRNAs) against 89 target sites on the ANGPTL3 locus were tested in Huh7 cells. Cells were treated with 400 ng of mRNA encoding the CRISPR-off construct and 50 ng of the respective gRNA. Huh7 cells were seeded at a density of 25,000 cells per well in 96-well plates. Epigenetic silencing with the CRISPR-off construct was observed to be stable over 7 to 14 days with the top guides (FIGs. 1A and IB). Controls included WT Cas9 + ANGPTL3 gRNA (gRNAl 137 or gRNAl 138), and CRISPR-off with no gRNA.
Figure imgf000087_0001
[0243] Relative ANGPTL3 expression in Huh7 cells treated with CRISPR-off (DNMT3A-DNMT3L-dCas9-KRAB) epigenetic repressors and various gRNAs was measured at days 7 and 14, and expression results were visualized to illustrate the silencing observed for each gRNA as well as the target site of the respective gRNA in the ANGPTL3 locus (FIG. 2). ANGPTL3 silencing for 25 exemplary gRNAs at day 7 and day 14 is shown in FIG. 3.
[0244] End-modified sgRNAs purified using standard desalting were obtained from Integrated DNA Technologies (Table 8; SEQ: SEQ ID NO). The three nucleotides at the 5’ end and the three nucleotides at the 3’ end of each guide are 2’-O-methyl modified. The three intemucleoside linkages at the 3’ end and the three internucleoside linkages at the 5’ end are phosphorothioate intemucleoside linkages. In the Table, mX (i.e., mA, mC, mG, or mU) represents a 2’-O-methyl modified ribonucleoside, rX (i.e., rA, rC, rG, or rU) indicates a natural ribonucleoside, and * indicates a phosphorothioate linkage. All internucleoside linkages that are not phosphorothioate linkages are phosphate linkages.
Table 8. Exemplary End-Modified gRNA Sequences Targeting ANGPTL3
Figure imgf000088_0001
Figure imgf000089_0001
Figure imgf000090_0001
Figure imgf000091_0001
Figure imgf000092_0001
Figure imgf000093_0001
Figure imgf000094_0001
Figure imgf000095_0001
Figure imgf000096_0001
Figure imgf000097_0001
Figure imgf000098_0001
Figure imgf000099_0001
Figure imgf000100_0001
Figure imgf000101_0001
Figure imgf000102_0001
Example 5: Guide RNA Repression of ANGPTL3 with CRISPR-off in Primary Human Hepatocytes for Analysis of Specificity and Durability
[0245] Primary human PXB cells from PhoenixBio are used to test the efficacy of the gRNAs in primary hepatocytes. PXB cultures are maintained according to manufacturer recommendations. Briefly, PXB maintenance media is thawed and made up within 30 minutes of the cells’ arrival. Upon media change, cells are allowed to acclimate for two days in a 37°C, 5% CO2 incubator. On the second day after receipt, LNPs are formulated with CRISPR-off + sgRNA, GFP-mRNA and WT CRISPR Cas9 in various concentrations using the SPARK™ (Precision Nanosystems) and the Genvoy-ILM™ mRNA LNP formulation kit. Subsequently, the LNPs are characterized for encapsulation efficiency and total mRNA pay load delivery via the Quant- it™ RiboGreen RNA assay kit. LNPs are then added to the media in specified quantities of total mRNA to achieve clinically relevant levels of ANGPTL3 silencing. Media is changed every other day for a duration of up to four weeks to assess durability and/or inheritability of silencing. ANGPTL3 silencing is assessed every seven days by ELISA to measure secreted ANGPTL3 levels in the media. ANGPTL3 concentration will be controlled to total hepatocytes using a human albumin ELISA (Thermo Fisher®) and CellTiter-Glo®. Data is then presented as total ANGPTL3 secretion as percent of CRISPR-off -mRNA only negative control. Cells are then processed for RNAseq evaluation and genome-wide bisulfite sequencing.
