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WO2025235563A1 - Modulation épigénétique pour la régulation positive de gènes - Google Patents

Modulation épigénétique pour la régulation positive de gènes

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Publication number
WO2025235563A1
WO2025235563A1 PCT/US2025/028069 US2025028069W WO2025235563A1 WO 2025235563 A1 WO2025235563 A1 WO 2025235563A1 US 2025028069 W US2025028069 W US 2025028069W WO 2025235563 A1 WO2025235563 A1 WO 2025235563A1
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WO
WIPO (PCT)
Prior art keywords
expression
activator
dna
sequence
gene
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Pending
Application number
PCT/US2025/028069
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English (en)
Inventor
Chevaun Danielle MORRISON-SMITH
Jeremiah D. FARELLI
Charles W. O'DONNELL
Marcus Ian GIBSON
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Omega Therapeutics Inc
Original Assignee
Omega Therapeutics Inc
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Filing date
Publication date
Application filed by Omega Therapeutics Inc filed Critical Omega Therapeutics Inc
Publication of WO2025235563A1 publication Critical patent/WO2025235563A1/fr
Pending legal-status Critical Current
Anticipated expiration legal-status Critical

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    • 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
    • C12N15/113—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing
    • 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
    • C12N9/14—Hydrolases (3)
    • C12N9/16—Hydrolases (3) acting on ester bonds (3.1)
    • C12N9/22—Ribonucleases [RNase]; Deoxyribonucleases [DNase]
    • C12N9/222—Clustered regularly interspaced short palindromic repeats [CRISPR]-associated [CAS] enzymes
    • 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
    • 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
    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/10—Type of nucleic acid
    • C12N2310/20—Type of nucleic acid involving clustered regularly interspaced short palindromic repeats [CRISPR]

Definitions

  • ECs epigenomic controllers
  • PK pharmacokinetics
  • PD pharmacodynamics
  • ECs consist of one or more protein domains (e.g., one or more activating domains and/or effector domains) selected from a validated library and tethered to custom, site-specific DNA-binding-domains to induce a desired regulatory effect.
  • this fusion is optimally encoded in an mRNA therapeutic producing one or several effector-DBD fusion proteins, delivered tissue-specifically using custom lipid nanoparticles (LNPs).
  • LNPs custom lipid nanoparticles
  • the disclosure provides an expression activator for increasing expression of a target gene comprising: (i) a DNA-targeting moiety that binds to a target sequence of 15-20 nucleotides in or near the target gene; and (ii) an effector domain comprising an epigenetic activating domain encoded by a fragment of a gene selected from MYBL1, DUX4, MYB, CEPBPD, ATF4, NCOA2, TP53, 1 FH12877464.1 Attorney Docket: OGY-02925 FOXO1, NFE2, E2F2, UBN1, CREB3L1, FOXO3, KLF15, MLXIPL, LEUTX, INO80D, CR3L1, ATF6A, CSN9, FOXO6, CACTIN, C1QBP, KLF4, MYC, NUTM1, VP16, VP64, YLR278C, PIP2 and STB3.
  • a DNA-targeting moiety that binds to a target sequence of 15-20 nucleotides
  • the disclosure provides an expression activator for increasing expression of a target gene comprising: (i) a DNA-targeting moiety that binds to a target sequence of 15-20 nucleotides in or near the target gene; and (ii) an effector domain comprising an epigenetic activating domain encoded by a fragment of a gene selected from MYBL1, DUX4, MYB, CEPBPD, ATF4, NCOA2, TP53, FOXO1, NFE2, E2F2, UBN1, CREB3L1, FOXO3, KLF15, MLXIPL, LEUTX, INO80D, CR3L1, ATF6A, CSN9, FOXO6, CACTIN, C1QBP, KLF4, MYC, NUTM1, VP16, VP64, YLR278C, PIP2, PA.v1b and STB3.
  • a DNA-targeting moiety that binds to a target sequence of 15-20 nucleotides in or near the target gene
  • an effector domain compris
  • the DNA-targeting moiety comprises a zinc finger (ZF) domain. In some embodiments, the DNA-targeting moiety comprises a transcription activator-like effector (TALE) domain. In some embodiments, the DNA-targeting moiety comprises a nuclease inactive polypeptide and a gRNA comprising a sequence complementary to the target sequence. In some embodiments, the nuclease inactive polypeptide is a nuclease inactive Cas (dCas) polypeptide. In some embodiments, the nuclease inactive polypeptide is operably linked to at least one GCN4 peptide, and wherein the effector domain is operably linked to an antibody or antibody fragment specific for the at least one GCN4 peptide.
  • ZF zinc finger
  • TALE transcription activator-like effector
  • the antibody or antibody fragment is a scFv.
  • the effector domain comprises an amino acid sequence having at least 90% identity to an amino acid sequence selected from SEQ ID Nos: 1-35. In some embodiments, the effector domain comprises an amino acid sequence selected from SEQ ID Nos: 1-35. In some embodiments, the effector domain comprises an amino acid sequence having at least 90% identity to an amino acid sequence selected from SEQ ID Nos: 1-35 and 116. In some embodiments, the effector domain comprises an amino acid sequence selected from SEQ ID Nos: 1-35 and 116. In some embodiments, the target sequence is in or near a promoter region of the gene. In some embodiments, target sequence is in or near an enhancer region of the gene.
  • the effector domain demethylates a CpG island, methylates a histone, adds an acetyl group to a histone, binds to a methylated CpG island, binds to a protein to form heterochromatin, and/or binds a GATA sequence in DNA.
  • FH12877464.1 Attorney Docket: OGY-02925
  • the DNA-targeting moiety and the effector domain do not naturally occur in the same gene product.
  • the DNA binding moiety comprises a first polypeptide and the effector domain comprises a second polypeptide that is not covalently linked to the first polypeptide.
  • the DNA binding moiety and the effector domain comprise a single polypeptide.
  • the disclosure provides a method of increasing expression of a target gene in a cell, comprising contacting the cell with an expression activator described herein.
  • the disclosure provides a nucleic acid encoding an expression activator described herein.
  • the disclosure provides a recombinant expression vector comprising ta nucleic acid described herein.
  • the disclosure provide a messenger RNA (mRNA) encoding an expression activator described herein.
  • mRNA messenger RNA
  • the disclosure provides a lipid nanoparticle (LNP) comprising an expression activator, a nucleic acid, a recombinant expression vector, or an mRNA, described herein.
  • LNP lipid nanoparticle
  • the disclosure provides a pharmaceutical composition comprising an expression activator, a nucleic acid, a recombinant expression vector, an mRNA, or an LNP, described herein, and a pharmaceutically acceptable carrier.
  • the disclosure provides a method of altering expression of a target gene in a cell, comprising contacting the cell with an expression activator, a nucleic acid, a recombinant expression vector, an mRNA, an LNP, or a pharmaceutical composition, described herein.
  • FIGs.1A-1B provide graphs depicting Target Gene 1 mRNA expression following treatment with lipid nanoparticles (LNPs) having separate mRNAs encoding dCas9-SunTag and an scFv fused to an epigenetic activating domain of a polypeptide (VPR, VP64, NCOA2, LEUTX, E2F2, KLF15, INO80D, FOXO6, TP53, or PIP2), and gRNAs designed to target the distal promoter (FIG.
  • LNPs lipid nanoparticles
  • FIGs.2A-2B provide graphs depicting Target Gene 2 mRNA expression following FH12877464.1 Attorney Docket: OGY-02925 treatment with LNPs having separate mRNAs encoding a dCas9-SunTag and an scFv fused to an epigenetic activating domain of a polypeptide (VPR, VP64, VP16, NCOA2, MYBL1, LEUTX, CREB3L1, E2F2, KLF15, UBN1, INO80D, FOXO6, or TP53), and gRNAs designed to target the enhancer, promoter, or enhancer and promoter of Target Gene 2 in HEP3B cells as measured by RT-qPCR.
  • VPR VP64, VP16, NCOA2, MYBL1, LEUTX, CREB3L1, E2F2, KLF15, UBN1, INO80D, FOXO6, or TP53
  • FIGs.3A-3B provide graphs depicting Target Gene 3 mRNA expression following treatment with LNPs having separate mRNAs encoding a dCas9-SunTag and an scFv fused to an epigenetic activating domain of a polypeptide (VPR, VP64, VP16, NCOA2, MYBL1, LEUTX, CREB3L1, E2F2, KLF15, UBN1, PIP2, INO80D, FOXO6, or TP53), and gRNAs designed to target the enhancer, promoter, or enhancer and promoter of Target Gene 3 in HEP3B cells as measured by RT-qPCR.
  • FIGs.4A-4B provide graphs depicting Target Gene 4 mRNA expression following treatment with LNPs having separate mRNAs encoding a dCas9-SunTag and an scFv fused to an epigenetic activating domain of a polypeptide (MYBL1, E2F2, NCOA2, LEUTX, or CREB3L1), and gRNAs designed to target Site 1, Site 2, Site 3, Site 4, and Site 5 of Target Gene 4 in Hepa1-6 cells as measured by RT-qPCR. Control cells were untreated.
  • MYBL1, E2F2, NCOA2, LEUTX, or CREB3L1 polypeptide
  • FIG.5 provides a graph depicting Target Gene 4 protein expression following treatment with LNPs having separate mRNAs encoding a dCas9-SunTag and an scFv fused to an epigenetic activating domain of a polypeptide (MYBL1, or E2F2), and gRNAs designed to target Site 1, Site 2, Site 3, Site 4, Site 5, or a combination thereof of Target Gene 4 in Hepa1-6 cells as measured by ELISA. Control cells were treated with mRNAs encoding a PIP2 polypeptide. Target Gene 4 protein expression was determined on Day 2.
  • FIGs.6A-6D provide graphs depicting Target Gene 5 mRNA expression following treatment with LNPs having: an mRNA encoding a fusion polypeptide of a TAL designed to target Target Gene 5 and effector domain TET2 (TAL01-TET2) (control); mRNA encoding dCas9-SunTag and pooled gRNA targeting Target Gene 5 (control); mRNA encoding dCas9- SunTag, pooled gRNA, and mRNA encoding an scFv fused to an epigenetic activating domain of a polypeptide (NCOA2 or LEUTX), with or without mRNA encoding TAL01- TET2.
  • TAL01-TET2 effector domain
  • NCOA2 or LEUTX epigenetic activating domain of a polypeptide
  • Target Gene 5 mRNA expression was determined by RT-PCR either before (FIG.6A) or after (FIG.6B) cells were sorted for GFP expression. After 15 days, unsorted (FIG.6C) or sorted (FIG.6D) cells were re-transfected and mRNA expression was determined.
  • FIGs.7A-7B provide graphs depicting Target Gene 6 mRNA expression following treatment with LNPs having separate mRNAs encoding a dCas9-SunTag and an scFv fused to an epigenetic activating domain of a polypeptide (VP64, VP16, NCOA2, MYBL1, LEUTX, CREB3L1, E2F2, KLF15, FOXO3, UBN1, INO80D, or FOXO6), and gRNAs designed to target a site in Target Gene 6 in K-562 cells as measured by RT-qPCR. Control cells were untreated.
  • a polypeptide VP64, VP16, NCOA2, MYBL1, LEUTX, CREB3L1, E2F2, KLF15, FOXO3, UBN1, INO80D, or FOXO6
  • FIGs.8A-8B provide graphs depicting Target Gene 7 mRNA expression following treatment with LNPs having separate mRNAs encoding a dCas9-VPR and an scFv fused to an epigenetic activating domain of a polypeptide (NCOA2, MYBL1, LEUTX, CREB3L1, or E2F2), and gRNAs designed to target Site 1 and Site 2 of Target Gene 7 in Hepa1-6 cells as measured by RT-qPCR. Control cells were untreated. mRNA expression was determined on Days 4, 12, and 18.
  • FIGs.9A-9B provide graphs depicting Target Gene 8 mRNA expression following treatment with LNPs having separate mRNAs encoding an scFv fused to an epigenetic activating domain of a NCOA2 or a PA.v1b polypeptide, and gRNAs designed to target Site 1, Site 2, Site 3, and Site 4 of Target Gene 8 in Hepa1-6 cells as measured by RT-qPCR. Control cells were untreated. mRNA expression was determined on Days 3, and 7. mRNA expression was determined using gRNAs targeting Site 1, Site 2, Site 3, and Site 4 of Target Gene 8 either in combination (FIG.9A) or individually (FIG.9B).
  • FIG.10 provides graphs depicting Target Gene 9 mRNA expression following treatment with LNPs having separate mRNAs encoding a dCas9-SunTag and an scFv fused to an epigenetic activating domain of a polypeptide (LEUTX, or NOCA2), and gRNAs designed to target the promoter, enhancer or both regions of Target Gene 9 in Hepa1-6 cells as measured by RT-qPCR. Control cells were untreated. mRNA expression was determined on Day 2.
  • FIG.11A provides a graph depicting Target Gene 4 protein expression in serum extracted from C57BL/J6 mice following treatment with LNPs having separate mRNAs encoding a dCas9-SunTag and an scFv fused to an epigenetic activating domain of a polypeptide (NCOA2, or LEUTX), and a gRNA designed to target Site 1 of Target Gene 4.
  • Target Gene 4 protein levels were measured by ELISA at 0, 3, and 8 days post- administration.
  • Buffer (PBS) containing no LNPs were used as a control.
  • FIG.11B provides a graph depicting normalized serum Target Gene 4 protein levels, expressed as a percentage of Day 0 baseline, in male C57BL/J6 mice treated with PBS, FH12877464.1 Attorney Docket: OGY-02925 NCOA2, or LEUTX LNPs as described in FIG.11A.
  • FIG.11C provides a graph depicting relative Target Gene 4 mRNA expression in liver tissue collected 8 days after treatment with PBS, NCOA2, or LEUTX LNPs as described in FIG.11A. The expression levels were normalized to the housekeeping gene 36b4 and measured using RT-qPCR.
  • FIG.12 provides a graph depicting Target Gene 4 protein expression in serum extracted from C57BL/J6 mice following treatment with LNPs having separate mRNAs encoding a dCas9-SunTag and an scFv fused to an epigenetic activating domain of a polypeptide (NCOA2, TET2-XTEN80-dCas9+NCOA2, PA.v1b, E2F2, MYBL1, KLF15, or PRDM9), and a gRNA designed to target Site 1 of Target Gene 4.
  • Target Gene 4 protein levels were measured by ELISA at -1, 2, and 6 days post-administration. Buffer (PBS) containing no LNPs was used as a control.
  • FIG.13A provides a graph depicting Target Gene 4 protein expression in serum extracted from C57BL/J6 mice following treatment with LNPs having separate mRNAs encoding a dCas9-SunTag and an scFv fused to an epigenetic activating domain of a polypeptide (NCOA2, PA.v1b, E2F2, MYBL1, or KLF15), and a gRNA designed to target Site 1 of Target Gene 4.
  • Target Gene 4 protein levels were measured by ELISA at -1, 2, and 6 days post-administration.
  • Buffer (PBS) containing no LNPs was used as a control and data was normalized as a percentage of Day 0.
  • FIG.13B provides a graph depicting Target Gene 4 protein expression in serum extracted from C57BL/J6 mice following treatment with LNPs having an mRNAs encoding a dCas9-SunTag, an scFv fused to an epigenetic activating domain of a TET2-XTEN80- dCas9+NCOA2 polypeptide, and a gRNA designed to target Site 1 of Target Gene 4.
  • Target Gene 4 protein levels were measured by ELISA at -1, 2, and 6 days post-administration.
  • Buffer (PBS) containing no LNPs was used as a control and data was normalized as a percentage of Day 0.
  • FIG.13C provides a graph depicting Target Gene 4 protein expression in serum extracted from C57BL/J6 mice following treatment with LNPs having an mRNAs encoding a dCas9-SunTag, an scFv fused to an epigenetic activating domain of a PRDM9 polypeptide, and a gRNA designed to target Site 1 of Target Gene 4.
  • Target Gene 4 protein levels were measured by ELISA at -1, 2, and 6 days post-administration.
  • Buffer (PBS) containing no LNPs was used as a control and data was normalized as a percentage of Day 0.
  • FIG.14 provides a graph depicting relative Target Gene 8 mRNA expression in liver tissue from C57BL/J6 mice following treatment with LNPs having separate mRNAs FH12877464.1 Attorney Docket: OGY-02925 encoding a an scFv fused to an epigenetic activating domain of a NCOA2 polypeptide, and a gRNA designed to target Site 4 of Target Gene 8.
  • Target Gene 8 mRNA levels were measured by RT-qPCR at 3, 22, and 36 days after administration of the first dose.
  • Buffer (PBS) containing no LNPs was used as a control.
  • FIG.15 provides a graph depicting relative body weight data C57BL/J6 mice following treatment with LNPs having separate mRNAs encoding an scFv fused to an epigenetic activating domain of a NCOA2 polypeptide, and a gRNA designed to target Site 4 of Target Gene 8. Body weight was measured out to 36-days after administration of the first dose. Buffer (PBS) containing no LNPs was used as a control and data was normalized as a percentage of Day 0.
  • PBS Buffer
  • FIG.16 provides a graph depicting the levels of Target Gene 6 mRNA, following treatment of human K562 cells with MC3 LNP-formulated ZF-NCOA2 or ZF-LEUTX (ZF: engineered zinc finger protein targeting the promoter DNA sequence of Target Gene 6 and fused to a transcriptional co-activator NCOA2 or LEUTX), as measured by RT-qPCR.
  • mRNA expression was normalized based upon ACTB (housekeeper) mRNA levels. Control cells were untreated. mRNA expression was determined on Days 1, 2, 7, and 14.
  • DETAILED DESCRIPTION The present disclosure is based, at least in part, on the discovery of epigenetic activating domains of polypeptides.
  • the present disclosure provides epigenomic controllers (ECs) comprising a DNA-targeting moiety and an epigenetic activating domain encoded by a fragment of a gene selected from MYBL1, DUX4, MYB, CEPBPD, ATF4, NCOA2, TP53, FOXO1, NFE2, E2F2, UBN1, CREB3L1, FOXO3, KLF15, MLXIPL, LEUTX, INO80D, CR3L1, ATF6A, CSN9, FOXO6, CACTIN, C1QBP, KLF4, MYC, NUTM1, VP16, VP64, YLR278C, PIP2 and STB3.
  • ECs epigenomic controllers
  • the present disclosure provides epigenomic controllers (ECs) comprising a DNA- targeting moiety and an epigenetic activating domain encoded by a fragment of a gene selected from MYBL1, DUX4, MYB, CEPBPD, ATF4, NCOA2, TP53, FOXO1, NFE2, E2F2, UBN1, CREB3L1, FOXO3, KLF15, MLXIPL, LEUTX, INO80D, CR3L1, ATF6A, CSN9, FOXO6, CACTIN, C1QBP, KLF4, MYC, NUTM1, VP16, VP64, YLR278C, PIP2, STB3, and PA.v1b.
  • ECs epigenomic controllers
  • expression activator refers to an agent or entity with one or more functionalities that increases expression of a target gene in a cell and FH12877464.1 Attorney Docket: OGY-02925 that specifically binds to a DNA sequence (e.g., a DNA sequence associated with a target gene or a transcription control element operably linked to a target gene).
  • a DNA sequence e.g., a DNA sequence associated with a target gene or a transcription control element operably linked to a target gene.
  • the present disclosure provides an expression activator comprising (i) a DNA-targeting moiety that binds a target sequence; and (ii) an effector domain comprising an epigenetic activating domain.
  • the expression activator comprises a single DNA-targeting moiety.
