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WO2024145615A2 - Compositions et méthodes de régulation épigénétique de l'expression de angptl3 - Google Patents

Compositions et méthodes de régulation épigénétique de l'expression de angptl3 Download PDF

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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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WO2024145615A3 (fr
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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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Chroma Medicine Inc
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/10—Transferases (2.)
    • C12N9/1003—Transferases (2.) transferring one-carbon groups (2.1)
    • C12N9/1007—Methyltransferases (general) (2.1.1.)
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • 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

La présente divulgation concerne des compositions et des méthodes comprenant des éditeurs épigénétiques pour la modification épigénétique de l'ANGPTL3, ainsi que des acides nucléiques et des vecteurs codant ceux-ci. Sont également divulguées des cellules épigénétiquement modifiées par ces éditeurs épigénétiques.
PCT/US2023/086491 2022-12-30 2023-12-29 Compositions et méthodes de régulation épigénétique de l'expression de angptl3 Ceased WO2024145615A2 (fr)

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