[0246] The top 19 gRNAs (Table 9) are selected to be tested in primary human hepatocytes (PHH). The top gRNAs are selected based on (i) ANGPTL3 silencing efficiency and durability in Huh7 cells, and (ii) whether they have a perfect alignment with the human ANGPTL3 gene and up to one mismatch with the non-human primate ANGPTL3 gene. Combinations of gRNAs are also tested to determine their efficacy and durability. The negative control is CRISPR-off only (no gRNA). The positive control is CRISPRi and WT Cas9 co-transfected with a gRNA targeted to the ANGPTL3 locus. All tested gRNAs are predicted to bind to both human and cynomolgus ANGPTL3.
[0247] Robust ANGPTL3 silencing is observed. For some gRNAs, >70% reduction in secreted ANGPTL3 at day 7 is observed (depending on transfection efficiency).
Table 9. gRNAs Being Screened in PHH
Figure imgf000103_0001
Figure imgf000104_0001
Example 6: ZF-off guide screening of ANGPTL3 in Huh7 cells
[0248] A total of 84 zinc finger proteins were designed from the ZF library to nine ANGPTL3 target sites (selected from GRCh38 chromosome 1 between 62596520 and 62598520). The target sites had no other exact matches in the human genome (GRCh38). [0249] Huh7 cells were transfected with ZF-off constructs (DNMT3A-DNMT3L-ZF- KRAB) in mRNA format (Table 10) using messenger max lipid transfection reagent (Invitrogen). Wild-type Cas9 nuclease together with a high performing AAGPTZ -targeting gRNA (gRNA 1138) was used as a positive control. Negative controls were lipid transfection reagent only and CRISPR-off with no gRNA. Cells were maintained in culture for 14 days and media was assayed at day 7 and 14 for ANGPTL3 secretion using an ELISA assay (Fisher Scientific).
[0250] The ANGPTL3 repression results of the primary ZF-off screen are shown in FIG. 4, illustrating the silencing activity of the various constructs tested in the context of the position of their respective target site in the ANGPTL3 locus. The ZF-off screen yielded hits with silencing activity comparable to WT Cas9/gRNA at day 7 (FIG. 4). ZF-off constructs with high silencing efficiency were further examined in follow-up experiments.
[0251] In total, 80 ZF-off constructs were screened, with their ANGPTL3 knockdown activity relative to the negative control shown in Table 10 below (“TxF” refers to a transfection reagent).
Table 10. ZF-off Construct Activity
Figure imgf000104_0002
Figure imgf000105_0001
Figure imgf000106_0001
Figure imgf000107_0001
Example 7: Multiplex guide screening for CRISPR-off silencing otANGPTL3
[0252] The top 19 gRNAs from the primary screen (Table 9) were tested in Huh7 cells to assess durability in an unbiased 19 x 19 gRNA duplex screen, allowing for the assessment of all possible gRNA pairs of the top 19 hits. The top gRNAs were selected based on ANGPTL3 silencing efficiency and durability in Huh7 cells. Briefly, Huh7 cells were treated with mRNA encoding a CRISPR-off construct (SEQ ID NO: 1013) alongside all single gRNAs with every combination of duplex gRNA pairs. Cells were maintained in culture for 14 days and ANGPTL3 secretion was measured in the media by ELISA (Fisher Scientific) on day 7 and day 14. Silencing data was normalized to lipid-only controls and to cell number (measured using CellTiter-Glo®, Promega). Positive controls consisted of CRISPRi and WT Cas9 nuclease paired with a high-performing gRNA targeted to the ANGPTL3 locus.
Negative controls were lipid only and a CRISPR-off construct paired with a gRNA targeted to a different (non-AAGPTL3) genomic locus. Increased potency and durability were observed in dual gRNA treated cells (cells treated with CRISPR-off construct together with paired gRNAs) as compared to single-gRNA mediated CRISPR-off repression (FIG. 5). A number of gRNA pairs were screened, with the screening data for days 7 and 14 shown in Table 11 below.