  • the expression activator comprises more than one DNA-targeting moiety. In some embodiments, the expression activator comprises a single effector domain. In some embodiments, the expression activator comprises one or more effector domains. In some embodiments, the expression activator comprises more than one effector domain. In some embodiments, the expression activator comprises one to four effector domains. In some embodiments, the expression activator comprises two effector domains.
  • the target sequence is a span of nucleotides (e.g., 10-50, 10-40, 10-30, 15-30, 15-25, or 15-20 nucleotides) in or near an insulated genomic domain (IGD) comprising a target gene.
  • IGD insulated genomic domain
  • IGDs are units of genomic space with boundaries defined by factors that mechanistically drive functional insulation between gene transcription activities.
  • IGDs are physical units that serve to parse chromosomes into discrete functional segments.
  • an IGD comprises a DNA loop formed by interactions between two DNA sites bound by homodimerized CTCF and cohesin (see Dowen, et al (2014) Cell 159:374-87). In such an IGD, occupation of each of the DNA sites bound by CTCF and cohesin inhibits DNA-bound components on one chromosomal side of the DNA site from interacting with DNA-bound components on the opposite chromosomal side.
  • the DNA sites occupied by CTCF and cohesin in such DNA loops act as boundaries for the IGD.
  • the formation of such DNA loops facilitates (i) enhancer-promoter interactions in which both the enhancer and promoter are within the loop, (ii) inhibition of enhancer-promoter interactions in which one of those elements is within the loop and the other is outside the loop, or (iii) both (i) and (ii).
  • FH12877464.1 Attorney Docket: OGY-02925
  • the target sequence is in or near a promoter.
  • the target sequence is in or near an enhancer element.
  • the DNA-targeting moiety comprises a polypeptide that binds the target sequence.
  • the DNA-targeting moiety comprises a zinc finger (ZF) domain that binds the target sequence. In some embodiments, the DNA-targeting moiety comprises a transcription activator-like effector (TALE) domain that binds the target sequence. In some embodiments, the DNA-targeting moiety comprises a catalytically inactive site-directed nuclease (e.g., a catalytically inactive Cas nuclease) and a guide sequence, wherein the guide sequence is complementary, or substantially complementary, to the target sequence. In some embodiments, the effector domain induces one or more epigenetic changes. In some embodiments, the effector domain recruits components of the endogenous transcriptional machinery to increase expression of the target gene.
  • ZF zinc finger
  • TALE transcription activator-like effector
  • the effector domain is a polypeptide, that upon binding to a transcriptional control element, recruits one or more activator proteins and/or transcription factors to activate, or substantially activate, gene transcription. In some embodiments, the effector domain promotes recruitment of transcription factors, thereby increasing expression of the target gene.
  • the effector domain comprises an epigenetic modifying moiety (e.g., a moiety for introducing an epigenetic modification in or near the target gene).
  • the effector domain is an enzyme, that upon binding to a transcriptional control element, catalyzes one or more modifications of a genomic region comprising the transcriptional control element, wherein the one or more modifications activates, or substantially activates, gene transcription.
  • the one or more modifications are selected from a DNA modification and a histone modification.
  • the disclosure provides a nucleic acid encoding an expression activator described herein.
  • the nucleic acid is an mRNA.
  • the disclosure provides a recombinant expression vector comprising the nucleic acid.
  • the expression activator , the nucleic acid (e.g., mRNA), or the recombinant expression vector is formulated in a lipid nanoparticle (LNP).
  • LNP lipid nanoparticle
  • the disclosure provides a system comprising two or more expression activators described herein. In some embodiments, the system comprises 2, 3, 4, 5, 6, 7, 8, 9, or 10 expression activators described herein.
  • the system comprises two or more nucleic acids, wherein each nucleic acid encodes an expression activator described herein.
  • the two or more nucleic acids are each mRNAs.
  • the system comprises two or more recombinant expression vectors, FH12877464.1 Attorney Docket: OGY-02925 wherein each recombinant expression vector comprises a nucleic acid encoding an expression activator described herein.
  • the two or more expression activators, the two or more nucleic acids, or the two or more recombinant expression vectors are formulated in the same LNP or in different LNPs.
  • the disclosure provides a nucleic acid encoding two or more expression activators (e.g., 2, 3, 4, 5, 6, 7, 8, 9, or 10 expression activators) described herein.
  • the nucleic acid is an mRNA.
  • the disclosure provides a recombinant expression vector comprising the nucleic acid.
  • the nucleic acid or the recombinant expression vector is formulated in an LNP.
  • the disclosure provides a pharmaceutical composition comprising an expression activator described herein, a nucleic acid described herein, a recombinant expression vector described herein, an LNP described herein, or a system described herein, and a pharmaceutically acceptable carrier.
  • the disclosure provides a method of altering (e.g., increasing) expression of a target gene in a cell, comprising contacting the cell with an expression activator described herein, a nucleic acid described herein, a recombinant expression vector described herein, an LNP described herein, a system described herein, or a pharmaceutical composition described herein.
  • expression of target gene is increased compared to a control cell not contacted with the expression activator, nucleic acid, recombinant expression vector, LNP, system, or pharmaceutical composition.
  • the disclosure provides a method of introducing one or more epigenetic modifications to a region comprising a transcriptional control element of a target gene in a cell, the method comprising contacting the cell with an expression activator described herein, a nucleic acid described herein, a recombinant expression vector described herein, an LNP described herein, a system described herein, or a pharmaceutical composition described herein.
  • the transcriptional control element comprises a promoter of the target gene.
  • the one or more epigenetic modifications comprises DNA demethylation and/or histone modification.
  • an expression activator of the disclosure has a targeting function and an effector function.
  • the targeting function localizes the effector function of the expression activator to a region of the genome.
  • the region of the genome comprises an IGD of a target gene.
  • the region of the genome is in the IGD of the target gene.
  • the effector function comprises introducing one or more epigenetic modifications to the region of the genome.
  • the expression activator comprises a DNA-targeting moiety and an effector domain.
  • the targeting function of the expression activator is mediated by the DNA-targeting moiety.
  • the targeting function is mediated by the DNA-targeting moiety binding to a target sequence in the region of the genome.
  • the effector domain is a transcriptional activator moiety described herein.
  • the DNA-targeting moiety binds to a target sequence in the target gene, whereupon the effector domain of the expression activator functions to introduce one or more epigenetic modifications to a region in the target gene.
  • the DNA-targeting moiety binds to a target sequence in a genomic region comprising the target gene IGD (e.g., the human target gene IGD), whereupon the effector domain of the expression activator functions to introduce one or more epigenetic modifications to a region in or near the target gene IGD (e.g., the human target gene IGD).
  • the DNA-targeting moiety binds to a target sequence in the target gene IGD (e.g., the human target gene IGD), whereupon the effector domain of the expression activator functions to introduce one or more epigenetic modifications to a region in the target gene IGD (e.g., the human target gene IGD).
  • one or more epigenetic modifications is introduced to a transcriptional control element (e.g., promoter or enhancer) of a target gene (e.g., a human target gene), or a portion thereof.
  • the one or more epigenetic modifications results in increased expression of a target gene (e.g., a human target gene), e.g., as compared to a control cell not contacted with the expression activator.
  • Target Sequences In some embodiments, the DNA-targeting moiety binds to a target sequence in a target gene.
  • the DNA-targeting moiety binds to a target sequence in a FH12877464.1 Attorney Docket: OGY-02925 genomic region comprising the IGD of the target gene . In some embodiments, the DNA- targeting moiety binds to a target sequence in the IGD of the target gene. In some embodiments, the DNA-targeting moiety comprises a ZF that binds the target sequence in a genomic region comprising the IGD of the target gene. In some embodiments, the DNA-targeting moiety comprises a ZF that binds the target sequence in the IGD. In some embodiments, the DNA-targeting moiety comprises a TALE that binds the target sequence in a genomic region comprising the IGD of the target gene.
  • the DNA- targeting moiety comprises a TALE that binds the target sequence in the IGD.
  • the DNA-targeting moiety comprises a site-directed nuclease (e.g., a catalytically inactive site-directed nuclease) that binds the target sequence in a genomic region comprising the IGD of the target gene.
  • the DNA-targeting moiety comprises a site-directed nuclease (e.g., a catalytically inactive site-directed nuclease) that binds the target sequence in the IGD.
  • the site-directed nuclease comprises a Cas nuclease described herein (e.g., a catalytically inactive Cas nuclease) and a gRNA comprising a spacer sequence corresponding to the target sequence.
  • the spacer sequence is a sequence that defines the target sequence.
  • the target sequence is present in a double-stranded genomic DNA having one strand comprising the target sequence comprising a protospacer sequence adjacent to a PAM sequence that is referred to as the “PAM strand,” and a second strand that is referred to as the “non-PAM strand” and is complementary to the PAM strand.
  • gRNA spacer sequence and the target sequence are complementary to the non-PAM strand of the genomic DNA molecule.
  • a spacer sequence “corresponding to” a target sequence refers to a guide sequence that binds to the non-PAM strand of the target sequence by Watson-Crick base-pairing, wherein the spacer sequence has sufficient complementarity to the non-PAM strand as to enable targeting of the Cas nuclease to the target sequence in the genomic DNA molecule.
  • the spacer sequence has up to 1, 2, or 3 mismatches relative to the target sequence in the genomic DNA molecule, wherein the spacer sequence has sufficient complementarity to the non-PAM strand as to enable targeting of the Cas nuclease to the target sequence in the genomic DNA molecule.
  • the DNA-targeting moiety binds to a target sequence in a genomic region comprising the IGD, wherein the target sequence is upstream of or in a 5 ⁇ boundary of the IGD. In some embodiments, the target sequence is between a 5 ⁇ and 3 ⁇ boundary of the IGD. In some embodiments, the target sequence is downstream of or in the 3 ⁇ boundary of the IGD.
  • the DNA-targeting moiety binds to a target FH12877464.1 Attorney Docket: OGY-02925 sequence in the IGD, wherein the target sequence is in a region (e.g., a 0.5-2kb region) comprising a transcriptional control element (e.g., a promoter or enhancer).
  • the region comprises a promoter.
  • the target sequence is in a promoter.
  • the region comprises an enhancer.
  • the target sequence is in an enhancer.
  • the target sequence is in or near a CpG island in the IGD.
  • the target is in a region of about 100 bases, about 200 bases, about 300 bases, about 400 bases, about 500 bases, about 600 bases, about 700 bases, about 800 bases, about 900 bases, about 1,000 bases, about 1,100 bases, about 1,200 bases, about 1,300 bases, about 1,400 bases, about 1,500 bases, about 1,600 bases, about 1,700 bases, about 1,800 bases, about 1,900 bases, or about 2,000 bases comprising the CpG island.
  • the target is in a region of about 100 bases, about 200 bases, about 300 bases, about 400 bases, about 500 bases, about 600 bases, about 700 bases, about 800 bases, about 900 bases, or about 1,000 bases comprising the CpG island.
  • the target sequence is not more than about 300 bases, about 400 bases, or about 500 bases upstream or downstream the CpG island. In some embodiments, the target sequence is in the CpG island.
  • the DNA-targeting moiety binds to a target sequence in the IGD, wherein the target sequence is in a region (e.g., a 0.5-2kb region) comprising a transcriptional control element (e.g., a promoter) of the gene. In some embodiments, the target sequence is in a region comprising a promoter of the gene.
  • promoter refers to a genomic region upstream of a transcriptional start sequence (TSS) of a transcript of interest.
  • the promoter may include 50 bp, 100 bp, 150 bp, 200 bp, 250 bp, 300 bp, 400 bp, 500 bp, 600 bp, 700 bp, 800 bp, 900 bp, or 1000 bp upstream of a TSS.
  • the target sequence is in a region comprising an enhancer of the gene.
  • the target sequence is in a coding region of the gene. The length of the target sequence depends on the DNA-targeting moiety used.
  • the DNA-targeting moiety comprises a ZF and the target sequence is about 10 to about 50 nucleotides, about 10 to about 40 nucleotides, about 10 to about 30 nucleotides, about 10 to about 20 nucleotides, or about 15 to about 20 nucleotides. In some embodiments, the DNA-targeting moiety comprises a ZF and the target sequence is about 10, about 11, about 12, about 13, about 14, about 15, about 16, about 17, about 18, about 19, or about 20 nucleotides. In some embodiments, the DNA-targeting moiety comprises a ZF and the target sequence is 15 nucleotides. In some embodiments, the DNA-targeting moiety comprises a ZF and the target sequence is 16 nucleotides.
  • the DNA- FH12877464.1 Attorney Docket: OGY-02925 targeting moiety comprises a ZF and the target sequence is 17 nucleotides. In some embodiments, the DNA-targeting moiety comprises a ZF and the target sequence is 18 nucleotides. In some embodiments, the DNA-targeting moiety comprises a ZF and the target sequence is 19 nucleotides. In some embodiments, the DNA-targeting moiety comprises a ZF and the target sequence is 20 nucleotides.
  • the DNA-targeting moiety comprises a TALE and the target sequence is about 10 to about 50 nucleotides, about 10 to about 40 nucleotides, about 10 to about 30 nucleotides, about 10 to about 20 nucleotides, or about 15 to about 20 nucleotides. In some embodiments, the DNA-targeting moiety comprises a TALE and the target sequence is about 10, about 11, about 12, about 13, about 14, about 15, about 16, about 17, about 18, about 19, or about 20 nucleotides. In some embodiments, the DNA-targeting moiety comprises a TALE and the target sequence is 15 nucleotides. In some embodiments, the DNA-targeting moiety comprises a TALE and the target sequence is 16 nucleotides.
  • the DNA-targeting moiety comprises a TALE and the target sequence is 17 nucleotides. In some embodiments, the DNA-targeting moiety comprises a TALE and the target sequence is 18 nucleotides. In some embodiments, the DNA-targeting moiety comprises a TALE and the target sequence is 19 nucleotides. In some embodiments, the DNA-targeting moiety comprises a TALE and the target sequence is 20 nucleotides.
  • the DNA-targeting moiety comprises a site-directed nuclease (e.g., a catalytically inactive site-directed nuclease) and the target sequence is about 10 to about 50 nucleotides, about 10 to about 40 nucleotides, about 10 to about 30 nucleotides, about 10 to about 20 nucleotides, or about 15 to about 20 nucleotides.
  • the DNA-targeting moiety comprises a site-directed nuclease (e.g., a catalytically inactive site-directed nuclease) and the target sequence is about 10, about 11, about 12, about 13, about 14, about 15, about 16, about 17, about 18, about 19, or about 20 nucleotides.
  • the DNA-targeting moiety comprises a site-directed nuclease (e.g., a catalytically inactive site-directed nuclease) and the target sequence is 15 nucleotides. In some embodiments, the DNA-targeting moiety comprises a site-directed nuclease (e.g., a catalytically inactive site-directed nuclease) and the target sequence is 16 nucleotides. In some embodiments, the DNA-targeting moiety comprises a site-directed nuclease (e.g., a catalytically inactive site-directed nuclease) and the target sequence is 17 nucleotides.
  • the DNA-targeting moiety comprises a site-directed nuclease (e.g., a catalytically inactive site-directed nuclease) and the target sequence is 18 nucleotides. In some embodiments, the DNA-targeting moiety comprises a site-directed nuclease (e.g., a FH12877464.1 Attorney Docket: OGY-02925 catalytically inactive site-directed nuclease) and the target sequence is 19 nucleotides. In some embodiments, the DNA-targeting moiety comprises a site-directed nuclease (e.g., a catalytically inactive site-directed nuclease) and the target sequence is 20 nucleotides.
  • a site-directed nuclease e.g., a catalytically inactive site-directed nuclease
  • the target sequence is 20 nucleotides.
  • the target sequence is 10-50 nucleotides (e.g., 10-40, 10-30, 15-30, 15-25, or 15-20 nucleotides) in a genomic region comprising an IGD. In some embodiments, the target sequence is about 10 to about 50 nucleotides, about 10 to about 40 nucleotides, about 10 to about 30 nucleotides, about 10 to about 20 nucleotides, or about 15 to about 20 nucleotides in a genomic region comprising the IGD. In some embodiments, the target sequence is about 10, about 11, about 12, about 13, about 14, about 15, about 16, about 17, about 18, about 19, or about 20 nucleotides in a genomic region comprising the IGD.
  • the target sequence is 10-50 nucleotides (e.g., 10-40, 10-30, 15-30, 15-25, or 15-20 nucleotides) in the IGD. In some embodiments, the target sequence is about 10 to about 50 nucleotides, about 10 to about 40 nucleotides, about 10 to about 30 nucleotides, about 10 to about 20 nucleotides, or about 15 to about 20 nucleotides in the IGD. In some embodiments, the target sequence is about 10, about 11, about 12, about 13, about 14, about 15, about 16, about 17, about 18, about 19, or about 20 nucleotides in the IGD.
  • the target sequence is 10-50 nucleotides (e.g., 10-40, 10-30, 15-30, 15-25, or 15-20 nucleotides) in a region (e.g., a 0.1-2kb region) of the IGD, wherein the region comprises a transcriptional control element (e.g., a promoter or enhancer).
  • a transcriptional control element e.g., a promoter or enhancer
  • the target sequence is about 10 to about 50 nucleotides, about 10 to about 40 nucleotides, about 10 to about 30 nucleotides, about 10 to about 20 nucleotides, or about 15 to about 20 nucleotides in a region (e.g., a 0.1-2 kb region) of the IGD, wherein the region comprises a transcriptional control element (e.g., a promoter or enhancer).
  • a transcriptional control element e.g., a promoter or enhancer
  • the target sequence is about 10, about 11, about 12, about 13, about 14, about 15, about 16, about 17, about 18, about 19, or about 20 nucleotides in a region (e.g., a 0.1-2 kb region) of the IGD, wherein the region comprises a transcriptional control element (e.g., a promoter or enhancer).
  • the target sequence is 10-50 nucleotides (e.g., 10-40, 10-30, 15-30, 15-25, or 15-20 nucleotides) in a transcriptional control element (e.g., a promoter or enhancer) in the IGD.
  • the target sequence is about 10 to about 50 nucleotides, about 10 to about 40 nucleotides, about 10 to about 30 nucleotides, about 10 to about 20 nucleotides, or about 15 to about 20 nucleotides in a transcriptional control element (e.g., a promoter or enhancer) in the IGD.
  • the target sequence is about 10, about 11, about 12, about 13, about 14, about 15, about 16, about 17, about 18, about 19, FH12877464.1 Attorney Docket: OGY-02925 or about 20 nucleotides in a transcriptional control element (e.g., a promoter or enhancer) in the IGD.
  • the target sequence is 10-50 nucleotides (e.g., 10-40, 10-30, 15-30, 15-25, or 15-20 nucleotides) in a region (e.g., a 0.1-2 kb region) of the IGD, wherein the region comprises a promoter. In some embodiments, the target sequence is within or overlapping the promoter. In some embodiments, the target sequence is about 10 to about 50 nucleotides, about 10 to about 40 nucleotides, about 10 to about 30 nucleotides, about 10 to about 20 nucleotides, or about 15 to about 20 nucleotides in a region (e.g., a 0.1-2 kb region) of the IGD, wherein the region comprises a promoter.
  • the target sequence is 10-50 nucleotides (e.g., 10-40, 10-30, 15-30, 15-25, or 15-20 nucleotides) in a region (e.g., a 0.1-2 kb region) of the IGD, wherein the region comprises
  • the target sequence is about 10, about 11, about 12, about 13, about 14, about 15, about 16, about 17, about 18, about 19, or about 20 nucleotides in a region (e.g., a 0.1-2 kb region) of the IGD, wherein the region comprises a promoter.