Table 11. Dual gRNA Screening Data
Figure imgf000107_0002
Figure imgf000108_0001
Figure imgf000109_0001
Figure imgf000110_0001
Figure imgf000111_0001
Example 8: Multiplex CRISPR-off Silencing of ANGPTL3 and PCSK9
[0253] Huh7 cells were co-transfected with a CRISPR-off construct (SEQ ID NO: 1011) and either a potent gRNA against PCSK9 (gRNA41; targeting sequence of SEQ ID NO: 1024), a potent gRNA against ANGPTL3 (RNA1094; targeting sequence of SEQ ID NO: 940), or a combination of these guides, using MessengerMAX™ (Invitrogen). Cells were then grown for 7 days in culture. ANGPTL3 and PCSK9 secretion were measured for each cell population on day 6 after transfection. Data were normalized to transfection reagent only controls and cell number (CellTiter-Glo®, Promega). Controls included WT Cas9 with gRNA79 (targeting sequence of SEQ ID NO: 1025; targets PCSK9) and gRNA1138 (targeting sequence of SEQ ID NO: 1023; targets ANGPTL3). as well as CRISPRi (dCas9- KRAB) with gRNA41 (targeting sequence of SEQ ID NO: 1024; targets PCSK9) and RNA486 (targeting sequence of SEQ ID NO: 935; targets ANGPTL3). Cells were transfected with different ratios of CRISPR-off effector:gRNA(PCSX9):gRNA(A/VGPTL3). Robust multiplex silencing of PCSK9 and ANGPTL3 was observed in various transfected cell populations, while relative cell number was not significantly affected (FIG. 6).
[0254] Multiplex dose response experiments were performed with different CRISPR-off constructs (SEQ ID NO: 1013, SEQ ID NO: 1009, and SEQ ID NO: 1011, respectively) paired with a potent gRNA against PCSK9 (gRNA41; targeting sequence of SEQ ID NO: 1024) and a potent guide against ANGPTL3 (RNA1096; targeting sequence of SEQ ID NO: 962)). PCSK9 and ANGPTL3 expression was measured on day 7 after transfection. Dose response results are shown in FIG. 7.
Figure imgf000111_0002
Example 9: Guide RNA Assays in Primary Human and Cynomolgus Hepatocytes for Assessment of Specificity and Cross-Reactivity
[0255] Primary human and cynomolgus hepatocyte cultures from BioIVT are used to test the efficacy of the gRNAs in primary hepatocytes. Hepatocyte cultures are maintained according to manufacturer recommendations. Briefly, hepatocyte maintenance media is thawed and made up within 30 minutes of the cells’ arrival. Upon media change, cells are allowed to acclimate for two days in a 37 °C, 5% CO2 incubator. On the second day after receipt, the manufacturer’s instructions are followed to formulate LNPs with CRISPR-off + sgRNA, GFP-mRNA and WT CRISPR Cas9 in various concentrations using the SPARK™ (Precision Nanosystems) and the GenVoy-ILM™ mRNA LNP formulation kit.
Subsequently, the LNPs are characterized for encapsulation efficiency and total mRNA payload delivery via the Quant-iT™ RiboGreen RNA assay kit. LNPs are then added to the media in specified quantities of total mRNA to achieve clinically relevant levels of silencing of ANGPTL3. ANGPTL3 silencing will be assessed at 3 days by ELISA to measure secreted ANGPTL3 levels in the media. ANGPTL3 concentration will be controlled to total hepatocytes using a human albumin ELISA (Thermo Fisher®) and CellTiter-Glo®. Data is then presented as total ANGPTL3 secretion as percent of CRISPR-off + off-target gRNA negative control. Specificity may be assessed by isolating and processing the cells for RNAseq evaluation and genome-wide bisulfite sequencing.
[0256] The top 19 gRNAs (Table 9) are selected to be tested in primary human hepatocytes (PHH). The top gRNAs are selected based on ANGPTL3 silencing efficiency and durability in Huh7 cells. Combinations of gRNAs are also tested to determine their efficacy and durability. The negative controls are CRISPR-off only (without gRNA) and vehicle only. The positive control is CRISPRi co-transfected with gRNA1096 (SEQ ID NO: 962) targeted to the ANGPTL3 locus. All tested gRNAs are predicted to bind to human ANGPTL3.
[0257] Robust ANGPTL3 silencing is observed. For some gRNAs, >70% reduction in secreted ANGPTL3 at day 7 is observed.
Example 10: Full Specificity Screen of Constructs in Primary Human Hepatocytes [0258] The specificity of CRISPR-off and ZF-off constructs for silencing ANGPTL3 is tested in primary human hepatocytes. The readouts to assess specificity are RNAseq, methylation array and whole genome bisulfite sequencing assays. Genome-wide expression and methylation changes after epigenetic editing compared to negative controls are profiled. Robust ANGPTL3 silencing is observed. For some gRNAs and some ZF-off constructs, >70% reduction in secreted ANGPTL3 at day 7 is observed while off-target activity is not detectable.
Example 11: CpG Methylation Patterns
[0259] The CpG methylation patterns in human hepatocytes (e.g., primary cells or cell lines) treated with CRISPR-off or ZF-off are investigated. Hybrid capture assays are performed on bisulfite treated DNA to investigate methylation patterns at CpG sites that are induced by CRISPR-off or ZF-off at the Ikb region around the ANGPTL3 TSS.
Example 12: Stable ANGPTL3 Silencing via Epigenetic Editing in Liver Humanized Mouse Model
[0260] Liver humanized mice are a special mouse model with functional engraftment of primary human hepatocytes in the host liver (see, e.g., Tateno and Kojima, Lab Anim Res. (2020) 36:2). Such a model has been used to evaluate the efficacy of liver-targeted gene therapies with better predictability than rodent models (see, e.g., Paulk et al., Mol Ther. (2018) 26(l):289-303).
[0261] CRISRP-off and ZF-off constructs targeting the human ANGPTL3 genomic locus as described herein are formulated into lipid nanoparticles (LNPs) and intravenously administered to liver humanized mice. Circulating human ANGPTL3 level reduction post treatment is measured, as well as human ANGPTL3 gene expression from liver and human ANGPTL3 locus methylation status. The assays employed are species specific such that residual rodent cells will not interfere with the assay results. A single administration of CRISPR-off or ZF-off targeting the human ANGPTL3 locus leads to durable (>2 months) epigenetic silencing of human ANGPTL3 (inside the primary human hepatocytes) in liver humanized mice.
Example 13: Fusion Proteins with Variant NLS Configurations
[0262] Several improved fusion protein constructs were developed using variant nuclear localization sequence (NLS) configurations to have significantly higher epi-silencing activity [0263] Constructs with variant configurations of NLS domains (FIGs. 8A and 8B) were constructed and tested in ANGPTL3 loci in Huh7 cells. Exemplary fusion protein construct amino acid and DNA sequences are shown below: Table 12: Exemplary Fusion Protein Construct Amino Acid and DNA Sequences
Figure imgf000114_0001
Figure imgf000115_0001
Figure imgf000116_0001
Figure imgf000117_0001
Figure imgf000118_0001
Figure imgf000119_0001
Figure imgf000120_0001
Figure imgf000121_0001
Figure imgf000122_0001
Figure imgf000123_0001
Figure imgf000124_0001
Figure imgf000125_0001
Figure imgf000126_0001
Figure imgf000127_0001
Figure imgf000128_0001
Figure imgf000129_0001
Figure imgf000130_0001
Figure imgf000131_0001
Figure imgf000132_0001
Figure imgf000133_0001
Figure imgf000134_0001
Figure imgf000135_0001
Figure imgf000136_0001
Figure imgf000137_0001
Figure imgf000138_0001
Figure imgf000139_0001
Figure imgf000140_0001
Figure imgf000141_0001
Figure imgf000142_0001
Figure imgf000143_0001
Figure imgf000144_0001
Figure imgf000145_0001
Figure imgf000146_0001
Figure imgf000147_0001
Cell Culture and Transfection
[0264] Huh7 cells (Sekisui XenoTech, LLC) were cultured in DMEM with 10% FBS. All experiments in Huh7 cells were done using chemically synthesized guide RNA and in vitro transcribed effector constructs. Huh7 cells were reverse transfected using MessengerMAX™ reagent from Invitrogen. Secreted ANGPTL3 levels were measured at the indicated time points using the Human ANGPTL3 ELISA Kit from Fisher Scientific. All ELISA data was normalized for cell numbers using a CellTiter-Glo® kit from Promega.