  • the target sequence is 10-50 nucleotides (e.g., 10-40, 10-30, 15-30, 15-25, or 15-20 nucleotides) in a promoter in the IGD.
  • the target sequence is about 10 to about 50 nucleotides, about 10 to about 40 nucleotides, about 10 to about 30 nucleotides, about 10 to about 20 nucleotides, or about 15 to about 20 nucleotides in a promoter in the IGD. In some embodiments, the target sequence is about 10, about 11, about 12, about 13, about 14, about 15, about 16, about 17, about 18, about 19, or about 20 nucleotides in a promoter in the IGD.
  • the target sequence is 10-50 nucleotides (e.g., 10-40, 10-30, 15-30, 15-25, or 15-20 nucleotides) in a region (e.g., a 0.1-2kb region) of the IGD, wherein the region comprises an enhancer. In some embodiments, the target sequence is within or overlapping the enhancer. In some embodiments, the target sequence is about 10 to about 50 nucleotides, about 10 to about 40 nucleotides, about 10 to about 30 nucleotides, about 10 to about 20 nucleotides, or about 15 to about 20 nucleotides in a region (e.g., a 0.1-2kb region) of the IGD, wherein the region comprises an enhancer.
  • the target sequence is 10-50 nucleotides (e.g., 10-40, 10-30, 15-30, 15-25, or 15-20 nucleotides) in a region (e.g., a 0.1-2kb region) of the IGD, wherein the region comprises an enhancer.
  • the target sequence is about 10, about 11, about 12, about 13, about 14, about 15, about 16, about 17, about 18, about 19, or about 20 nucleotides in a region (e.g., a 0.1-2kb region) of the IGD, wherein the region comprises an enhancer.
  • the target sequence is 10-50 nucleotides (e.g., 10-40, 10-30, 15-30, 15-25, or 15-20 nucleotides) in an enhancer in the IGD.
  • the target sequence is about 10 to about 50 nucleotides, about 10 to about 40 nucleotides, about 10 to about 30 nucleotides, about 10 to about 20 nucleotides, or about 15 to about 20 nucleotides in an enhancer in the IGD. In some embodiments, the target sequence is about 10, FH12877464.1 Attorney Docket: OGY-02925 about 11, about 12, about 13, about 14, about 15, about 16, about 17, about 18, about 19, or about 20 nucleotides in an enhancer in the IGD.
  • the target sequence is 10-50 nucleotides (e.g., 10-40, 10-30, 15-30, 15-25, or 15-20 nucleotides) in a region (e.g., a 0.1-2kb region) of the IGD, wherein the region comprises a CTCF binding site (e.g., a CTCF binding site at a boundary of the IGD or a CTCF binding site in the IGD).
  • the target sequence is within or overlapping the CTCF binding site.
  • the target sequence is about 10 to about 50 nucleotides, about 10 to about 40 nucleotides, about 10 to about 30 nucleotides, about 10 to about 20 nucleotides, or about 15 to about 20 nucleotides in a region (e.g., a 0.1- 2kb region) of the IGD, wherein the region comprises a CTCF binding site (e.g., a CTCF binding site at a boundary of the IGD or a CTCF binding site in the IGD).
  • a CTCF binding site e.g., a CTCF binding site at a boundary of the IGD or a CTCF binding site in the IGD.
  • the target sequence is about 10, about 11, about 12, about 13, about 14, about 15, about 16, about 17, about 18, about 19, or about 20 nucleotides in a region (e.g., a 0.1-2kb region) of the IGD, wherein the region comprises a CTCF binding site (e.g., a CTCF binding site at a boundary of the IGD or a CTCF binding site in the IGD).
  • the target sequence is 10-50 nucleotides (e.g., 10-40, 10-30, 15-30, 15-25, or 15-20 nucleotides) in a region (e.g., a 0.1-2 kb region) of the IGD, wherein the region comprises an enhancer.
  • the target sequence is within or overlapping the enhancer. In some embodiments, the target sequence is about 10 to about 50 nucleotides, about 10 to about 40 nucleotides, about 10 to about 30 nucleotides, about 10 to about 20 nucleotides, or about 15 to about 20 nucleotides in a region (e.g., a 0.1-2 kb region) of the IGD, wherein the region comprises an enhancer. In some embodiments, the target sequence is about 10, about 11, about 12, about 13, about 14, about 15, about 16, about 17, about 18, about 19, or about 20 nucleotides in a region (e.g., a 0.1-2 kb region) of the IGD, wherein the region comprises an enhancer.
  • the target sequence is 10-50 nucleotides (e.g., 10-40, 10-30, 15-30, 15-25, or 15-20 nucleotides) in a region (e.g., a 0.1-2 kb region) of the IGD, wherein the region comprises a CpG island. In some embodiments, the target sequence is within or overlapping the CpG island.
  • the target sequence is about 10 to about 50 nucleotides, about 10 to about 40 nucleotides, about 10 to about 30 nucleotides, about 10 to about 20 nucleotides, or about 15 to about 20 nucleotides in a region (e.g., a 0.1-2kb region) of the of the IGD, wherein the region comprises a CpG island.
  • a region e.g., a 0.1-2kb region
  • the target sequence is about 10, about 11, about 12, about 13, about 14, about 15, about 16, FH12877464.1 Attorney Docket: OGY-02925 about 17, about 18, about 19, or about 20 nucleotides in a region (e.g., a 0.1-2 kb region) of the of the IGD, wherein the region comprises a CpG island.
  • the target sequence is 10-50 nucleotides (e.g., 10-40, 10-30, 15-30, 15-25, or 15-20 nucleotides) in an enhancer.
  • the target sequence is about 10 to about 50 nucleotides, about 10 to about 40 nucleotides, about 10 to about 30 nucleotides, about 10 to about 20 nucleotides, or about 15 to about 20 nucleotides in an enhancer. In some embodiments, the target sequence is about 10, about 11, about 12, about 13, about 14, about 15, about 16, about 17, about 18, about 19, or about 20 nucleotides in an enhancer. In some embodiments, the target sequence is 10-50 nucleotides (e.g., 10-40, 10-30, 15-30, 15-25, or 15-20 nucleotides) in a region (e.g., a 0.1-2 kb region) of the IGD, wherein the region comprises a promoter.
  • a region e.g., a 0.1-2 kb region
  • the target sequence is within or overlapping a target gene promoter.
  • the target sequence is about 10 to about 50 nucleotides, about 10 to about 40 nucleotides, about 10 to about 30 nucleotides, about 10 to about 20 nucleotides, or about 15 to about 20 nucleotides in a region (e.g., a 0.1-2 kb region) of the IGD, wherein the region comprises a promoter.
  • the target sequence is about 10, about 11, about 12, about 13, about 14, about 15, about 16, about 17, about 18, about 19, or about 20 nucleotides in a region (e.g., a 0.1-2 kb region) of the IGD, wherein the region comprises a promoter.
  • the target sequence is 10-50 nucleotides (e.g., 10-40, 10-30, 15-30, 15-25, or 15-20 nucleotides) in a promoter. In some embodiments, the target sequence is about 10 to about 50 nucleotides, about 10 to about 40 nucleotides, about 10 to about 30 nucleotides, about 10 to about 20 nucleotides, or about 15 to about 20 nucleotides in a promoter. In some embodiments, the target sequence is about 10, about 11, about 12, about 13, about 14, about 15, about 16, about 17, about 18, about 19, or about 20 nucleotides in a promote).
  • the target sequence is 10-50 nucleotides (e.g., 10-40, 10-30, 15-30, 15-25, or 15-20 nucleotides) in a region (e.g., a 0.1-2 kb region) of the target gene. In some embodiments, the target sequence is about 10 to about 50 nucleotides, about 10 to about 40 nucleotides, about 10 to about 30 nucleotides, about 10 to about 20 nucleotides, or about 15 to about 20 nucleotides in a region (e.g., a 0.1-2 kb region) of the target gene.
  • the target sequence is about 10, about 11, about 12, about 13, about 14, about 15, about 16, about 17, about 18, about 19, or about 20 nucleotides in a region (e.g., a 0.1-2 kb region) of the target gene.
  • FH12877464.1 Attorney Docket: OGY-02925
  • the target sequence is 10-50 nucleotides (e.g., 10-40, 10-30, 15-30, 15-25, or 15-20 nucleotides).
  • the target sequence is about 10- 50 nucleotides (e.g., 10-40, 10-30, 15-30, 15-25, or 15-20 nucleotides) within a region of about 200 or fewer nucleotides.
  • the target sequence is about 10 to about 50 nucleotides, about 10 to about 40 nucleotides, about 10 to about 30 nucleotides, about 10 to about 20 nucleotides, or about 15 to about 20 nucleotides within a region of about 200 or fewer nucleotides. In some embodiments, the target sequence is about 10, about 11, about 12, about 13, about 14, about 15, about 16, about 17, about 18, about 19, or about 20 nucleotides within a region of about 200 or fewer nucleotides. In some embodiments, the region comprises 10, 11, 12, 13, 14, 15, 16, 17, or 18 contiguous nucleotides of the sequence.
  • the region is about 175, about 150, about 125, about 100, about 90, about 80, about 70, about 60, about 50, about 45, about 40, about 35, about 30, about 25, or about 20 nucleotides.
  • the target sequence is 15, 16, 17, 18, 19, or 20 nucleotides.
  • the DNA-targeting moiety binds to the target sequence with submicromolar or nanomolar binding affinity (KD). Binding affinity is typically measured and reported by the equilibrium dissociation constant (KD), which is used to evaluated and rank strengths of bimolecular interactions.
  • KD refers to the equilibrium dissociation constant of a binding reaction between an DNA-targeting moiety and a target sequence.
  • the value of KD is a numeric representation of the ratio of the DNA- targeting moiety off-rate constant (kd) to the on-rate constant (ka).
  • the value of KD is inversely related to the binding affinity of the DNA-targeting moiety and target sequence. The smaller the KD value the greater the affinity.
  • kd or “kd” (alternatively “koff” or “koff”) is intended to refer to the off- rate constant for the dissociation of the DNA-targeting moiety from a complex of the DNA-targeting moiety and the target sequence.
  • kd is a numeric representation of the fraction of complexes that decay or dissociate per second, and is expressed in units sec -1 .
  • ka or “ka” (alternatively “kon” or “kon”) is intended to refer to the on-rate constant for the association of the DNA-targeting moiety and the target sequence.
  • the value of ka is a numeric representation of the number of DNA-targeting moiety/target DNA complexes formed per second in a 1 molar (1M) solution of the DNA-targeting moiety and the target DNA, and is expressed in units M -1 sec -1 .
  • Methods to measure binding affinity (KD) of a DNA-targeting moiety to the target sequence include, but are not limited to, DNA electrophoretic mobility shift assay (EMSA) and surface plasmon resonance.
  • ESA DNA electrophoretic mobility shift assay
  • FH12877464.1 Attorney Docket: OGY-02925
  • the DNA-targeting moiety e.g., TALE, ZF, dCas9/gRNA
  • the DNA-targeting moiety binds to the target sequence with an affinity (KD) that is no greater than about 950, 900, 850, 800, 750, 700, 650, 600, 500, 500, 450, 400, 350, 300, 250, 200, 250, 200, 175, 150, 125, 110, 100, 90, 80, 70, 60, 50, 40, 30, 20, 10, 5, 4, 3, 2, or 1 nM.
  • KD affinity
  • the DNA-targeting moiety binds to the target sequence with an affinity (KD) of about 1 nM to about 100 nM, about 10 nM to about 500 nM, about 100 nM to about 1 ⁇ M, about 500 nM to about 1 ⁇ M, about 500 nM to about 2 ⁇ M, about 1 ⁇ M to about 2 ⁇ M, about 1 ⁇ M to about 3 ⁇ M, about 1 ⁇ M to about 4 ⁇ M, or about 1 ⁇ M to about 5 ⁇ M.
  • KD affinity
  • the DNA-targeting moiety binds to the target sequence with an affinity (KD) of about 1 nM to about 10 nM, about 1 nM to about 20 nM, about 1 nM to about 30 nM, about 1 nM to about 40 nM, about 1 nM to about 50 nM, about 10 nM to about 50 nM, about 10 nM to about 100 nM, about 10 nM to about 200 nM, about 50 nM to about 200 nM, about 50 nM to about 300 nM, about 50 nM to about 400 nM, or about 50 nM to about 50 nM.
  • KD affinity
  • DNA-targeting moiety The present disclosure provides, e.g., expression activators comprising a DNA- targeting moiety that specifically targets, e.g., binds, a genomic sequence element (e.g., a promoter, a TSS, or an anchor sequence) in, proximal to, and/or operably linked to a target gene.
  • the DNA-targeting moiety specifically binds to a DNA sequence, e.g., a DNA sequence associated with a target gene. Any molecule or compound that specifically binds a DNA sequence may be used as a DNA-targeting moiety.
  • the DNA-targeting moiety targets, e.g., binds, a component of a genomic complex.
  • the DNA-targeting moiety targets, e.g., binds, a transcriptional control sequence (e.g., a promoter or enhancer) operably linked to the target gene.
  • the DNA-targeting moiety targets, e.g., binds, a target gene or a part of a target gene.
  • the target of a DNA-targeting moiety may be referred to as its targeted component.
  • a targeted component may be any genomic sequence element operably linked to a target gene, or the target gene itself, including but not limited to a promoter, enhancer, anchor sequence, exon, intron, UTR encoding sequence, a splice site, or a transcription start site.
  • the DNA-targeting moiety binds specifically to one or more FH12877464.1 Attorney Docket: OGY-02925 target anchor sequences (e.g., within a cell) and not to non-targeted anchor sequences (e.g., within the same cell).
  • the DNA-targeting moiety comprises a CRISPR/Cas domain (e.g., a catalytically inactive CRISPR/Cas domain), a TAL effector domain, a Zn finger domain, a peptide nucleic acid (PNA), or a nucleic acid molecule.
  • an expression activator of the disclosure comprises one DNA- targeting moiety.
  • the expression activator comprises a plurality of DNA targeting moieties, wherein each DNA-targeting moiety does not detectably bind, e.g., does not bind, to another DNA-targeting moiety.
  • the DNA-targeting moiety binds to its target sequence with a KD of less than or equal to 500, 450, 400, 350, 300, 250, 200, 150, 100, 50, 40, 30, 20, 10, 9, 8, 7, 6, 5, 4, 3, 2, 1, 0.9, 0.8, 0.7, 0.6, 0.5, 0.4, 0.3, 0.2, 0.1, 0.09, 0.08, 0.07, 0.06, 0.05, 0.04, 0.03, 0.02, 0.01, 0.005, 0.002, or 0.001 nM (and optionally, a KD of at least 50, 40, 30, 20, 10, 9, 8, 7, 6, 5, 4, 3, 2, 1, 0.9, 0.8, 0.7, 0.6, 0.5, 0.4, 0.3, 0.2, 0.1, 0.09, 0.08, 0.07, 0.06, 0.05, 0.04,
  • the DNA-targeting moiety binds to its target sequence with a KD of 0.001 nM to 500 nM, e.g., 0.1 nM to 5 nM, e.g., about 0.5 nM.
  • a DNA-targeting moiety binds to a non-target sequence with a KD of at least 500, 600, 700, 800, 900, 1000, 2000, 5000, 10,000, or 100,000 nM (and optionally, does not appreciably bind to a non-target sequence).
  • the DNA-targeting moiety does not substantially bind to a non-target sequence.
  • the DNA-targeting moiety comprises a CRISPR/Cas domain.
  • a CRISPR/Cas protein can comprise a CRISPR/Cas effector and optionally one or more other domains.
  • a CRISPR/Cas domain typically has structural and/or functional similarity to a protein involved in the clustered regulatory interspaced short palindromic repeat (CRISPR) system, e.g., a Cas protein.
  • the CRISPR/Cas domain optionally comprises a guide RNA, e.g., single guide RNA (sgRNA).
  • the gRNA comprised by the CRISPR/Cas domain is noncovalently bound by the CRISPR/Cas domain.
  • CRISPR systems are adaptive defense systems originally discovered in bacteria and archaea.
  • CRISPR systems use RNA-guided nucleases termed CRISPR-associated or “Cas” endonucleases (e.g., Cas9 or Cpfl) to cleave foreign DNA.
  • CRISPR-associated or Cas endonucleases e.g., Cas9 or Cpfl
  • an endonuclease is directed to a target nucleotide sequence (e.
  • CRISPR systems use a single Cas endonuclease (rather than multiple Cas proteins).
  • One class II CRISPR system includes a type II Cas endonuclease such as Cas9, a CRISPR RNA (“crRNA”), and a trans-activating crRNA (“tracrRNA”).
  • the crRNA contains a “guide RNA”, typically about 20-nucleotide RNA sequence that corresponds to a target DNA sequence.
  • crRNA also contains a region that binds to the tracrRNA to form a partially double-stranded structure which is cleaved by Rnase III, resulting in a crRNA/tracrRNA hybrid.
  • a crRNA/tracrRNA hybrid then directs Cas9 endonuclease to recognize and cleave a target DNA sequence.
  • a target DNA sequence must generally be adjacent to a “protospacer adjacent motif’ (“PAM”) that is specific for a given Cas endonuclease; however, PAM sequences appear throughout a given genome.
  • PAM protospacer adjacent motif
  • CRISPR endonucleases identified from various prokaryotic species have unique PAM sequence requirements; examples of PAM sequences include 5’-NGG (Streptococcus pyogenes), 5’- NNAGAA (Streptococcus thermophilus CRISPR1), 5’-NGGNG (Streptococcus thermophilus CRISPR3), and 5’-NNNGATT (Neisseria meningiditis).
  • Some endonucleases e.g., Cas9 endonucleases, are associated with G-rich PAM sites, e.
  • Another class II CRISPR system includes the type V endonuclease Cpfl , which is smaller than Cas9; examples include AsCpfl (from Acidaminococcus sp.) and LbCpfl (from Lachnospiraceae sp.).
  • Cpfl -associated CRISPR arrays are processed into mature crRNAs without the requirement of a tracrRNA; in other words, a Cpfl system requires only Cpfl nuclease and a crRNA to cleave a target DNA sequence.
  • Cpfl endonucleases are associated with T-rich PAM sites, e.g., 5’-TTN. Cpfl can also recognize a 5’-CTA PAM motif.
  • Cpfl cleaves a target DNA by introducing an offset or staggered double-strand break with a 4- or 5-nucleotide 5’ overhang, for example, cleaving a target DNA with a 5-nucleotide offset or staggered cut located 18 nucleotides downstream from (3 ⁇ from) from a PAM site on the coding strand and 23 nucleotides downstream from the PAM site on the complimentary strand; the 5-nucleotide overhang that results from such offset cleavage allows more precise genome editing by DNA insertion by homologous recombination than by insertion at blunt- end cleaved DNA.
  • Cas protein e.g., a Cas9 protein
  • a Cas protein may be from any of a variety of prokaryotic species.
  • a particular Cas protein e.g., a particular Cas9 protein
  • a DNA-targeting moiety includes a sequence targeting polypeptide, such as a Cas protein, e.g., Cas9.
  • a Cas protein e.g., a Cas9 protein
  • a Cas protein may be obtained from a bacteria or archaea or synthesized using known methods.
  • a Cas protein may be from a gram-positive bacteria or a gram-negative bacteria.
  • a Cas protein may be from a Streptococcus (e.g., an S.