In Vitro Transcription of Effector Constructs and Synthetic gRNA [0265] 1 pg of linearized effector template was used to set up in vitro transcription reactions using T7 mScript™ 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 ’-poly adenylated. End-modified sgRNA that had three 2’0- methyl modified nucleotides with phosphorothioate linkages on both 5’ and 3’ ends were obtained from Integrated DNA Technologies.
Methylation Profiling
[0266] Genomic DNA was 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 was bisulfite converted using an EZ-96 DNA Methylation- Gold™ MagPrep kit (Zymo Research) according to manufacturer’s instructions. For hybridization capture experiments, DNA libraries were prepared using the xGen™ Methyl-Seq DNA Library Prep Kit (IDT) and hybrid capture was conducted using the xGen™ Hybridization Capture of DNA Libraries kit (IDT). For amplicon sequencing experiments, DNA libraries were prepared using the xGen™ Methyl-Seq DNA Library Prep Kit (IDT) and hybrid capture was conducted using the xGen™ Hybridization Capture of DNA Libraries kit (IDT). Bisulfite-converted DNA from each sample was used to seed PCR corresponding to each of the two VIM amplicons using a Platinum™ Taq kit (Invitrogen). Pooled products were cleaned using the AMPure XP kit (Beckman Coulter) and fragment size was assessed via D1000 screentape on a Tapestation 4200 (Agilent) prior to sequencing by commercial service (Azenta).
Example 14: Alternative KRAB Domains
[0267] In this example, fusion proteins were constructed with alternative KRAB domains (Table 13) and showed improved activity as compared to CRISPR-off when tested using the experimental procedures of Example 1 (FIGs. 9A-9D).
Table 13: Alternative KRAB Domains
Figure imgf000148_0001
Figure imgf000149_0001
Example 15: ZIM 3 Fusion Constructs
[0268] Novel fusions of ZIM3 and KOX1KRAB are generated. Both ZIM 3 and KOX1KRAB are KRAB family proteins with extensive homology. Thus, sequences are designed which represent halfway points between ZIM3 and KOX1KRAB. These 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 K0X1KRAB variants were both assembled by first, BLAST of ZIM3 or K0X1KRAB 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 14).
Table 14: ZIM-KOX1KRAB Chimera Proteins
Figure imgf000150_0001
SEQUENCES
[0269] The SEQ ID NOs (SEQ) of nucleotide (nt) and amino acid (aa) sequences described in the present disclosure are listed below.