  • pyogenes or an S. thermophilus
  • Francisella e.g., an F. novicida
  • Staphylococcus e.g., an S. aureus
  • Acidaminococcus e.g., an Acidaminococcus sp. BV3L6
  • Neisseria e.g., an N. meningitidis
  • Cryptococcus a Corynebacterium, a Haemophilus, a Eubacterium, a Pasteurella, a Prevotella, a Veillonella, or a Marinobacter.
  • a Cas protein requires a protospacer adjacent motif (PAM) to be present in or adjacent to a target DNA sequence for the Cas protein to bind and/or function.
  • the PAM is or comprises, from 5 ⁇ to 3 ⁇ , NGG, YG, NNGRRT, NNNRRT, NGA, TYCV, TATV, NTTN, or NNNGATT, where N stands for any nucleotide, Y stands for C or T, R stands for A or G, and V stands for A or C or G.
  • a Cas protein is a protein listed in Table 1.
  • a Cas protein comprises one or more mutations altering its PAM.
  • a Cas protein comprises E1369R, E1449H, and R1556A mutations or analogous substitutions to the amino acids corresponding to said positions. In some embodiments, a Cas protein comprises E782K, N968K, and R1015H mutations or analogous substitutions to the amino acids corresponding to said positions. In some embodiments, a Cas protein comprises DI 135V, R1335Q, and T1337R mutations or analogous substitutions to the amino acids corresponding to said positions. In some embodiments, a Cas protein comprises S542R and K607R mutations or analogous substitutions to the amino acids corresponding to said positions.
  • a Cas protein comprises S542R, K548V, and N552R mutations or analogous substitutions to the amino acids corresponding to said positions.
  • Table 1 Exemplary Cas Proteins of the Disclosure FH12877464.1 Attorney Docket: OGY-02925
  • the Cas protein is modified to deactivate the nuclease, e.g., nuclease-deficient Cas.
  • the Cas protein is a Cas9 protein.
  • a number of CRISPR endonucleases having modified functionalities are available, for example: a “nickase” version of Cas9 generates only a single-strand break; a catalytically inactive Cas9 (“dCas9”) does not cut target DNA.
  • dCas binding to a DNA sequence may interfere with transcription at that site by steric hindrance.
  • a DNA-targeting moiety is or comprises a catalytically inactive Cas, e.g., dCas. Many catalytically inactive Cas proteins are known in the art.
  • dCas9 comprises mutations in each endonuclease domain of the Cas protein, e.g., D10A and H840A mutations.
  • a catalytically inactive Cas9 protein e.g., dCas9, comprises a D11A mutation or an analogous substitution to the amino acid corresponding to said position.
  • FH12877464.1 Attorney Docket: OGY-02925
  • a catalytically inactive Cas9 protein e.g., dCas9, comprises a H969A mutation or an analogous substitution to the amino acid corresponding to said position.
  • a catalytically inactive Cas9 protein e.g., dCas9
  • a catalytically inactive Cas9 protein comprises a N995A mutation or an analogous substitution to the amino acid corresponding to said position.
  • a catalytically inactive Cas9 protein e.g., dCas9
  • a catalytically inactive Cas9 protein e.g., dCas9, comprises a D10A mutation or an analogous substitution to the amino acid corresponding to said position.
  • a catalytically inactive Cas9 protein e.g., dCas9
  • a catalytically inactive Cas9 protein comprises a H557A mutation or an analogous substitution to the amino acid corresponding to said position.
  • a catalytically inactive Cas9 protein e.g., dCas9
  • a catalytically inactive Cas9 protein e.g., dCas9, comprises a D839A mutation or an analogous substitution to the amino acid corresponding to said position.
  • a catalytically inactive Cas9 protein e.g., dCas9
  • a catalytically inactive Cas9 protein comprises a H840A mutation or an analogous substitution to the amino acid corresponding to said position.
  • a catalytically inactive Cas9 protein e.g., dCas9
  • a catalytically inactive Cas9 protein, e.g., dCas9 comprises D10A, D839A, H840A, and N863A mutations or analogous substitutions to the amino acids corresponding to said positions.
  • a catalytically inactive Cas9 protein e.g., dCas9
  • a catalytically inactive Cas9 protein e.g., dCas9
  • a catalytically inactive Cas9 protein e.g., dCas9, comprises a E1006A mutation or an analogous substitution to the amino acid corresponding to said position.
  • a catalytically inactive Cas9 protein e.g., dCas9
  • a catalytically inactive Cas9 protein e.g., dCas9
  • a catalytically inactive Cas9 protein e.g., dCas9
  • a catalytically inactive Cas9 protein e.g., dCas9
  • a catalytically inactive Cas9 protein comprises a D587A mutation or an analogous substitution to the amino acid corresponding to said position.
  • a catalytically inactive Cas9 protein e.g., dCas9
  • a catalytically inactive Cas9 protein, e.g., dCas9 comprises an N611A mutation or an analogous substitution to the amino acid corresponding to said position.
  • a catalytically inactive Cas9 protein comprises D16A, D587A, H588A, and N611A mutations or analogous substitutions to the amino acids corresponding to said positions.
  • the disclosure is directed to an expression activator or a polypeptide comprising one or more (e.g., one) DNA-targeting moiety and one or more effector domain, wherein the one or more DNA-targeting moiety is or comprises a CRISPR/Cas domain comprising a Cas protein, e.g., catalytically inactive Cas9 protein, e.g., dCas9, or a functional variant or fragment thereof.
  • dCas9 comprises an amino acid sequence of SEQ ID NO:108. In some embodiments, the dCas9 is encoded by a nucleic acid sequence of SEQ ID NO:109.
  • a DNA-targeting moiety comprises a Cas domain comprising or linked (e.g., covalently linked) to a gRNA.
  • a gRNA is a short synthetic RNA composed of a “scaffold” sequence necessary for Cas-protein binding and a user-defined about 20 nucleotide targeting sequence for a genomic target.
  • guide RNA sequences are generally designed to have a length of between 17-24 nucleotides (e.g., 19, 20, or 21 nucleotides) and be complementary to the targeted nucleic acid sequence.
  • Custom gRNA generators and algorithms are available commercially for use in the design of effective guide RNAs.
  • Gene editing has also been achieved using a chimeric “single guide RNA” (“sgRNA”), an engineered (synthetic) single RNA molecule that mimics a naturally occurring crRNA-tracrRNA complex and contains both a tracrRNA (for binding the nuclease) and at least one crRNA (to guide the nuclease to the sequence targeted for editing).
  • sgRNA single guide RNA
  • a gRNA comprises a nucleic acid sequence that is complementary to a target sequence described herein.
  • a gRNA comprises a nucleic acid sequence that is at least 90, 95, 99, or 100% complementary to a target sequence described herein.
  • a gRNA for use with a DNA- targeting moiety that comprises a Cas molecule is an sgRNA.
  • a gRNA is used with a dCas9 fused to one or more peptides (e.g., GCN4 peptides) and an expression activator comprising an antibody or antibody fragment specific for the one or more peptides, and operably linked to an effector domain.
  • a DNA-targeting moiety is or comprises a TAL effector (also sometimes referred to herein as a “TALE”) domain.
  • a TAL effector domain e.g., a TAL effector domain that specifically binds a DNA sequence, comprises a plurality of TAL effector repeats or fragments thereof, and optionally one or more additional portions of naturally occurring TAL effector repeats (e.g., N- and/or C-terminal of the plurality of TAL effector domains) wherein each TAL effector repeat recognizes a nucleotide.
  • a TAL effector protein can comprise a TAL effector domain and optionally one or more other domains.
  • Many TAL effector domains are known to those of skill in the art and are commercially available, e.g., from Thermo Fisher Scientific.
  • TALEs are natural effector proteins secreted by numerous species of bacterial pathogens including the plant pathogen Xanthomonas which modulates gene expression in host plants and facilitates bacterial colonization and survival.
  • the specific binding of TAL effectors is based on a central repeat domain of tandemly arranged nearly identical repeats of typically 33 or 34 amino acids (the repeat variable di-residues, RVD domain).
  • Members of the TAL effectors family differ mainly in the number and order of their repeats. The number of repeats ranges from 1.5 to 33.5 repeats and the C-terminal repeat is usually shorter in length (e.g., about 20 amino acids) and is generally referred to as a “half- repeat”.
  • Each repeat of the TAL effector features a one-repeat-to-one-base-pair correlation with different repeat types exhibiting different base-pair specificity (one repeat recognizes one base-pair on the target gene sequence).
  • the smaller the number of repeats the weaker the protein-DNA interactions.
  • a number of 6.5 repeats has been shown to be sufficient to activate transcription of a reporter gene (Scholze et al., 2010).
  • RVD repeat variable di-residues
  • TAL effectors from Xanthomonas include, Hax2, Hax3, Hax4, AvrXa7, AvrXa10 and AvrBs3.
  • the TAL effector repeat of the TAL effector domain of the present disclosure may be derived from a TAL effector from any bacterial species (e.g., Xanthomonas species such as the African strain of Xanthomonas oryzae pv. Oryzae (Yu et al.2011), Xanthomonas campestris pv.
  • the TAL effector domain in accordance with the present disclosure comprises an RVD domain as well as flanking sequence(s) (sequences on the N-terminal and/or C-terminal side of the RVD domain) also from the naturally occurring TAL effector. In some embodiments, it may comprise more or fewer repeats than the RVD of the naturally occurring TAL effector domain.
  • the TAL effector domain of the present disclosure is designed to target a given DNA sequence based on the above code and others known in the art.
  • the number of TAL effector repeats are selected based on the desired DNA target sequence. For example, TAL effector repeats may be removed or added in order to suit a specific target sequence.
  • the TAL effector domain of the present disclosure comprises between 6.5 and 33.5 TAL effector repeats.
  • TAL effector domain of the present disclosure comprises between 8 and 33.5 FH12877464.1 Attorney Docket: OGY-02925 TAL effector repeats, e.g., between 10 and 25 TAL effector repeats, e.g., between 10 and 14 TAL effector repeats.
  • the TAL effector domain comprises TAL effector repeats that correspond to a perfect match to the DNA target sequence.
  • a mismatch between a repeat and a target base-pair on the DNA target sequence is permitted as along as it allows for the function of the expression activation system, e.g., the expression activator comprising the TAL effector domain.
  • TALE binding is inversely correlated with the number of mismatches.
  • the TAL effector domain of an expression activator of the present disclosure comprises no more than 7 mismatches, 6 mismatches, 5 mismatches, 4 mismatches, 3 mismatches, 2 mismatches, or 1 mismatch, and optionally no mismatch, with the target DNA sequence.
  • the binding affinity is thought to depend on the sum of matching repeat-DNA combinations.
  • TAL effector domains having 25 TAL effector repeats or more may be able to tolerate up to 7 mismatches.
  • the TAL effector domain of the present disclosure may comprise additional sequences derived from a naturally occurring TAL effector.
  • the length of the C-terminal and/or N-terminal sequence(s) included on each side of the TAL effector repeat portion of the TAL effector domain can vary and be selected by one skilled in the art, for example based on the studies of Zhang et al. (2011). Zhang et al., have characterized a number of C-terminal and N-terminal truncation mutants in Hax3 derived TAL-effector based proteins and have identified key elements, which contribute to optimal binding to the target sequence and thus activation of transcription. Generally, it was found that transcriptional activity is inversely correlated with the length of the N-terminus. Regarding the C-terminus, an important element for DNA binding residues within the first 68 amino acids of the Hax 3 sequence was identified.
  • a TAL effector domain of the present disclosure comprises 1) one or more TAL effector repeats derived from a naturally occurring TAL effector; 2) at least 70, 80, 90, 100, 110, 120, 130, 140, 150, 170, 180, 190, 200, 220, 230, 240, 250, 260, 270, 280 or more amino acids from the FH12877464.1 Attorney Docket: OGY-02925 naturally occurring TAL effector on the N-terminal side of the TAL effector repeats; and/or 3) at least 68, 80, 90, 100, 110, 120, 130, 140, 150, 170, 180, 190, 200, 220, 230, 240, 250, 260 or more amino acids from the naturally occurring TAL effector on the
  • a modulating agent comprises a DNA-targeting moiety comprising an engineered DNA binding domain (DBD), e.g., a TAL effector comprising a TAL effector repeat that binds to a target sequence, e.g., a promoter or transcription start site (TSS)) sequence operably linked to a target gene, e.g., a sequence proximal to the transcription regulatory element, e.g., an anchor sequence of an anchor sequence mediated conjunction (ASMC) comprising a target gene , e.g., a sequence proximal to the anchor sequence.
  • the TAL effector binds to a target sequence described herein.
  • a DNA-targeting moiety is or comprises a Zn finger domain.
  • a Zn finger domain comprises a Zn finger, e.g., a naturally occurring Zn finger or engineered Zn finger, or fragment thereof. Many Zn fingers are known to those of skill in the art and are commercially available, e.g., from Sigma-Aldrich.
  • a Zn finger domain comprises a plurality of Zn fingers, wherein each Zn finger recognizes three nucleotides.
  • a Zn finger protein can comprise a Zn finger domain and optionally one or more other domains.
  • a Zn finger molecule comprises a non-naturally occurring Zn finger protein that is engineered to bind to a target DNA sequence of choice. See, for example, Beerli, et al. (2002) Nature Biotechnol.20:135-141; Pabo, et al. (2001) Ann. Rev. Biochem.70:313-340; Isalan, et al. (2001) Nature Biotechnol.19:656-660; Segal, et al. (2001) Curr. Opin. Biotechnol.12:632-637; Choo, et al. (2000) Curr. Opin. Struct. Biol. 10:411-416; U.S. Pat.
  • An engineered Zn finger may have a novel binding specificity, compared to a naturally-occurring Zn finger.
  • Engineering methods include, but are not limited to, rational design and various types of selection.
  • Rational design includes, for example, using databases comprising triplet (or quadruplet) nucleotide sequences and individual Zn finger amino acid FH12877464.1 Attorney Docket: OGY-02925 sequences, in which each triplet or quadruplet nucleotide sequence is associated with one or more amino acid sequences of zinc fingers which bind the particular triplet or quadruplet sequence. See, for example, U.S. Pat. Nos.6,453,242 and 6,534,261, incorporated by reference herein in their entireties. Exemplary selection methods, including phage display and two-hybrid systems, are disclosed in U.S. Pat.
  • the DNA-targeting moiety comprises a Zn finger domain comprising an engineered zinc finger that binds (in a sequence-specific manner) to a target DNA sequence.
  • the Zn finger domain comprises one Zn finger or fragment thereof.
  • the Zn finger domain comprises a plurality of Zn fingers (or fragments thereof), e.g., 2, 3, 4, 5, 6 or more Zn fingers (and optionally no more than 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, or 2 Zn fingers).
  • the Zn finger domain comprises at least three Zn fingers.
  • the Zn finger domain comprises four, five or six Zn fingers.
  • the Zn finger domain comprises 8, 9, 10, 11 or 12 Zn fingers.
  • a Zn finger domain comprising three Zn FH12877464.1 Attorney Docket: OGY-02925 fingers recognizes a target DNA sequence comprising 9 or 10 nucleotides.
  • a Zn finger domain comprising four Zn fingers recognizes a target DNA sequence comprising 12 to 14 nucleotides.
  • a Zn finger domain comprising six Zn fingers recognizes a target DNA sequence comprising 18 to 21 nucleotides.
  • a DNA targeting domain comprises a two-handed Zn finger protein. Two handed zinc finger proteins are those proteins in which two clusters of zinc fingers are separated by intervening amino acids so that the two zinc finger domains bind to two discontinuous target DNA sequences.
  • SIP1 An example of a two-handed type of zinc finger binding protein is SIP1, where a cluster of four zinc fingers is located at the amino terminus of the protein and a cluster of three Zn fingers is located at the carboxyl terminus (see Remade, et al. (1999) EMBO Journal 18(18):5073-5084). Each cluster of zinc fingers in these domains is able to bind to a unique target sequence and the spacing between the two target sequences can comprise many nucleotides.
  • an expression activator comprises a DNA-targeting moiety comprising an engineered DNA binding domain (DBD), e.g., a Zn finger domain comprising a Zn finger (ZFN) that binds to a target sequence, e.g., a promoter or transcription start site (TSS)) sequence operably linked to a target gene, e.g., a sequence proximal to the transcription regulatory element, e.g., an anchor sequence of an anchor sequence mediated conjunction (ASMC) comprising a target gene, e.g., a sequence proximal to the anchor sequence.
  • the ZFN binds to a target sequence described herein.
  • the ZFN can be engineered to carry epigenetic effector molecules to target sites.
  • Effector Domain expression activators of the present disclosure comprise one or more effector domains.
  • an effector domain when used as part of an expressor activator or an expression activation system described herein, increases expression of a target gene in a cell.
  • the expression activator comprises a single effector domain.
  • the expression activator comprises one or more effector domains.
  • the expression activator comprises more than one effector domain.
  • the expression activator comprises one to four effector domains.
  • the expression activator comprises two effector FH12877464.1 Attorney Docket: OGY-02925 domains. In some embodiments, the expression activator comprises three effector domains. In some embodiments, the expression activator comprises four effector domains. In some embodiments, the effector domain has functionality unrelated to the binding of the DNA-targeting moiety. For example, effector domains may target, e.g., bind, a genomic sequence element or genomic complex component proximal to the genomic sequence element targeted by the DNA-targeting moiety or recruit a transcription factor. As a further example, an effector domain may comprise an enzymatic activity, e.g., a genetic modification functionality. In some embodiments, the effector domain induces DNA demethylation.
  • the effector domain induces DNA demethylation of a CpG island (i.e., a region of the genome comprising a high concentration of CpG residues).
  • the effector domain induces histone modification.
  • the effector domain comprises a histone modifying enzyme.
  • the histone modifying enzyme is selected from a histone acetyltransferase, a histone deacetylase (HDAC), a histone lysine methyl-transferase, and a histone lysine demethylase.
  • the effector domain forms a complex for epigenetic modification.
  • an effector domain forms a complex that induces DNA modification and/or histone modification.
  • an effector domain comprises an epigenetic activating domain.
  • an “epigenetic activating domain” refers to a polypeptide capable of inducing an epigenetic change such that expression of a target gene is increased.
  • an epigenetic activating domain is encoded by a fragment of a gene.
  • the fragment of a gene is of a gene selected from MYBL1, DUX4, MYB, CEPBPD, ATF4, NCOA2, TP53, FOXO1, NFE2, E2F2, UBN1, CREB3L1, FOXO3, KLF15, MLXIPL, LEUTX, INO80D, CR3L1, ATF6A, CSN9, FOXO6, CACTIN, C1QBP, KLF4, MYC, NUTM1, VP16, VP64, YLR278C, PIP2 and STB3.
  • the fragment of a gene is of a gene selected from MYBL1, DUX4, MYB, CEPBPD, ATF4, NCOA2, TP53, FOXO1, NFE2, E2F2, UBN1, CREB3L1, FOXO3, KLF15, MLXIPL, LEUTX, INO80D, CR3L1, ATF6A, CSN9, FOXO6, CACTIN, C1QBP, KLF4, MYC, NUTM1, VP16, VP64, YLR278C, PIP2, STB3, and PA.v1b.
  • the epigenetic activating domain is encoded by a fragment of gene MYBL1.
  • MYBL1 is a transcription factor involved in regulating gene expression, cell cycle progression, differentiation, and proliferation, and belongs to the MYB family characterized by DNA-binding domains that mediate sequence-specific DNA interactions.
  • the epigenetic activating domain encoded by a fragment of gene MYBL1 is set forth in SEQ ID NO: 1.
  • the epigenetic activating domain comprises an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO: 1.
  • the epigenetic activating domain comprises the amino acid sequence of SEQ ID NO: 1. In some embodiments, the epigenetic activating domain consists of the amino acid sequence of SEQ ID NO: 1. In some embodiments, the epigenetic activating domain encoded by a fragment of gene MYBL1 is set forth in SEQ ID NO: 2. In some embodiments, the epigenetic activating domain comprises an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO: 2. In some embodiments, the epigenetic activating domain comprises the amino acid sequence of SEQ ID NO: 2.
  • the epigenetic activating domain consists of the amino acid sequence of SEQ ID NO: 2.
  • the epigenetic activating domain is encoded by a fragment of gene DUX4.
  • DUX4 is a transcription factor encoded by the double homeobox 4 gene, characterized by its role in regulating early embryonic gene expression, and involvement in epigenetic reprogramming.
  • the epigenetic activating domain encoded by a fragment of gene DUX4 is set forth in SEQ ID NO: 3.
  • the epigenetic activating domain comprises an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO: 3.
  • the epigenetic activating domain comprises the amino acid sequence of SEQ ID NO: 3.
  • the epigenetic activating domain consists of the amino acid sequence of SEQ ID NO: 3.
  • the epigenetic activating domain is encoded by a fragment of gene MYB.
  • the epigenetic activating domain encoded by a fragment of gene MYB is set forth in SEQ ID NO: 4.
  • the epigenetic activating domain comprises an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO: 4.
  • the epigenetic activating domain comprises the amino acid sequence of SEQ ID NO: 4.
  • the epigenetic activating domain consists of the amino acid sequence of SEQ ID NO: 4.
  • FH12877464.1 Attorney Docket: OGY-02925
  • the epigenetic activating domain is encoded by a fragment of gene CEBPD.
  • CEBPD is a member of the CCAAT/enhancer-binding protein (C/EBP) family of transcription factors, and plays a key role in regulating genes involved in immune and inflammatory responses, cell cycle arrest, and differentiation.
  • the epigenetic activating domain encoded by a fragment of gene CEBPD is set forth in SEQ ID NO: 5.
  • the epigenetic activating domain comprises an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO: 5.
  • the epigenetic activating domain comprises the amino acid sequence of SEQ ID NO: 5.
  • the epigenetic activating domain consists of the amino acid sequence of SEQ ID NO: 5.
  • the epigenetic activating domain is encoded by a fragment of gene ATF4.
  • ATF4 is a member of the activating transcription factor (ATF) family and functions as a stress-responsive transcription factor that regulates gene expression programs involved in amino acid metabolism, oxidative stress response, and the integrated stress response (ISR).
  • the epigenetic activating domain encoded by a fragment of gene ATF4 is set forth in SEQ ID NO: 6.
  • the epigenetic activating domain comprises an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO: 6.
  • the epigenetic activating domain comprises the amino acid sequence of SEQ ID NO: 6.
  • the epigenetic activating domain consists of the amino acid sequence of SEQ ID NO: 6.
  • the epigenetic activating domain is encoded by a fragment of gene NCOA2.
  • NCOA2 is a nuclear receptor coactivator that interacts with ligand-bound nuclear receptors to enhance transcription of target genes, functioning as part of a multiprotein coactivator complex and possessing intrinsic histone acetyltransferase activity that contributes to chromatin remodeling and epigenetic regulation of gene expression.
  • the epigenetic activating domain encoded by a fragment of gene of NCOA2 is set forth in SEQ ID NO: 7.
  • the epigenetic activating domain comprises an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO: 7.
  • the epigenetic activating domain comprises the amino acid sequence of SEQ ID NO: 7.
  • the epigenetic activating domain consists of the amino acid sequence of SEQ ID NO: 7. FH12877464.1 Attorney Docket: OGY-02925
  • the epigenetic activating domain is encoded by a fragment of gene TP53.
  • TP53 is a critical transcription factor that regulates the expression of genes involved in cell cycle arrest, DNA repair, apoptosis, and senescence in response to cellular stress and DNA damage.
  • the epigenetic activating domain encoded by a fragment of gene TP53 is set forth in SEQ ID NO: 8.
  • the epigenetic activating domain comprises an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO: 8.
  • the epigenetic activating domain comprises the amino acid sequence of SEQ ID NO: 8.
  • the epigenetic activating domain consists of the amino acid sequence of SEQ ID NO: 8.
  • the epigenetic activating domain is encoded by a fragment of gene FOXO1.
  • FOXO1 is a member of the forkhead box O (FOXO) family of transcription factors, which plays a pivotal role in regulating gene expression programs involved in cell cycle control, apoptosis, oxidative stress resistance, and metabolism.
  • the epigenetic activating domain encoded by a fragment of gene FOXO1 is set forth in SEQ ID NO: 9.
  • the epigenetic activating domain comprises an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO: 9.
  • the epigenetic activating domain comprises the amino acid sequence of SEQ ID NO: 9.
  • the epigenetic activating domain consists of the amino acid sequence of SEQ ID NO: 9.
  • the epigenetic activating domain is encoded by a fragment of gene NFE2.
  • NFE2 is a transcription factor that is part of the cap ‘n’ collar-basic leucine zipper (CNC-bZIP) family and functions primarily in regulating gene expression programs related to erythroid and megakaryocytic differentiation, as well as oxidative stress responses.
  • the epigenetic activating domain encoded by a fragment of gene NFE2 is set forth in SEQ ID NO: 10.
  • the epigenetic activating domain comprises an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO: 10.
  • the epigenetic activating domain comprises the amino acid sequence of SEQ ID NO: 10. In some embodiments, the epigenetic activating domain consists of the amino acid sequence of SEQ ID NO: 10. In some embodiments, the epigenetic activating domain is encoded by a fragment of gene E2F2.
  • E2F2 is a member of the E2F family of transcription factors, which play essential FH12877464.1 Attorney Docket: OGY-02925 roles in regulating the expression of genes involved in cell cycle progression, DNA replication, and apoptosis. In some embodiments, the epigenetic activating domain encoded by a fragment of gene E2F2 is set forth in SEQ ID NO: 11.
  • the epigenetic activating domain comprises an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO: 11.
  • the epigenetic activating domain comprises the amino acid sequence of SEQ ID NO: 11.
  • the epigenetic activating domain consists of the amino acid sequence of SEQ ID NO: 11.
  • the epigenetic activating domain is encoded by a fragment of gene UBN1.
  • the epigenetic activating domain encoded by a fragment of gene UBN1 is set forth in SEQ ID NO: 12.
  • the epigenetic activating domain comprises an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO: 12.
  • the epigenetic activating domain comprises the amino acid sequence of SEQ ID NO: 12. In some embodiments, the epigenetic activating domain consists of the amino acid sequence of SEQ ID NO: 12. In some embodiments, the epigenetic activating domain is encoded by a fragment of gene CREB3L1.
  • CREB3L1 is a transcription factor belonging to the CREB3 subfamily of basic leucine zipper (bZIP) proteins, which is activated through regulated intramembrane proteolysis in response to cellular stress, and regulates genes involved in secretion, extracellular matrix production, and cellular differentiation.
  • the epigenetic activating domain encoded by a fragment of gene of CREB3L1 is set forth in SEQ ID NO: 13.
  • the epigenetic activating domain comprises an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO: 13.
  • the epigenetic activating domain comprises the amino acid sequence of SEQ ID NO: 13.
  • the epigenetic activating domain consists of the amino acid sequence of SEQ ID NO: 13.
  • the epigenetic activating domain is encoded by a fragment of gene FOXO3.
  • FOXO3 is a transcription factor belonging to the forkhead box O (FOXO) FH12877464.1 Attorney Docket: OGY-02925 family, which regulates the expression of genes involved in apoptosis, cell cycle arrest, oxidative stress resistance, DNA repair, and longevity.
  • the epigenetic activating domain encoded by a fragment of gene FOXO3 is set forth in SEQ ID NO: 14.
  • the epigenetic activating domain comprises an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO: 14.
  • the epigenetic activating domain comprises the amino acid sequence of SEQ ID NO: 14. In some embodiments, the epigenetic activating domain consists of the amino acid sequence of SEQ ID NO: 14. In some embodiments, the epigenetic activating domain is encoded by a fragment of gene KLF15.
  • KLF15 is a member of the Krüppel-like factor (KLF) family of zinc finger transcription factors, and it regulates the expression of genes involved in metabolism, circadian rhythm, cellular differentiation, and plays a critical role in glucose and lipid homeostasis. In some embodiments, the epigenetic activating domain encoded by a fragment of gene KLF15 is set forth in SEQ ID NO: 15.
  • the epigenetic activating domain comprises an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO: 15.
  • the epigenetic activating domain comprises the amino acid sequence of SEQ ID NO: 15.
  • the epigenetic activating domain consists of the amino acid sequence of SEQ ID NO: 15.
  • the epigenetic activating domain is encoded by a fragment of gene MLXIPL.
  • MLXIPL is a transcription factor that forms a heterodimer with MLX and regulates the expression of genes involved in glycolysis and lipogenesis in response to glucose levels, thereby playing a key role in metabolic homeostasis, particularly in the liver and adipose tissue.
  • the epigenetic activating domain encoded by a fragment of gene of MLXIPL is set forth in SEQ ID NO: 16.
  • the epigenetic activating domain comprises an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO: 16.
  • the epigenetic activating domain comprises the amino acid sequence of SEQ ID NO: 16. In some embodiments, the epigenetic activating domain consists of the amino acid sequence of SEQ ID NO: 16. In some embodiments, the epigenetic activating domain is encoded by a fragment of gene LEUTX.
  • LEUTX is a homeobox-containing transcription factor expressed transiently during early embryonic development, where it plays a role in activating genes associated with zygotic genome activation and early cell fate determination.
  • the FH12877464.1 Attorney Docket: OGY-02925 epigenetic activating domain encoded by a fragment of gene LEUTX is set forth in SEQ ID NO: 17.
  • the epigenetic activating domain comprises an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO: 17.
  • the epigenetic activating domain comprises the amino acid sequence of SEQ ID NO: 17.
  • the epigenetic activating domain consists of the amino acid sequence of SEQ ID NO: 17.
  • the epigenetic activating domain is encoded by a fragment of gene INO80D.
  • INO80D is a component of the INO80 chromatin remodeling complex, which plays a critical role in regulating DNA accessibility by repositioning or restructuring nucleosomes, thereby contributing to processes such as transcriptional regulation, DNA replication, and DNA damage repair through ATP-dependent chromatin remodeling activity.
  • the epigenetic activating domain encoded by a fragment of gene INO80D is set forth in SEQ ID NO: 18.
  • the epigenetic activating domain comprises an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO: 18.
  • the epigenetic activating domain comprises the amino acid sequence of SEQ ID NO: 18. In some embodiments, the epigenetic activating domain consists of the amino acid sequence of SEQ ID NO: 18. In some embodiments, the epigenetic activating domain is encoded by a fragment of gene CR3L1.
  • CR3L1 also known as CREB3L1
  • bZIP basic leucine zipper
  • the epigenetic activating domain encoded by a fragment of gene CR3L1 is set forth in SEQ ID NO: 19.
  • the epigenetic activating domain comprises an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO: 19.
  • the epigenetic activating domain comprises the amino acid sequence of SEQ ID NO: 19.
  • the epigenetic activating domain consists of the amino acid sequence of SEQ ID NO: 19.
  • the epigenetic activating domain is encoded by a fragment of gene ATF6A.
  • ATF6A is a transcription factor that regulate genes involved in protein folding, endoplasmic reticulum-associated degradation (ERAD), and restoration of ER homeostasis
  • ESD endoplasmic reticulum-associated degradation
  • the epigenetic activating domain encoded by a fragment of gene ATF6A is set forth in SEQ ID NO: 20.
  • the epigenetic activating domain comprises an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO: 20.
  • the epigenetic activating domain comprises the amino acid sequence of SEQ ID NO: 20.
  • the epigenetic activating domain consists of the amino acid sequence of SEQ ID NO: 20.
  • the epigenetic activating domain is encoded by a fragment of gene CSN9.
  • CSN9 is a protein component associated with the COP9 signalosome, a conserved multi-subunit complex involved in the regulation of protein degradation via the ubiquitin-proteasome system, particularly through modulation of cullin-RING E3 ubiquitin ligases.
  • the epigenetic activating domain encoded by a fragment of gene CSN9 is set forth in SEQ ID NO: 21.
  • the epigenetic activating domain comprises an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO: 21.
  • the epigenetic activating domain comprises the amino acid sequence of SEQ ID NO: 21.
  • the epigenetic activating domain consists of the amino acid sequence of SEQ ID NO: 21.
  • the epigenetic activating domain is encoded by a fragment of gene FOXO6.
  • FOXO6 is a member of the forkhead box O (FOXO) family of transcription factors, which regulate gene expression programs involved in various cellular processes, including metabolism, stress response, and neurogenesis.
  • the epigenetic activating domain encoded by a fragment of gene FOXO6 is set forth in SEQ ID NO: 22.
  • the epigenetic activating domain comprises an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO: 22.
  • the epigenetic activating domain comprises the amino acid sequence of SEQ ID NO: 22.
  • the epigenetic activating domain consists of the amino acid sequence of SEQ ID NO: 22.
  • the epigenetic activating domain is encoded by a fragment of gene CACTIN.
  • CACTIN is a protein subunit of the spliceosome C complex which is involved in pre-mRNA splicing, ensuring accurate removal of introns during transcription.
  • the epigenetic activating domain encoded by a fragment of gene CACTIN is set forth in SEQ ID NO: 23.
  • the epigenetic activating FH12877464.1 Attorney Docket: OGY-02925 domain comprises an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO: 23.
  • the epigenetic activating domain comprises the amino acid sequence of SEQ ID NO: 23.
  • the epigenetic activating domain consists of the amino acid sequence of SEQ ID NO: 23.
  • the epigenetic activating domain is encoded by a fragment of gene C1QBP.
  • the epigenetic activating domain encoded by a fragment of gene C1QBP is set forth in SEQ ID NO: 24.
  • the epigenetic activating domain comprises an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO: 24.
  • the epigenetic activating domain comprises the amino acid sequence of SEQ ID NO: 24.
  • the epigenetic activating domain consists of the amino acid sequence of SEQ ID NO: 24. In some embodiments, the epigenetic activating domain is encoded by a fragment of gene FOXO3. In some embodiments, the epigenetic activating domain encoded by a fragment of gene FOXO3 is set forth in SEQ ID NO: 25. In some embodiments, the epigenetic activating domain comprises an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO: 25. In some embodiments, the epigenetic activating domain comprises the amino acid sequence of SEQ ID NO: 25.
  • the epigenetic activating domain consists of the amino acid sequence of SEQ ID NO: 25.
  • the epigenetic activating domain is encoded by a fragment of gene KLF4.
  • KLF4 is a member of the Krüppel-like factor (KLF) family of zinc finger transcription factors, and it regulates the expression of genes involved in various cellular processes, including proliferation, differentiation, and apoptosis.
  • the epigenetic activating domain encoded by a fragment of gene KLF4 is set forth in SEQ ID NO: 26.
  • the epigenetic activating domain comprises an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO: 26.
  • the epigenetic activating domain comprises the amino acid sequence of SEQ ID NO: 26.
  • the epigenetic activating domain consists of the amino acid sequence of SEQ ID NO: 26.
  • the epigenetic activating domain is encoded by a fragment of gene MYC.
  • the epigenetic activating domain encoded by a fragment of gene MYC is set forth in SEQ ID NO: 27.
  • the epigenetic activating domain comprises an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO: 27.
  • the epigenetic activating domain comprises the amino acid sequence of SEQ ID NO: 27.
  • the epigenetic activating domain consists of the amino acid sequence of SEQ ID NO: 27.
  • the epigenetic activating domain encoded by a fragment of gene MYC is set forth in SEQ ID NO: 28.
  • the epigenetic activating domain comprises an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO: 28.
  • the epigenetic activating domain comprises the amino acid sequence of SEQ ID NO: 28.
  • the epigenetic activating domain consists of the amino acid sequence of SEQ ID NO: 28.
  • the epigenetic activating domain is encoded by a fragment of gene NUTM1.
  • NUTM1 is a nuclear protein involved in chromatin regulation and transcription. NUTM1 is best known for forming oncogenic fusion proteins, such as BRD4- NUT, which drive aggressive cancers like NUT carcinoma by promoting widespread histone acetylation and blocking cellular differentiation.
  • the epigenetic activating domain encoded by a fragment of gene NUTM1 is set forth in SEQ ID NO: 29.
  • the epigenetic activating domain comprises an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO: 29.
  • the epigenetic activating domain comprises the amino acid sequence of SEQ ID NO: 29. In some embodiments, the epigenetic activating domain consists of the amino acid sequence of SEQ ID NO: 29. In some embodiments, the epigenetic activating domain is encoded by a fragment of gene E2F2.
  • E2F2 is a member of the E2F family of transcription factors, which play essential roles in regulating the expression of genes involved in cell cycle progression, DNA replication, and apoptosis.
  • the epigenetic activating domain encoded FH12877464.1 Attorney Docket: OGY-02925 by a fragment of gene E2F2 is set forth in SEQ ID NO: 30.
  • the epigenetic activating domain comprises an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO: 30.
  • the epigenetic activating domain comprises the amino acid sequence of SEQ ID NO: 30.
  • the epigenetic activating domain consists of the amino acid sequence of SEQ ID NO: 30.
  • the epigenetic activating domain is encoded by a fragment of gene VP16.
  • VP16 is a potent transcriptional activator encoded by the UL48 gene of herpes simplex virus (HSV) and plays a crucial role in initiating the viral lytic cycle by forming a complex with host transcription factors to activate immediate-early viral gene expression.
  • the epigenetic activating domain encoded by a fragment of gene VP16 is set forth in SEQ ID NO: 31.
  • the epigenetic activating domain comprises an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO: 31.
  • the epigenetic activating domain comprises the amino acid sequence of SEQ ID NO: 31. In some embodiments, the epigenetic activating domain consists of the amino acid sequence of SEQ ID NO: 31. In some embodiments, the epigenetic activating domain is encoded by a fragment of gene VP64.
  • VP64 is a synthetic transcriptional activation domain composed of four tandem repeats of the herpes simplex virus VP16 protein's activation domain. VP64 is commonly fused to catalytically inactive Cas9 (dCas9) in CRISPR activation (CRISPRa) systems to upregulate target gene expression.
  • the epigenetic activating domain encoded by a fragment of gene VP64 is set forth in SEQ ID NO: 32.
  • the epigenetic activating domain comprises an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO: 32.
  • the epigenetic activating domain comprises the amino acid sequence of SEQ ID NO: 32.
  • the epigenetic activating domain consists of the amino acid sequence of SEQ ID NO: 32.
  • the epigenetic activating domain is encoded by a fragment of gene YLR278C.
  • YLR278C is a predicted zinc-cluster DNA-binding transcription factor active in the nucleus, with roles in sequence-specific transcriptional activation by RNA polymerase II.
  • the epigenetic activating domain encoded by a fragment of gene YLR278C is set forth in SEQ ID NO: 33.
  • the epigenetic activating domain comprises an amino acid sequence having at least 70%, 75%, FH12877464.1 Attorney Docket: OGY-02925 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO: 33.
  • the epigenetic activating domain comprises the amino acid sequence of SEQ ID NO: 33. In some embodiments, the epigenetic activating domain consists of the amino acid sequence of SEQ ID NO: 33. In some embodiments, the epigenetic activating domain is encoded by a fragment of gene PIP2.
  • PIP2 also known as Pip2p or Oaf2
  • Pip2p or Oaf2 is a transcription factor in Saccharomyces cerevisiae that forms a heterodimer with Oaf1p to regulate genes involved in peroxisome proliferation and fatty acid metabolism.