Figure imgf000151_0001
Figure imgf000152_0001
Figure imgf000153_0001
Figure imgf000154_0001
Figure imgf000155_0001
Figure imgf000156_0001
Figure imgf000157_0001
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Figure imgf000161_0001
Figure imgf000162_0001
Figure imgf000163_0001
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Figure imgf000166_0001
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Figure imgf000168_0001
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Figure imgf000170_0001
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Figure imgf000175_0001
Figure imgf000176_0001
Figure imgf000177_0001
Figure imgf000178_0001
Figure imgf000179_0001
Figure imgf000180_0001
Figure imgf000181_0001
Figure imgf000182_0001
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Figure imgf000184_0001
Figure imgf000185_0001
Figure imgf000186_0001
Figure imgf000187_0001
Figure imgf000188_0001
Figure imgf000189_0001
Figure imgf000190_0001
Figure imgf000191_0001
Figure imgf000192_0001
Figure imgf000193_0001
Figure imgf000194_0001
Figure imgf000195_0001
Figure imgf000196_0001
Figure imgf000197_0001
Figure imgf000198_0001
Figure imgf000199_0001
Figure imgf000200_0001
Figure imgf000201_0001
Figure imgf000202_0001
Figure imgf000203_0001
Figure imgf000204_0001
Figure imgf000205_0001
Figure imgf000206_0001
Figure imgf000207_0001
Figure imgf000208_0001
Figure imgf000209_0001
Figure imgf000210_0001
Figure imgf000211_0001

Claims

1. A system for repressing transcription of a human ANGPTL3 gene in a human cell, optionally a human hepatocyte, comprising a) one or more fusion proteins that collectively comprise a DNA methyltransferase (DNMT) domain and/or a domain that recruits a DNMT, optionally wherein the DNMT domain and/or the recruiter domain comprise a DNMT3A domain and/or a DNMT3L domain, and optionally wherein the recruited DNMT is DNMT3A, and a transcriptional repressor domain, each domain being linked to a DNA-binding domain that binds to a target region in the human ANGPTL3 gene; or b) one or more nucleic acid molecules encoding the one or more fusion proteins.
2. The system of claim 1, wherein the DNA-binding domain binds to a target sequence in SEQ ID NO: 1019, 1020, or 1021.
3. The system of claim 1 or 2, wherein the DNA-binding domain targets the fusion protein(s) to one or more sequences in the ANGPTL3 gene selected from SEQ ID NOs: 700-708 and 811-899.
4. The system of any one of claims 1-3, wherein the DNA-binding domain comprises a dead CRISPR Cas (dCas) domain, a ZFP domain, or a TALE domain.
5. The system of claim 4, wherein the DNA-binding domain comprises a dCas9 domain and the system further comprises (i) one or more guide RNAs comprising any one of SEQ ID NOs: 900-988, or (ii) nucleic acid molecules coding for the one or more guide RNAs.
6. The system of claim 4 or 5, wherein the dCas domain comprises a dCas9 sequence, optionally a sequence with at least 90% identity to SEQ ID NO: 12 or 13.
7. The system of claim 4, wherein the ZFP domain targets a nucleotide sequence selected from SEQ ID NOs: 700-708.
8. The system of claim 7, wherein the ZFP domain comprises the F1-F6 amino acid sequences of any one of ZFP810 through ZFP893 as shown in Table 1.
9. The system of any one of claims 1-8, wherein the DNMT3A domain comprises a sequence with at least 90% identity to SEQ ID NO: 574 or 575.
10. The system of any one of claims 1-9, wherein the DNMT3L domain comprises a sequence with at least 90% identity to a sequence selected from SEQ ID NOs: 578- 581.
11. The system of any one of claims 1-9, wherein the DNMT3L domain comprises a sequence with at least 90% identity to a sequence selected from SEQ ID NOs: 582- 603.
12. The system of any one of claims 1-8, wherein the DNMT domain comprises a sequence with at least 90% identity to a sequence selected from SEQ ID NOs: 601- 603.
13. The system of any one of claims 1-12, wherein the transcriptional repressor domain comprises a sequence with at least 90% identity to a sequence selected from SEQ ID NOs: 33-570.
14. The system of any one of claims 1-12, wherein the transcriptional repressor domain comprises a KRAB domain derived from KOX1, ZIM3, ZFP28, or ZN627.
15. The system of claim 14, wherein the KRAB domain comprises a sequence with at least 90% identity to a sequence selected from SEQ ID NOs: 89, 116, 245, and 255.
16. The system of any one of claims 1-12, wherein the transcriptional repressor domain comprises a fusion of the N- and C-terminal regions of ZIM3 and KOX1 KRAB, and optionally comprises the amino acid sequence of SEQ ID NO: 571 or 572.
17. The system of any one of claims 1-12, wherein the transcriptional repressor domain is derived from KAP1, MECP2, HPla/CBX5, HPlb, CBX8, CDYL2, TOX, T0X3, T0X4, EED, EZH2, RBBP4, RC0R1, or SCML2.