  • the epigenetic activating domain encoded by a fragment of gene PIP2 is set forth in SEQ ID NO: 34.
  • the epigenetic activating domain comprises an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO: 34.
  • the epigenetic activating domain comprises the amino acid sequence of SEQ ID NO: 34.
  • the epigenetic activating domain consists of the amino acid sequence of SEQ ID NO: 34.
  • the epigenetic activating domain is encoded by a fragment of gene STB3.
  • STB3 is a gene in Saccharomyces cerevisiae encoding a transcriptional regulator that binds to ribosomal RNA processing element (RRPE) motifs, playing a role in the glucose-induced transition from quiescence to growth.
  • the epigenetic activating domain encoded by a fragment of gene STB3 is set forth in SEQ ID NO: 35.
  • the epigenetic activating domain comprises an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO: 35.
  • the epigenetic activating domain comprises the amino acid sequence of SEQ ID NO: 35.
  • the epigenetic activating domain consists of the amino acid sequence of SEQ ID NO: 35.
  • the epigenetic activating domain is encoded by PA.v1b, a fragment of gene vIRF-2.
  • PA.v1b is a synthetic construct derived from vIRF-2, a viral interferon regulatory factor encoded by Human gamma herpesvirus 8 (HHV-8), known to modulate host transcription and interfere with immune signaling pathways.
  • the epigenetic activating domain encoded by a fragment of gene vIRF-2, PA.v1b is set forth in SEQ ID NO: 116.
  • the epigenetic activating domain comprises an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO: 116.
  • the epigenetic activating domain comprises the amino acid sequence of SEQ FH12877464.1 Attorney Docket: OGY-02925 ID NO: 116.
  • the epigenetic activating domain consists of the amino acid sequence of SEQ ID NO: 116.
  • an effector domain stimulates or promotes transcription, e.g., of the target gene.
  • the effector domain recruits a factor that induces transcription, e.g., of the target gene.
  • the effector domain promotes epigenetic modification, e.g., directly or indirectly.
  • an effector domain can indirectly promote epigenetic modification by recruiting an endogenous protein that epigenetically modifies the chromatin.
  • An effector domain can directly promote epigenetic modification by catalyzing epigenetic modification, wherein the effector domain comprises enzymatic activity and directly places an epigenetic mark on the chromatin.
  • an effector domain may comprise a peptide ligand, a full- length protein, a protein fragment.
  • an effector domain comprises a dominant negative component (e.g., dominant negative moiety), e.g., a protein that recognizes and binds a sequence (e.g., an anchor sequence, e.g., a CTCF binding motif), but with an inactive (e.g., mutated) dimerization domain, e.g., a dimerization domain that is unable to form a functional anchor sequence-mediated conjunction), or binds to a component of a genomic complex (e.g., a transcription factor subunit, etc.) preventing formation of a functional transcription factor, etc.
  • a dominant negative component e.g., dominant negative moiety
  • a protein that recognizes and binds a sequence e.g., an anchor sequence, e.g., a CTCF binding motif
  • an inactive dimerization domain e.g., mutated dimerization domain that is unable to form a functional anchor sequence-mediated conjunction
  • a component of a genomic complex e.
  • the Zinc Finger domain of CTCF can be altered so that it binds a specific anchor sequence (by adding zinc fingers that recognize flanking nucleic acids), while the homo-dimerization domain is altered to prevent the interaction between engineered CTCF and endogenous forms of CTCF.
  • a dominant negative component comprises a synthetic nucleating polypeptide with a selected binding affinity for an anchor sequence within a target anchor sequence-mediated conjunction.
  • binding affinity may be at least 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or higher or lower than binding affinity of an endogenous nucleating polypeptide (e.g., CTCF) that associates with a target anchor sequence.
  • an endogenous nucleating polypeptide e.g., CTCF
  • a synthetic nucleating polypeptide may have between 30- 90%, 30-85%, 30-80%, 30-70%, 50-80%, 50-90% amino acid sequence identity to a corresponding endogenous nucleating polypeptide.
  • a nucleating polypeptide may modulate (e.g., disrupt), such as through competitive binding, e.g., competing with binding of an endogenous nucleating polypeptide to its anchor sequence.
  • FH12877464.1 Attorney Docket: OGY-02925
  • a candidate effector domain may be determined to be suitable for use as an effector domain by methods known to those of skill in the art.
  • a candidate effector domain may be tested by assaying whether, when the candidate effector domain is present in the nucleus of a cell and appropriately localized (e.g., to a target gene or transcription control element operably linked to said target gene, e.g., via a DNA-targeting moiety), the candidate effector domain increases expression of the target gene in the cell, e.g., increases the level of RNA transcript encoded by the target gene (e.g., as measured by RNASeq or Northern blot) or increases the level of protein encoded by the target gene (e.g., as measured by ELISA).
  • the candidate effector domain increases expression of the target gene in the cell, e.g., increases the level of RNA transcript encoded by the target gene (e.g., as measured by RNASeq or Northern blot) or increases the level of protein encoded by the target gene (e.g., as measured by ELISA).
  • an expression activator comprises a plurality of effector domains, wherein each effector domain does not detectably bind, e.g., does not bind, to another effector domain.
  • an expression activation system comprises a first expression activator comprising a first effector domain and a second expression activator comprising a second effector domain, wherein the first effector domain does not detectably bind, e.g., does not bind, to the second effector domain.
  • an expression activation system comprises a plurality of expression activators, wherein each member of the plurality of expression activators comprises an effector domain, wherein each effector domain does not detectably bind, e.g., does not bind, to another effector domain.
  • an expression activation system comprises a first expression activator comprising a first effector domain and a second expression activator comprising a second effector domain, wherein the first effector domain does not detectably bind, e.g., does not bind, to the second effector domain.
  • an expression activation system comprises a first expression activator comprising a first effector domain and a second expression activator comprising a second effector domain, wherein the first effector domain does not detectably bind, e.g., does not bind, to another first effector domain, and the second effector domain does not detectably bind, e.g., does not bind, to another second effector domain.
  • an effector domain for use in the compositions and methods described herein is functional in a monomeric, e.g., non-dimeric, state.
  • the epigenetic activating domain modulates the two- dimensional structure of chromatin (i.e., modulates structure of chromatin in a way that would alter its two-dimensional representation).
  • an effector domain comprises a component of a gene editing system, e.g., a CRISPR/Cas domain, e.g., a Zn Finger domain, e.g., a TAL effector domain.
  • an epigenetic activating domain may comprise a polypeptide (e.g., peptide or protein moiety) linked to a gRNA and a targeted nuclease, e.g., a Cas9, e.g., a wild type Cas9, a nickase Cas9 (e.g., Cas9 D10A), a catalytically inactive Cas9 (dCas9), eSpCas9, Cpfl, C2C1, or C2C3, or a nucleic acid encoding such a nuclease.
  • a Cas9 e.g., a wild type Cas9, a nickase Cas9 (e.g., Cas9 D10A)
  • dCas9 catalytically inactive Cas9
  • eSpCas9 eSpCas9
  • Cpfl C2C1
  • C2C3 nucleic acid encoding such
  • an expression activator may further comprise one or more additional moieties (e.g., in addition to one or more targeting moieties and one or more effector domains).
  • an additional moiety is selected from a tagging or monitoring moiety, a cleavable moiety (e.g., a cleavable moiety positioned between a DNA-targeting moiety and an effector domain or at the N- or C-terminal end of a polypeptide), a small molecule, a membrane translocating polypeptide, or a pharmaco-agent moiety.
  • Linkers An expression activator or an expression activation system as disclosed herein may comprise one or more linkers.
  • a linker may connect a targeting moiety to an effector moiety, an effector moiety to another effector moiety, or a targeting moiety to another targeting moiety.
  • a linker may be a chemical bond, e.g., one or more covalent bonds or non-covalent bonds.
  • a linker is covalent.
  • a linker is non- covalent.
  • a linker is a peptide linker.
  • Such a linker may be between 2-30, 5-30, 10-30, 15-30, 20-30, 25-30, 2-25, 5-25, 10-25, 15-25, 20-25, 2-20, 5-20, 10-20, 15-20, 2-15, 5-15, 10-15, 2-10, 5-10, or 2-5 amino acids in length, or greater than or equal to 2, 5, 10, 15, 20, 25, or 30 amino acids in length (and optionally up to 50, 40, 30, 25, 20, 15, 10, or 5 amino acids in length).
  • a linker can be used to space a first domain or moiety from a second domain or moiety, e.g., a DNA-targeting moiety from an effector moiety.
  • a linker can be positioned between a DNA-targeting moiety and an effector moiety, e.g., to provide molecular flexibility of secondary and tertiary structures.
  • a linker may comprise flexible, rigid, and/or cleavable linkers described herein.
  • a linker includes at least one glycine, alanine, and serine amino acids to provide for flexibility.
  • a linker is a hydrophobic linker, such as including a negatively charged sulfonate group, polyethylene glycol (PEG) group, or pyrophosphate diester group.
  • a linker is FH12877464.1 Attorney Docket: OGY-02925 cleavable to selectively release a moiety (e.g., polypeptide) from a modulating agent, but sufficiently stable to prevent premature cleavage.
  • a moiety e.g., polypeptide
  • one or more moieties and/or domains of an expression activator described herein are linked with one or more linkers.
  • an expression activator may comprise a linker situated between the targeting moiety and the effector moiety.
  • the linker may have a sequence of ASGSGGGSGGARD (SEQ ID NO:112), or ASGSGGGSGG (SEQ ID NO:110).
  • a system comprising a first and second activator may comprise a first linker situated between the first targeting moiety and the first effector moiety, and a second linker situated between the second targeting moiety and the second effector moiety.
  • the first and the second linker may be identical.
  • the first and the second linker may be different.
  • the first linker may comprise an amino acid sequence according to SEQ ID NO:110 or a sequence with at least 80, 85, 90, 95, 99, or 100% identity thereto and the second linker may comprise an amino acid sequence according to SEQ ID NO:112 or a sequence with at least 80, 85, 90, 95, 99, or 100% identity thereto.
  • GS linker As will be known by one of skill in the art, commonly used flexible linkers have sequences consisting primarily of stretches of Gly and Ser residues (“GS” linker). Flexible linkers may be useful for joining domains/moieties that require a certain degree of movement or interaction and may include small, non-polar (e.g., Gly) or polar (e.g., Ser or Thr) amino acids. Incorporation of Ser or Thr can also maintain the stability of a linker in aqueous solutions by forming hydrogen bonds with water molecules, and therefore reduce unfavorable interactions between a linker and moieties/domains. In some embodiments, the linker is a GS linker or a variant thereof e.g., G4S (SEQ ID NO:111).
  • Rigid linkers are useful to keep a fixed distance between domains/moieties and to maintain their independent functions. Rigid linkers may also be useful when a spatial separation of domains is critical to preserve the stability or bioactivity of one or more components in the fusion.
  • Rigid linkers may have an alpha helix-structure or Pro-rich sequence, (XP)n, with X designating any amino acid, preferably Ala, Lys, or Glu.
  • Cleavable linkers may release free functional domains in vivo.
  • linkers may be cleaved under specific conditions, such as presence of reducing reagents or proteases. In vivo cleavable linkers may utilize reversible nature of a disulfide bond.
  • One example includes a thrombin-sensitive sequence (e.g., PRS) between the two Cys residues.
  • PRS thrombin-sensitive sequence
  • In vitro thrombin treatment of CPRSC results in the cleavage of a thrombin-sensitive FH12877464.1 Attorney Docket: OGY-02925 sequence, while a reversible disulfide linkage remains intact.
  • linkers are known and described, e.g., in Chen et al.2013. Fusion Protein Linkers: Property, Design and Functionality. Adv Drug Deliv Rev.65(10): 1357–1369.
  • cleavable linker may be a self-cleaving linker, e.g., a T2A peptide linker.
  • the linker may comprise a “ribosome skipping” sequence, e.g., a tPT2A linker.
  • molecules suitable for use in linkers described herein include a negatively charged sulfonate group; lipids, such as a poly (--CH2--) hydrocarbon chains, such as polyethylene glycol (PEG) group, unsaturated variants thereof, hydroxylated variants thereof, amidated or otherwise N-containing variants thereof; noncarbon linkers; carbohydrate linkers; phosphodiester linkers, or other molecule capable of covalently linking two or more components of an expression activator.
  • lipids such as a poly (--CH2--) hydrocarbon chains, such as polyethylene glycol (PEG) group, unsaturated variants thereof, hydroxylated variants thereof, amidated or otherwise N-containing variants thereof
  • PEG polyethylene glycol
  • Non-covalent linkers are also included, such as hydrophobic lipid globules to which the polypeptide is linked, for example through a hydrophobic region of a polypeptide or a hydrophobic extension of a polypeptide, such as a series of residues rich in leucine, isoleucine, valine, or perhaps also alanine, phenylalanine, or even tyrosine, methionine, glycine, or other hydrophobic residues.
  • Components of an expression activator may be linked using charge-based chemistry, such that a positively charged component of an expression activator is linked to a negative charge of another component.
  • an expression activation system comprising two or more expression activators described herein.
  • an expression activation system comprises 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, or more expression activators (and optionally no more than 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, or 2).
  • the expression activation system comprises a first expression activator comprising (i) a first DNA-targeting moiety that binds a first target sequence described herein, and (ii) a first effector domain; and at least one additional expression activator comprising (i) a second DNA-targeting moiety that binds a second target sequence described herein, and (ii) a second effector domain.
  • the expression activation system comprises a first expression activator comprising (i) a first DNA-targeting moiety that binds a first target sequence described herein, and (ii) a first effector domain; and at least one additional expression activator comprising (i) a second DNA-targeting moiety that binds a second target sequence described herein, and (ii) a second effector domain, wherein the first target sequence is different from the second target sequence.
  • the first effector domain is the same as the second effector domain. In some embodiments, the first effector domain is different from the second effector domain.
  • the expression activation system comprises at least 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 additional expression activators. In some embodiments, the expression activation system comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 additional expression activators. In some embodiments, the expression activation system comprises a first expression activator comprising (i) a first DNA-targeting moiety that binds a first target sequence described herein, and (ii) a first effector domain; and at least 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 additional expression activators, wherein each of the additional expression activators comprises (i) a DNA-targeting moiety that binds a target sequence described herein; and (ii) an effector domain, wherein the target sequence of each of the additional expression activators is different from one another and from the first target sequence.
  • the first effector domain and the effector domain of each of the additional expression activator s are the same or different.
  • each of the expression activators of the expression activation system binds to a different target sequence described herein.
  • each of the expression activators of the expression activation system are formulated in the same composition.
  • each of the expression activators of the expression activation system are formulated in different compositions.
  • the expression activators of the expression activation system each comprise a different DNA-targeting moiety (e.g., the first, second, third, or further expression activators each comprise different targeting moieties from one another).
  • an expression activation system may comprise a first expression activator and a second expression activator wherein the first expression activator comprises a first targeting moiety (e.g., a Cas9 domain, TAL effector domain, or Zn Finger domain), and the second expression activator comprises a second targeting moiety (e.g., a Cas9 domain, TAL effector domain, or Zn Finger domain) different from the first targeting moiety.
  • first targeting moiety e.g., a Cas9 domain, TAL effector domain, or Zn Finger domain
  • second targeting moiety e.g., a Cas9 domain, TAL effector domain, or Zn Finger domain
  • different is comprising distinct types of targeting moiety, e.g., the first targeting moiety comprises a Cas9 domain, and the second DNA-targeting moiety comprises a Zn finger domain.
  • the first targeting moiety comprises a first Cas9 domain (e.g., from a first species) and the second targeting moiety comprises a second Cas9 domain (e.g., from a second species).
  • the targeting moieties specifically bind two or more different target sequences.
  • an expression activation system comprising two or more Cas9 domains
  • the two or more Cas9 domains may be chosen or altered such that they only appreciably bind the gRNA corresponding to their target sequence (e.g., and do not appreciably bind the gRNA corresponding to the target of another Cas9 domain).
  • the two or more effector moieties may be chosen or altered such that they only appreciably bind to their target sequence (e.g., and do not appreciably bind the target sequence of another effector moiety).
  • an expression activation system comprises three or more expression activators and two or more expression activators comprise the same DNA- targeting moiety.
  • an expression activation system may comprise three expression activators, wherein the first and second expression activators both comprise a first DNA-targeting moiety and the third expression activator comprises a second different DNA- targeting moiety.
  • an expression activation system may comprise four expression activators, wherein the first and second expression activators both comprise a first DNA-targeting moiety and the third and fourth expression activators comprises a second different DNA-targeting moiety.
  • an expression activation system may comprise five expression activators, wherein the first and second expression activators both comprise a first DNA-targeting moiety, the third and fourth expression activators both comprise a second different DNA-targeting moiety, and the fifth expression activator comprises a third different DNA-targeting moiety.
  • the expression activators of the expression activation system each bind to a different target sequence described herein (e.g., the first, second, third, or further expression activators each bind DNA sequences that are different from one another).
  • an expression activation system may comprise a first expression activator and a FH12877464.1 Attorney Docket: OGY-02925 second expression activator wherein the first expression activator binds to a first target sequence described herein, and the second expression activator binds to a second target sequence described herein.
  • different can mean that: there is at least one position that is not identical between the target sequence bound by one expression activator and the target sequence bound by another expression activator, or that there is at least one position present in the target sequence bound by one expression activator that is not present in the target sequence bound by another expression activator.
  • the expression activators of the expression activation system each comprise a different effector domain (e.g., the first, second, third, or further expression activators each comprise a different effector domain from one another).
  • an expression activation system may comprise a first expression activator and a second expression activator wherein the first expression activator comprises a first effector moiety (e.g., comprising an epigenetic activating domain encoded by a fragment of gene MYC), and the second expression activator comprises a second effector moiety (e.g., comprising an epigenetic activating domain encoded by a fragment of gene MYBL1) or functional fragment thereof) different from the first effector moiety.
  • different can mean comprising distinct types of effector moiety.
  • different can mean comprising distinct variants of the same type of effector moiety.
  • an expression activation system comprises a first expression activator comprising a first effector moiety and a second expression activator comprising a second effector moiety, wherein the first effector moiety comprises an epigenetic activating domain encoded by a fragment of a gene selected from MYBL1, DUX4, MYB, CEPBPD, ATF4, NCOA2, TP53, FOXO1, NFE2, E2F2, UBN1, CREB3L1, FOXO3, KLF15, MLXIPL, LEUTX, INO80D, CR3L1, ATF6A, CSN9, FOXO6, CACTIN, C1QBP, KLF4, MYC, NUTM1, VP16, VP64, YLR278C, PIP2 and STB3, and the second effector moiety comprises a different epigenetic activating domain encoded by a fragment of a gene, wherein the gene is selected from MYBL1, DUX4, MYB, CEPBPD, ATF4, NCOA2, TP53
  • an expression activation system comprises a first expression activator comprising a first effector moiety and a second expression activator comprising a second effector moiety, wherein the first effector moiety comprises an epigenetic activating domain encoded by a fragment of a gene selected from MYBL1, DUX4, MYB, CEPBPD, FH12877464.1 Attorney Docket: OGY-02925 ATF4, NCOA2, TP53, FOXO1, NFE2, E2F2, UBN1, CREB3L1, FOXO3, KLF15, MLXIPL, LEUTX, INO80D, CR3L1, ATF6A, CSN9, FOXO6, CACTIN, C1QBP, KLF4, MYC, NUTM1, VP16, VP64, YLR278C, PIP2, STB3, and PA.v1b and the second effector moiety comprises a different epigenetic activating domain encoded by a fragment of a gene, wherein the gene is selected
  • an expression activation system comprises three or more expression activators and two or more expression activators comprise the same DNA- targeting moiety.
  • an expression activation system may comprise three expression activators, wherein the first and second expression activators both comprise a first effector moiety and the third expression activator comprises a second different effector moiety.
  • an expression activation system may comprise four expression activators, wherein the first and second expression activators both comprise a first effector moiety and the third and fourth expression activators comprises a second different effector moiety.
  • an expression activation system may comprise five expression activators, wherein the first and second expression activators both comprise a first effector moiety, the third and fourth expression activators both comprise a second different effector moiety, and the fifth expression activator comprises a third different effector moiety.
  • different can mean comprising different types of effector moiety or comprising distinct variants of the same type of effector moiety.
  • two or more (e.g., all) expression activators of an expression activation system are not covalently associated with each other, e.g., each expression activator is not covalently associated with any other expression activator.
  • two or more expression activators of an expression activation system are covalently associated with one another.
  • an expression activation system comprises a first expression activator and a second expression activator disposed on the same polypeptide, e.g., as a fusion molecule, e.g., connected by a peptide bond and optionally a linker.
  • the peptide is a self-cleaving peptide, e.g., a T2A self-cleaving peptide.
  • an expression activation system comprises a first expression activator and a second expression activator that are connected by a non-peptide bond, e.g., are conjugated to one another.
  • a protein or polypeptide of compositions of the present disclosure can be biochemically synthesized by employing standard solid phase techniques. Such methods include exclusive solid phase synthesis, partial solid phase synthesis methods, fragment condensation, classical solution synthesis. These methods can be used when a peptide is relatively short (e.g., 10 kDa) and/or when it cannot be produced by recombinant techniques (i.e., not encoded by a nucleic acid sequence) and therefore involves different chemistry.
  • Exemplary methods for producing an expression activator or polypeptide described herein involve expression in mammalian cells, although recombinant proteins can also be produced using insect cells, yeast, bacteria, or other cells under control of appropriate promoters.
  • Mammalian expression vectors may comprise non-transcribed elements such as an origin of replication, a suitable promoter, and other 5’ or 3’ flanking non-transcribed sequences, and 5’ or 3’ non-translated sequences such as necessary ribosome binding sites, a polyadenylation site, splice donor and acceptor sites, and termination sequences.
  • DNA sequences derived from the SV40 viral genome for example, SV40 origin, early promoter, splice, and polyadenylation sites may be used to provide other genetic elements required for expression of a heterologous DNA sequence.
  • Appropriate cloning and expression vectors for use with bacterial, fungal, yeast, and mammalian cellular hosts are described in Green & Sambrook, Molecular Cloning: A Laboratory Manual (Fourth Edition), Cold Spring Harbor Laboratory Press (2012).
  • large amounts of the expression activator or polypeptide are desired, it can be generated using techniques such as described by Brian Bray, Nature Reviews Drug Discovery, 2:587-593, 2003; and Weissbach & Weissbach, 1988, Methods for Plant Molecular Biology, Academic Press, NY, Section VIII, pp.421-463.
  • FH12877464.1 Attorney Docket: OGY-02925
  • mammalian cell culture systems can be employed to express and manufacture recombinant protein. Examples of mammalian expression systems include, without limitation, CHO cells, COS cells, HeLA and BHK cell lines.
  • compositions described herein may include a vector, such as a viral vector, e.g., a lentiviral vector, encoding a recombinant protein.
  • a vector e.g., a viral vector
  • compositions described herein may include a lipid nanoparticle encapsulating a vector, such as a viral vector, e.g., a lentiviral vector, encoding a recombinant protein.
  • a lipid nanoparticle encapsulating a vector e.g., a viral vector
  • Proteins comprise one or more amino acids.
  • Amino acids include any compound and/or substance that can be incorporated into a polypeptide chain, e.g., through formation of one or more peptide bonds.
  • an amino acid has the general structure H2N-C(H)I-COOH.
  • an amino acid is a naturally occurring amino acid.
  • an amino acid is a non-natural amino acid; in some embodiments, an amino acid is a D-amino acid; in some embodiments, an amino acid is an L- amino acid.
  • Standard amino acid refers to any of the twenty standard L-amino acids commonly found in naturally occurring peptides.
  • Nonstandard amino acid refers to any amino acid, other than the standard amino acids, regardless of whether it is prepared synthetically or obtained from a natural source.
  • an amino acid, including a carboxy- and/or amino-terminal amino acid in a polypeptide can contain a structural modification as compared with the general structure above.
  • an amino acid may be modified by methylation, amidation, acetylation, pegylation, glycosylation, phosphorylation, and/or substitution (e.g., of the amino group, the carboxylic acid group, one or more protons, and/or the hydroxyl group) as compared with the general structure.
  • such modification may, for example, alter the FH12877464.1 Attorney Docket: OGY-02925 circulating half-life of a polypeptide containing the modified amino acid as compared with one containing an otherwise identical unmodified amino acid.
  • such modification does not significantly alter a relevant activity of a polypeptide containing the modified amino acid, as compared with one containing an otherwise identical unmodified amino acid.
  • amino acid may be used to refer to a free amino acid; in some embodiments it may be used to refer to an amino acid residue of a polypeptide.
  • Nucleic Acids of the Disclosure In another aspect, provided herein are nucleic acids encoding an expression activator or an expression activation system of the present disclosure.
  • an expression activator may be provided via a composition comprising a nucleic acid encoding the expression activator, wherein the nucleic acid is associated with sufficient other sequences to achieve expression in a system of interest (e.g., in a particular cell, tissue, organism, etc.).
  • the present disclosure provides compositions of nucleic acids that encode an expression activator or fragment thereof.
  • nucleic acids may be or may include DNA, RNA, or any other nucleic acid moiety or entity as described herein and may be prepared by any technology described herein or otherwise available in the art (e.g., synthesis, cloning, amplification, in vitro or in vivo transcription, etc.).
  • provided nucleic acids that encode an expression activator or fragment thereof may be operationally associated with one or more replication, integration, and/or expression signals appropriate and/or sufficient to achieve integration, replication, and/or expression of the provided nucleic acid in a system of interest (e.g., in a particular cell, tissue, organism, etc.).
  • a composition for delivering an expression activator or an expression activation system described herein is or comprises a vector, e.g., a viral vector, comprising one or more nucleic acids encoding an expression activator or one or more components of an expression activator as described herein.
  • the present disclosure provides compositions of nucleic acids that encode an expression activator, one or more expression activators, or fragments thereof.
  • provided nucleic acids may be or include DNA, RNA, or any other nucleic acid moiety or entity as described herein, and may be prepared by any technology described herein or otherwise available in the art (e.g., synthesis, cloning, amplification, in FH12877464.1 Attorney Docket: OGY-02925 vitro or in vivo transcription, etc.).
  • the nucleic acid sequence may include, for example and without limitation, DNA, RNA, modified oligonucleotides (e.g., chemical modifications, such as modifications that alter the backbone linkages, sugar molecules, and/or nucleic acid bases), and artificial nucleic acids.
  • the nucleic acid sequence includes, for example and without limitation, genomic DNA, cDNA, peptide nucleic acids (PNA) or peptide oligonucleotide conjugates, locked nucleic acids (LNA), bridged nucleic acids (BNA), polyamides, triplex forming oligonucleotides, modified DNA, antisense DNA oligonucleotides, tRNA, mRNA, rRNA, modified RNA, miRNA, gRNA, and siRNA or other RNA or DNA molecules.
  • PNA peptide nucleic acids
  • LNA locked nucleic acids
  • BNA bridged nucleic acids
  • polyamides polyamides
  • provided nucleic acids encoding an expression activator, one or more expression activators, or polypeptide fragments thereof may be operationally associated with one or more replication, integration, and/or expression signals appropriate and/or sufficient to achieve integration, replication, and/or expression of the provided nucleic acid in a system of interest (e.g., in a particular cell, tissue, organism, etc.).
  • the nucleic acid sequence has a length from about 2 to about 5000 nts, about 10 to about 100 nts, about 50 to about 150 nts, about 100 to about 200 nts, about 150 to about 250 nts, about 200 to about 300 nts, about 250 to about 350 nts, about 300 to about 500 nts, about 10 to about 1000 nts, about 50 to about 1000 nts, about 100 to about 1000 nts, about 1000 to about 2000 nts, about 2000 to about 3000 nts, about 3000 to about 4000 nts, about 4000 to about 5000 nts, or any range therebetween.
  • a composition for delivering an expression activator or an expression activation system described herein is or comprises RNA, e.g., mRNA, comprising one or more nucleic acids encoding an expression activator as described herein.
  • a composition for delivering an expression activator or an expression activation system described herein is or comprises RNA, e.g., mRNA, comprising one or more components of an expression activator, as described herein.
  • a nucleic acid of the disclosure comprises nucleosides, e.g., purines or pyrimidines, e.g., adenine, cytosine, guanine, thymine, and uracil.
  • the nucleic acid sequence includes one or more nucleoside analogs.
  • the nucleoside analog includes, but is not limited to, a nucleoside analog, such as 5-fluorouracil; 5-bromouracil, 5-chlorouracil, 5-iodouracil, hypoxanthine, xanthine, 4-acetylcytosine, 4- methylbenzimidazole, 5-(carboxyhydroxylmethyl) uracil, 5-carboxymethylaminomethyl-2- thiouridine, 5-carboxymethylaminomethyluracil, dihydrouracil, dihydrouridine, beta-D- galactosylqueosine, inosine, N6-isopentenyladenine, 1-methylguanine, 1-methylinosine, 2,2- FH12877464.1 Attorney Docket: OGY-02925 dimethylguanine, 2-methyladenine, 2-methylguanine, 3-methylcytosine, 5-methylcytosine, N6-aden
  • RNA e.g., an mRNA, encoding an expression activator or an expression activation system as described herein.
  • an mRNA comprises an open reading frame (ORF), e.g., a sequence of codons that is translatable into a peptide or protein, e.g., into an expression activator or an expression activation system.
  • ORF open reading frame
  • Open Reading Frames An open reading frame includes a start codon at its 5'-end and a subsequent nucleotide region which usually exhibits a length which is a multiple of 3 nucleotides.
  • an ORF is terminated by a stop-codon (e.g., TAA, TAG, or TGA).
  • the ORF may be isolated, or it may be incorporated in a longer nucleic acid sequence, e.g., in a vector or an mRNA.
  • An ORF may also be known in the art as a protein coding region.
  • an mRNA of the disclosure comprises an ORF, e.g., encoding a DNA-targeting moiety and/or an effector domain of an expression activator or an expression activation system described herein.
  • an ORF comprises a sequence that has been sequence optimized. Sequence-optimized nucleotide sequences disclosed herein are distinct from the corresponding wild-type nucleotide acid sequences and from other known sequence-optimized nucleotide sequences, e.g., these sequence-optimized nucleic acids have unique compositional characteristics.
  • FH12877464.1 Attorney Docket: OGY-02925
  • the mRNA comprises a bicistronic RNA.
  • a bicistronic RNA is typically an RNA, preferably an mRNA, comprising two ORFs.
  • the mRNA comprises a multicistronic RNA.
  • a multicistronic RNA is typically an RNA, preferably an mRNA, comprising more than two ORFs.
  • the nucleic acid encoding the expression activation system is a multicistronic sequence.
  • the multicistronic sequence is a bicistronic sequence.
  • the multicistronic sequence comprises a sequence encoding the first expression activator and a sequence encoding the second expression activator.
  • the multicistronic sequence encodes a self-cleavable peptide sequence, e.g., a 2A peptide sequence, e.g., a T2A peptide sequence or a P2A sequence. In some embodiments, the multicistronic sequence encodes a T2A peptide sequence and a P2A peptide sequence. In some embodiments, a bicistronic construct further comprises a polyA tail.
  • a single mRNA transcript encoding the first expression activator, and the second expression activator are produced, which upon translation gets cleaved, e.g., after the glycine residue within the 2A peptide, to yield the first expression activator and the second expression activator as two separate proteins.
  • the first and the second expression activator are separated by “ribosome-skipping.”
  • the first expression activator and/ or the second expression activator retains a fragment of the 2A peptide after ribosome skipping.
  • the expression level of the first and second expression activator are equal.
  • the expression level of the first and the second expression activator are different.
  • the protein level of the first expression activator is within about 1%, 2%, 5%, or 10% of (greater than or less than) the protein level of the second expression activator.
  • a system encoded by a bicistronic nucleic acid increases expression of a target gene at least 1%, at least 2%, at least 3%, at least 4%, at least 5%, at least 10%, at least 20%, at least 30%, at least 40%, or at least 50%, in a cell, as compared to an otherwise similar system wherein the first and second expression activator are encoded by monocistronic nucleic acids.
  • a polynucleotide e.g., mRNA
  • a polynucleotide encoding an expression activator or an expression activation system of the present disclosure further comprises a 5' FH12877464.1 Attorney Docket: OGY-02925 UTR and/or a translation initiation sequence.
  • Natural 5 'UTRs bear features which function in initiation of protein translation. They harbor signatures, e.g., Kozak sequences, which are commonly involved in ribosomal initiation of translation of many genes.5 'UTRs also may form secondary structures that function in elongation factor binding to further facilitate translation.
  • Untranslated regions useful in the design and manufacture of polynucleotides include, for example and without limitation, those disclosed in International Patent Publication No. WO 2014/164253 (see also US 2016/0022840).
  • Other non-UTR sequences may be used as regions or subregions within the polynucleotides.
  • introns or fragments of introns sequences can be incorporated into regions of the polynucleotides.
  • incorporation of one or more intronic sequences may increase protein production and/or polynucleotide levels.
  • Combinations of features can be included in flanking regions and can be contained within other features.
  • an ORF can be flanked by a 5' UTR which can contain a strong Kozak translational initiation signal and/or a 3' UTR which can include an oligo(dT) sequence for templated addition of a poly-A tail.
  • 5' UTR can comprise a first polynucleotide fragment and a second polynucleotide fragment from the same and/or different genes such as the 5'UTRs described in U.S. Patent Application Publication No.2010/0293625.
  • a 5'UTR may comprise a sequence as set forth in SEQ ID NO:113.
  • a UTR, or a fragment thereof can be placed in the same orientation as in the transcript from which it was selected, or can be altered in orientation and/or location.
  • a 5' or 3' UTR can be inverted, shortened, lengthened, or made with one or more other 5' UTRs or 3' UTRs.
  • a UTR sequence can be changed in some way relative to a reference sequence, e.g., an endogenous UTR.
  • a 3' or 5' UTR can be altered relative to a wild-type or native UTR by a change in orientation or location, by inclusion of additional nucleotides, deletion of nucleotides, or swapping or transposition of nucleotides.
  • flanking regions e.g., flanking an ORF
  • flanking regions can be heterologous.
  • a 5' untranslated region can be derived from a different species than a FH12877464.1 Attorney Docket: OGY-02925 3' untranslated region.
  • the untranslated region can also include translation enhancer elements (TEE).
  • TEEs are described in U.S. Patent Application Publication No.2009/ 0226470.
  • a polynucleotide encoding an expression activator or an expression activation system further comprises a 3' UTR.
  • a 3'-UTR is the section of mRNA immediately following the translation termination codon.
  • a 3'-UTR includes regulatory regions that post-transcriptionally influence gene expression. Such regulatory regions within a 3'-UTR can influence polyadenylation, translation efficiency, localization, and/or stability of the mRNA.
  • a 3'- UTR comprises a binding site for regulatory proteins and/or microRNAs.
  • the 3'-UTR has a silencer region, which binds to activator proteins and inhibits the expression of the mRNA.
  • a 3'-UTR comprises an AU-rich element (ARE). Proteins may bind AREs to affect the stability and/or decay rate of mRNA.
  • a 3'-UTR comprises a sequence SEQ ID NO:114 that directs addition of adenine residues in a poly(A) tail to the end of the mRNA transcript.
  • a poly(A)tail comprises a sequence as set forth in SEQ ID NO:115. Terminal Modifications
  • an mRNA described herein comprises one or more terminal modifications, e.g., a 5 ⁇ Cap structure and/or a poly-A tail (e.g., between 100-200 nucleotides in length).
  • the 5 ⁇ cap structure may be selected from the group consisting of CapO, Capl, ARCA, inosine, Nl-methyl-guanosine, 2 ⁇ fluoro-guanosine, 7-deaza-guanosine, 8-oxo- guanosine, 2-amino-guanosine, LNA-guanosine, and 2-azido-guanosine.
  • the modified RNAs also contains a 5 ⁇ UTR comprising at least one Kozak sequence, and a 3 ⁇ UTR. Such modifications are known and are described, e.g., in WO 2012/135805 and WO 2013/052523.
  • the polynucleotide comprising an mRNA encoding an expression activator or an expression activation system of the present disclosure can further comprise a 5' cap.
  • the 5' cap can bind the mRNA Cap Binding Protein (CBP), thereby increasing mRNA stability.
  • CBP mRNA Cap Binding Protein
  • the cap can further assist the removal of 5' proximal introns removal during mRNA splicing.
  • a polynucleotide comprising an mRNA encoding an expression activator or an expression activation system of the present disclosure comprises a non-hydrolyzable cap structure preventing decapping.
  • a non- FH12877464.1 Attorney Docket: OGY-02925 hydrolyzable cap structure increases mRNA half-life. Cap structure hydrolysis requires cleavage of 5'-ppp-5' phosphorodiester linkages; thus, modified nucleotides can be used during the capping reaction.
  • Modified guanosine nucleotides may also be suitable for use in the present disclosure, e.g., a-thio-guanosine, a-methyl-phosphonate, and seleno-phosphate nucleotides.
  • a 5' cap comprises 2'-0-methylation of the ribose sugars at 5 '-terminal and/or 5'-anteterminal nucleotides at the 2'-hydroxyl group of the sugar ring.
  • a cap may include cap analogs, i.e., synthetic cap analogs, chemical caps, chemical cap analogs, or structural/functional cap analogs differing from naturally occurring (i.e., endogenous, wild-type, or physiological) 5'-caps in chemical structure.
  • Cap analogs may be chemically (i.e., non-enzymatically) or enzymatically synthesized and/or linked to the polynucleotides of the disclosure.
  • an mRNA encoding an expression activator or an expression activation system of the present disclosure can be capped after manufacture (e.g., IVT or chemical synthesis), using enzymes, to generate 5 '-cap structures.
  • 5' terminal caps can include endogenous caps or cap analogs.
  • a 5' terminal cap can comprise a guanine analog.
  • Suitable guanine analogs include, for example and without limitation, inosine, N1-methyl-guanosine, 2'fluoro- guanosine, 7-deaza-guanosine, 8-oxo-guanosine, 2-amino-guanosine, LNA-guanosine, and 2- azido- guanosine.
  • an mRNA encoding an expression activator or an expression activation system of the present disclosure further comprises a poly-A tail.
  • one or more terminal groups on the poly-A tail can be incorporated for stabilization.
  • Such poly-A tails can also include structural moieties or 2'-0-methyl modifications, for example, as taught by Li et al. (2005) Current Biology 15: 1501-1507.
  • a poly-A tail when present is greater than 30 nucleotides in length.
  • a poly-A tail is greater than 35 nucleotides in length (e.g., at least about 35, 40, 45, 50, 55, 60, 70, 80, 90, 100, 120, 140, 160, 180, 200, 250, 300, 350, 400, 450, 500, 600, 700, 800, 900, 1,000, 1,100, 1,200, 1,300, 1,400, 1,500, 1,600, 1,700, 1,800, 1,900, 2,000, 2,500, or 3,000 nucleotides)
  • a poly-A tail is designed relative to the length of the overall polynucleotide or the length of a particular region of the polynucleotide.