18. The system of any one of claims 1-17, wherein the system comprises a) a fusion protein comprising the DNMT3A domain, the DNMT3L domain, the transcriptional repressor domain, and the DNA-binding domain, optionally wherein one or both of the DNMT3A domain and the DNMT3L domain are human, and optionally wherein the DNA-binding domain is a dead CRISPR Cas domain or a ZFP domain; or b) a nucleic acid molecule encoding the fusion protein.
19. The system of claim 18, wherein the fusion protein comprises, from N-terminus to C- terminus, the DNMT3A domain, a first peptide linker, the DNMT3L domain, a second peptide linker, the DNA-binding domain, a third peptide linker, and the transcriptional repressor domain.
20. The system of claim 19, wherein the fusion protein comprises, from N-terminus to C- terminus, the DNMT3A domain, the first peptide linker, the DNMT3L domain, the second peptide linker, a first nuclear localization signal (NLS), the DNA-binding domain, a second NLS, the third peptide linker, and the transcriptional repressor domain.
21. The system of claim 18, wherein the fusion protein comprises, from N-terminus to C- terminus, a first nuclear localization signal (NLS), the DNMT3A domain, the first peptide linker, the DNMT3L domain, the second peptide linker, the DNA-binding domain, the third peptide linker, the transcriptional repressor domain, and a second NLS.
22. The system of claim 18, wherein the fusion protein comprises, from N-terminus to C- terminus, first and second nuclear localization signals (NLSs), the DNMT3A domain, the first peptide linker, the DNMT3L domain, the second peptide linker, the DNA- binding domain, the third peptide linker, the transcriptional repressor domain, and third and fourth NLSs.
23. The system of any one of claims 18-22, wherein the transcriptional repressor domain is a KRAB domain, optionally a human K0X1, ZFP28, ZN627, or ZIM3 KRAB domain.
24. The system of any one of claims 19-23, wherein one or both of the second and third peptide linkers are XTEN linkers, optionally selected from XTEN80 and XTEN16, and further optionally wherein the second peptide linker is XTEN80, and the third peptide linker is XTEN 16.
25. The system of claim 18, wherein the fusion protein comprises, from N-terminus to C- terminus, a human DNMT3A domain, a first peptide linker, a human DNMT3L domain, an XTEN80 peptide linker, a first NLS, a dSpCas9 domain, a second NLS, an XTEN 16 peptide linker, and a human K0X1 KRAB domain.
26. The system of claim 25, wherein the fusion protein comprises SEQ ID NO: 658 or a sequence at least 90% identical thereto.
27. The system of claim 18, wherein the fusion protein comprises, from N-terminus to C- terminus, a human DNMT3A domain, a first peptide linker, a human DNMT3L domain, an XTEN80 peptide linker, a first NLS, a ZFP domain, a second NLS, an XTEN 16 linker, and a human KOX1 KRAB domain.
28. The system of claim 27, wherein the fusion protein comprises SEQ ID NO: 659 or a sequence at least 90% identical thereto, optionally wherein the ZFP comprises the FIFO amino acid sequences of any one of ZFP810 through ZFP893 as shown in Table 1.
29. The system of claim 18, wherein the fusion protein comprises, from N-terminus to C- terminus, first and second NLSs, a human DNMT3A domain, a first peptide linker, a human DNMT3L domain, an XTEN80 peptide linker, a dSpCas9 domain, an XTEN 16 peptide linker, a human KOX1 KRAB domain, and third and fourth NLSs.
30. The system of claim 29, wherein the fusion protein comprises SEQ ID NO: 660 or a sequence at least 90% identical thereto.
31. The system of claim 18, wherein the fusion protein comprises, from N-terminus to C- terminus, first and second NLSs, a human DNMT3A domain, a first peptide linker, a human DNMT3L domain, an XTEN80 peptide linker, a ZFP domain, an XTEN16 peptide linker, a human KOX1 KRAB domain, and third and fourth NLSs.
32. The system of claim 18, wherein the fusion protein comprises, from N-terminus to C- terminus, first and second NLSs, a human DNMT3A domain, a first peptide linker, a human DNMT3L domain, an XTEN80 peptide linker, a dSpCas9 domain, an XTEN16 peptide linker, a human ZFP28 KRAB domain, and third and fourth NLSs.
33. The system of claim 32, wherein the fusion protein comprises SEQ ID NO: 661 or a sequence at least 90% identical thereto.
34. The system of claim 18, wherein the fusion protein comprises, from N-terminus to C- terminus, first and second NLSs, a human DNMT3A domain, a first peptide linker, a human DNMT3L domain, an XTEN80 peptide linker, a ZFP domain, an XTEN16 peptide linker, a human ZFP28 KRAB domain, and third and fourth NLSs.
35. The system of claim 18, wherein the fusion protein comprises, from N-terminus to C- terminus, first and second NLSs, a human DNMT3A domain, a first peptide linker, a human DNMT3L domain, an XTEN80 peptide linker, a dSpCas9 domain, an XTEN16 peptide linker, a human ZN627 KRAB domain, and third and fourth NLSs.
36. The system of claim 35, wherein the fusion protein comprises SEQ ID NO: 662 or a sequence at least 90% identical thereto.
37. The system of claim 18, wherein the fusion protein comprises, from N-terminus to C- terminus, first and second NLSs, a human DNMT3A domain, a first peptide linker, a human DNMT3L domain, an XTEN80 peptide linker, a ZFP domain, an XTEN16 peptide linker, a human ZN627 KRAB domain, and third and fourth NLSs.
38. The system of claim 18, wherein the fusion protein comprises, from N-terminus to C- terminus, first and second NLSs, a human DNMT3A domain, a first peptide linker, a human DNMT3L domain, an XTEN80 peptide linker, a dSpCas9 domain, an XTEN16 peptide linker, a human ZIM3 KRAB domain, and third and fourth NLSs.
39. The system of claim 38, wherein the fusion protein comprises SEQ ID NO: 663 or a sequence at least 90% identical thereto.
40. The system of claim 18, wherein the fusion protein comprises, from N-terminus to C- terminus, first and second NLSs, a human DNMT3A domain, a first peptide linker, a human DNMT3L domain, an XTEN80 peptide linker, a ZFP domain, an XTEN16 peptide linker, a human ZIM3 KRAB domain, and third and fourth NLSs.
41. The system of any one of claims 20-40, wherein at least one of the NLSs is an SV40 NLS.
42. The system of any one of claims 1-17, wherein the system comprises: a) a first fusion protein comprising a first DNA-binding domain and comprising or recruiting the DNMT3A domain, a second fusion protein comprising a second DNA-binding domain and comprising or recruiting the DNMT3L domain, and a third fusion protein comprising a third DNA-binding domain and comprising or recruiting the transcriptional repressor domain; or b) one or more nucleic acid molecules encoding the fusion proteins.
43. A human cell comprising the system of any one of claims 1-42, or progeny of the cell, optionally wherein the cell is a hepatocyte.
44. A pharmaceutical composition comprising the system of any one of claims 1-42 and a pharmaceutically acceptable excipient, optionally wherein the composition comprises lipid nanoparticles (LNPs) comprising the system, and/or the DNA-binding domain is a dCas domain and the LNPs further comprise one or more gRNAs.
45. A method of treating a patient in need thereof, comprising administering the system of any one of claims 1-42 or the pharmaceutical composition of claim 44 to the patient, optionally intravenously.
46. The method of claim 45, wherein the patient 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).
47. The system of any one of claims 1-42, or the pharmaceutical composition of claim 44, for use in treating a patient in need thereof, optionally in the method of claim 45 or 46.
48. Use of the system of any one of claims 1-42 in the manufacture of a medicament for treating a patient in need thereof, optionally in the method of claim 45 or 46.
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