  • a poly-A tail can be 10, 20, 30, 40, 50, 60, 70, 80, 90, or 100% greater in length than the polynucleotide or fragment thereof.
  • one or more polynucleotides may be linked together by a Poly- A binding protein (PABP) by the 3'-end of the PABP, using modified nucleotides at the 3'- terminus of a poly-A tail.
  • PABP Poly- A binding protein
  • an mRNA encoding an expression activator or an expression activator of the present disclosure comprises, consists essentially of, or consists of a 5' terminal cap, a 5' UTR, an open reading frame (ORF), a 3' UTR, and a polyA tail.
  • a modified mRNA may be cyclized, or concatemerized, to generate a translation competent molecule to assist interactions between poly-A binding proteins and 5 ⁇ -end binding proteins.
  • the mechanism of cyclization or concatemerization may occur through at least 3 different routes: 1) chemical, 2) enzymatic, and 3) ribozyme catalyzed.
  • the newly formed 5 ⁇ -/3 ⁇ -linkage may be intramolecular or intermolecular.
  • nucleic acids as described herein or nucleic acids encoding an expression activator or an expression activation system described herein may be incorporated into a vector.
  • Vectors including those derived from retroviruses such as lentivirus, are suitable tools to achieve long-term gene transfer since they allow long-term, stable integration of a transgene, and its propagation in daughter cells.
  • suitable vectors include expression vectors, replication vectors, probe generation vectors, and sequencing vectors.
  • an expression vector may be provided to a cell in the form of a viral vector.
  • Viruses that are useful as vectors include, but are not limited to, retroviruses, adenoviruses, adeno-associated viruses, herpes viruses, and lentiviruses.
  • a suitable vector contains an origin of replication functional in at least one organism, a promoter sequence, convenient restriction endonuclease sites, and one or more selectable markers. Expression of natural or synthetic nucleic acids is typically achieved by operably linking a nucleic acid encoding the gene of interest to a promoter and incorporating the construct into an expression vector.
  • Vectors can be suitable for replication and integration in eukaryotes.
  • Typical cloning vectors contain transcription and translation terminators, initiation sequences, and promoters useful for expression of the desired nucleic acid FH12877464.1 Attorney Docket: OGY-02925 sequence. Additional promoter elements, e.g., enhancing sequences, may regulate frequency of transcriptional initiation. Typically, these sequences are located in a region 30-110 bp upstream of a transcription start site, although a number of promoters have recently been shown to contain functional elements downstream of transcription start sites as well. In some embodiments, an expression activator or an expression activation system described herein acts at an enhancing sequence.
  • the enhancing sequence is an enhancer, a stretch enhancer, a shadow enhancer, a locus control region (LCR), or a super enhancer.
  • the super enhancer comprises a cluster of enhancers and other regulatory elements. In some embodiments, these sequences are located in a region .2- 2 Mb upstream or downstream of a transcription start site. In some embodiments, the region is a non-coding region. In some embodiments, the region is associated with long-range regulation of a target gene. In some embodiments, the regions are cell-type specific. In some embodiments, a super-enhancer modifies (e.g., increases) target gene expression by recruiting the target gene promoter.
  • the super enhancer interacts with a target gene promoterthrough an enhancer docking site.
  • the enhancer docking site is an anchor sequence.
  • the enhancer docking site is located at least 100 bp, 200 bp, 500 bp, 1000 bp, 1500 bp, 2000 bp, or 3000 bp away from the target gene promoter.
  • a super enhancer region is at least 100 bp, at least 200 bp, at least 300 bp, at least 500 bp, at least 1 kb, at least 2 kb, at least 3 kb, at least 5 kb, at least 10 kb, at least 15 kb, at least 20 kb, or at least 25 kb long. Spacing between promoter elements frequently is flexible, so that promoter function is preserved when elements are inverted or moved relative to one another. For example, in a thymidine kinase (tk) promoter, spacing between promoter elements can be increased to about 50 bp apart before activity begins to decline.
  • tk thymidine kinase
  • a suitable promoter for use in the present disclosure is the immediate early cytomegalovirus (CMV) promoter sequence.
  • CMV immediate early cytomegalovirus
  • This promoter sequence is a strong constitutive promoter sequence capable of driving high levels of expression of any polynucleotide sequence operatively linked thereto.
  • a suitable promoter is Elongation Growth Factor-1a (EF-1a).
  • constitutive promoter sequences may also be used, including, but not limited to, the simian virus 40 (SV40) early promoter, mouse mammary tumor virus (MMTV), human immunodeficiency virus (HIV) FH12877464.1 Attorney Docket: OGY-02925 long terminal repeat (LTR) promoter, MoMuLV promoter, an avian leukemia virus promoter, an Epstein-Barr virus immediate early promoter, a Rous sarcoma virus promoter, as well as human gene promoters including, but not limited to, an actin promoter, a myosin promoter, a hemoglobin promoter, and a creatine kinase promoter.
  • SV40 simian virus 40
  • MMTV mouse mammary tumor virus
  • HV human immunodeficiency virus
  • LTR long terminal repeat
  • MoMuLV promoter MoMuLV promoter
  • an avian leukemia virus promoter an Epstein-Barr virus immediate early promoter
  • inducible promoters are contemplated as part of the present disclosure.
  • use of an inducible promoter provides a molecular switch capable of turning on expression of a polynucleotide sequence to which it is operatively linked, when such expression is desired.
  • use of an inducible promoter provides a molecular switch capable of turning off expression when expression is not desired.
  • an expression vector to be introduced can also contain either a selectable marker gene or a reporter gene or both to facilitate identification and selection of expressing cells from the population of cells sought to be transfected or infected through viral vectors.
  • a selectable marker may be carried on a separate piece of DNA and used in a co-transfection procedure. Both selectable markers and reporter genes may be flanked with appropriate transcriptional control sequences to enable expression in the host cells.
  • reporter genes may be used for identifying potentially transfected cells and/or for evaluating the functionality of transcriptional control sequences.
  • a reporter gene is a gene that is not present in or expressed by a recipient source (of a reporter gene) and that encodes a polypeptide whose expression is manifested by some easily detectable property, e.g., enzymatic activity or visualizable fluorescence. Expression of a reporter gene is assayed at a suitable time after the DNA has been introduced into the recipient cells.
  • Suitable reporter genes may include genes encoding luciferase, beta- galactosidase, chloramphenicol acetyl transferase, secreted alkaline phosphatase, or the green fluorescent protein gene (e.g., Ui-Tei et al., 2000 FEBS Letters 479: 79-82).
  • Suitable expression systems are well known and may be prepared using known techniques or obtained commercially.
  • a construct with a minimal 5’ flanking region that shows highest level of expression of reporter gene is identified as a promoter.
  • Such promoter regions may FH12877464.1 Attorney Docket: OGY-02925 be linked to a reporter gene and used to evaluate agents for ability to modulate promoter- driven transcription.
  • Cells The present disclosure is further directed, in part, to cells comprising an expression activator or expression activation system described herein.
  • Any cell e.g., cell line, e.g., a cell line suitable for expression of a recombinant polypeptide, known to one of skill in the art is suitable to comprise an expression activator or an expression activation system described herein.
  • a cell e.g., cell line
  • a cell e.g., cell line
  • a cell may be used to express or amplify a nucleic acid, e.g., a vector, encoding an expression activator or an expression activation system, e.g., expression activator(s), described herein.
  • a cell comprises a nucleic acid encoding an expression activator or an expression activation system, e.g., expression activator(s), described herein.
  • a cell comprises a first nucleic acid encoding a first component of an expression activation system, e.g., a first expression activator, and a second nucleic acid encoding a second component of the expression activation system, e.g., a second expression activator.
  • a cell comprises nucleic acid encoding an expression activation system comprising two or more expression activators
  • the sequences encoding each expression activator are disposed on separate nucleic acid molecules, e.g., on different vectors, e.g., a first vector encoding a first expression activator and a second vector encoding a second expression activator.
  • the sequences encoding each expression activator are disposed on the same nucleic acid molecule, e.g., on the same vector. In some embodiments, some or all of the nucleic acid encoding the expression activation system is integrated into the genomic DNA of the cell. In some embodiments, the nucleic acid encoding a first expression activator of an expression activation system is integrated into the genomic DNA of a cell, and the nucleic acid encoding a second expression activator of an expression activation system is not integrated into the genomic DNA of a cell (e.g., is situated on a vector).
  • the nucleic acid(s) encoding a first and a second expression activator of an expression activation system are integrated into the genomic DNA of a cell, e.g., at the same (e.g., adjacent or colocalized) or different sites in the genomic DNA.
  • FH12877464.1 Attorney Docket: OGY-02925
  • Examples of cells that may comprise and/or express an expression activation system or an expression activator described herein include, but are not limited to, hepatocytes, neuronal cells, endothelial cells, myocytes, and lymphocytes.
  • modified RNAs are made using in vitro transcription (IVT) enzymatic synthesis.
  • IVT in vitro transcription
  • Methods of making IVT polynucleotides are known in the art and are described in WO 2013/151666, WO 2013/151668, WO 2013/151663, WO 2013/151669, WO 2013/151670, WO 2013/151664, WO 2013/151665, WO 2013/151671, WO 2013/151672, WO 2013/151667, and WO 2013/151736.
  • Methods of purification include purifying an RNA transcript comprising a polyA tail by contacting the sample with a surface linked to a plurality of thymidines or derivatives thereof and/or a plurality of uracils or derivatives thereof (polyT/U) under conditions such that the RNA transcript binds to the surface and eluting the purified RNA transcript from the surface (WO 2014/152031); using ion (e.g., anion) exchange chromatography that allows for separation of longer RNAs up to 10,000 nucleotides in length via a scalable method (WO 2014/144767); and subjecting a modified RMNA sample to DNAse treatment (WO 2014/152030).
  • ion e.g., anion
  • RNAs encoding proteins in the fields of human disease, antibodies, viruses, and a variety of in vivo settings are known and are disclosed in for example, Table 6 of International Publication Nos. WO 2013/151666, WO 2013/151668, WO 2013/151663, WO 2013/151669, WO 2013/151670, WO 2013/151664, WO 2013/151665, WO 2013/151736; Tables 6 and 7 International Publication No. WO 2013/151672; Tables 6, 178 and 179 of International Publication No. WO 2013/151671; and Tables 6, 185 and 186 of International Publication No WO 2013/151667.
  • an expression activator comprises or consists of a protein and may thus be produced by methods of making proteins as known in the art, for example. as provided in the present disclosure.
  • an expression activation system e.g., the expression activator(s) of an expression activation system, comprise one or more proteins and may thus be produced by methods of making proteins.
  • Nanoparticles include particles with a dimension (e.g., diameter) between about 1 and about 1000 nanometers, between about 1 and about 500 nanometers in size, between about 1 and about 100 nm, between about 30 nm and about 200 nm, between about 50 nm and about 300 nm, between about 75 nm and about 200 nm, between about 100 nm and about 200 nm, and any range therebetween.
  • a nanoparticle has a composite structure of nanoscale dimensions.
  • nanoparticles are typically spherical although different morphologies are possible depending on the nanoparticle composition. The portion of the nanoparticle contacting an environment external to the nanoparticle is generally identified as the surface of the nanoparticle.
  • nanoparticles have a greatest dimension ranging between 25 nm and 200 nm.
  • Nanoparticles as described herein comprise delivery systems that may be provided in any form, including but not limited to solid, semi-solid, emulsion, or colloidal nanoparticles.
  • a nanoparticle delivery system may include but is not limited to lipid-based systems, liposomes, micelles, micro-vesicles, exosomes, or gene gun.
  • the nanoparticle is a lipid nanoparticle (LNP).
  • the LNP is a particle that comprises a plurality of lipid molecules physically associated with each other by intermolecular forces.
  • an LNP may comprise multiple components, e.g., 3-4 components.
  • the expression activator or a pharmaceutical composition comprising said expression activator is encapsulated in an LNP.
  • the expression activation system or a pharmaceutical composition comprising said expression activation system is encapsulated in an LNP.
  • the nucleic acid encoding the first expression FH12877464.1 Attorney Docket: OGY-02925 activator and the nucleic acid encoding the second expression activator are present in the same LNP.
  • the nucleic acid encoding the first expression activator and the nucleic acid encoding the second expression activator are present in different LNPs. Preparation of LNPs and the modulating agent encapsulation may be used/and or adapted from Rosin et al, Molecular Therapy, vol.19, no.12, pages 1286-2200, December 2011.
  • lipid nanoparticle compositions disclosed herein are useful for expression of a protein encoded by mRNA.
  • nucleic acids when present in the lipid nanoparticles, are resistant in aqueous solution to degradation with a nuclease.
  • the LNP formulations may include a CCD lipid, a neutral lipid, and/or a helper lipid.
  • the LNP formulation comprises an ionizable lipid.
  • an ionizable lipid may be a cationic lipid, an ionizable cationic lipid, or an amine- containing lipid that can be readily protonated.
  • the lipid is a cationic lipid that can exist in a positively charged or neutral form depending on pH.
  • the cationic lipid is a lipid capable of being positively charged, e.g., under physiological conditions.
  • the lipid particle comprises a cationic lipid in formulation with one or more of neutral lipids, ionizable amine-containing lipids, biodegradable alkyl lipids, steroids, phospholipids including polyunsaturated lipids, structural lipids (e.g., sterols), PEG, cholesterol, and polymer conjugated lipids.
  • LNP formulation e.g., MC3 and/or SSOP
  • LNP formulation includes cholesterol, PEG, and/or a helper lipid.
  • the LNPs may be, e.g., microspheres (including unilamellar and multilamellar vesicles, lamellar phase lipid bilayers that, in some embodiments, are substantially spherical).
  • the LNP can comprise an aqueous core, e.g., comprising a nucleic acid encoding an expression activator or a system as disclosed herein and referred to herein as “cargo.”
  • the cargo for the LNP formulation includes at least one guide RNA.
  • the cargo e.g., a nucleic acid encoding an expression activator, or a system as disclosed herein
  • an LNP e.g., an LNP comprising a cationic lipid.
  • the cargo e.g., a nucleic acid encoding an expression activator, or a system as disclosed herein
  • the cargo may be associated with the LNP.
  • the cargo e.g., a nucleic acid encoding an expression activator, or a system as disclosed herein, may be encapsulated, e.g., fully encapsulated and/or partially encapsulated in an LNP.
  • an LNP comprising a cargo may be administered for systemic delivery, e.g., delivery of a therapeutically effective dose of cargo that can result in a broad exposure of an active agent within an organism.
  • Systemic delivery of lipid nanoparticles can be by any means known in the art including, for example and without limitation, intravenous, intraarterial, subcutaneous, and intraperitoneal delivery.
  • systemic delivery of lipid nanoparticles is by intravenous delivery.
  • an LNP comprising a cargo may be administered for local delivery, e.g., delivery of an active agent directly to a target site within an organism.
  • an LNP may be locally delivered into a disease site, e.g., a tumor, or other target site, e.g., a site of inflammation, or to a target organ, e.g., the liver, lung, stomach, colon, pancreas, uterus, breast, lymph nodes, and the like.
  • a target organ e.g., the liver, lung, stomach, colon, pancreas, uterus, breast, lymph nodes, and the like.
  • an LNP as disclosed herein may be locally delivered to a specific cell, e.g., lung, pancreas, and/or epithelial cells.
  • an LNP as disclosed herein may be locally delivered to a specific site, e.g., a tumor site, e.g., by subcutaneous or orthotopic administration.
  • the LNPs may be formulated as a dispersed phase in an emulsion, micelles, or an internal phase in a suspension.
  • the LNPs are biodegradable.
  • the LNPs do not accumulate to cytotoxic levels or cause toxicity in vivo at a therapeutically effective dose.
  • the LNPs do not accumulate to cytotoxic levels or cause toxicity in vivo after repeat administrations at a therapeutically effective dose.
  • the LNPs do not cause an innate immune response that leads to a substantially adverse effect at a therapeutically effective dose.
  • the LNP used comprises the formula (6Z,9Z,28Z,31Z)- heptatriacont-6,9,28,31-tetraene-19-y14-(dimethylamino)butanoate or ssPalm0-phenyl-P4C2 (ssPalmO-Phe, SS-OP).
  • the LNP formulation comprises the formula, (6Z,9Z,28Z,31Z)-heptatriacont-6,9,28,31-tetraene-19-yl4-(dimethylamino)butanoate(MC3), 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), Cholesterol, 1,2-dimyristoyl-rac-glycero- 3-methoxypolyethylene glycol-2000(PEG2k-DMG), e.g., MC3 LNP or ssPalmO-phenyl- P4C2 (ssPalmO-Phe, SS-OP), 1,2-dioleoyl-sn-glycero-3-phosphocholine(DOPC), Cholesterol, 1,2-dimyristoyl-rac-glycero-3-methoxypolyethylene glycol-2000(PEG2k-DMG), e.g., SSOP
  • Liposomes are spherical vesicle structures composed of a uni- or multilamellar lipid bilayer surrounding internal aqueous compartments and a relatively impermeable outer lipophilic phospholipid bilayer. Liposomes may be anionic, neutral, or cationic. Liposomes are biocompatible, nontoxic, can deliver both hydrophilic and lipophilic drug molecules, FH12877464.1 Attorney Docket: OGY-02925 protect their cargo from degradation by plasma enzymes, and transport their load across biological membranes and the blood brain barrier (BBB) (see, e.g., Spuch and Navarro, Journal of Drug Delivery, vol.2011, Article ID 469679, 12 pages, 2011).
  • BBB blood brain barrier
  • Vesicles can be made from several different types of lipids; however, phospholipids are most used to generate liposomes as drug carriers. Vesicles may comprise, for example and without limitation, DOTMA, DOTAP, DOTIM, DDAB, alone or together with cholesterol to yield DOTMA and cholesterol, DOTAP and cholesterol, DOTIM and cholesterol, and DDAB and cholesterol.
  • Methods for preparation of multilamellar vesicle lipids are known in the art (see, for example, U.S. Pat. No.6,693,086, the teachings of which relating to multilamellar vesicle lipid preparation are incorporated herein by reference).
  • vesicle formation can be spontaneous when a lipid film is mixed with an aqueous solution, it can also be expedited by applying force in the form of shaking by using a homogenizer, sonicator, or an extrusion apparatus (see, e.g., Spuch and Navarro, Journal of Drug Delivery, vol.2011, Article ID 469679, 12 pages, 2011).
  • Extruded lipids can be prepared by extruding through filters of decreasing size, as described in Templeton et al, Nature Biotech, 15:647-652, 1997, the teachings of which relate to extruded lipid preparation are incorporated herein by reference.
  • Viral Vectors In some embodiments, viral vector systems which can be utilized with the methods and compositions described herein.
  • Suitable viral vector systems for use include, for example and without limitation, (a) adenovirus vectors (e.g., an Ad5/F35 vector); (b) retrovirus vectors, including but not limited to lentiviral vectors (including integration competent or integration-defective lentiviral vectors), moloney murine leukemia virus, etc.; (c) adeno- associated virus vectors; (d) herpes simplex virus vectors; (e) SV 40 vectors; (f) polyoma virus vectors; (g) papilloma virus vectors; (h) picornavirus vectors; (i) pox virus vectors such as an orthopox, e.g., vaccinia virus vectors or avipox, e.g.
  • adenovirus vectors e.g., an Ad5/F35 vector
  • retrovirus vectors including but not limited to lentiviral vectors (including integration competent or integration-defective lentiviral vectors),
  • the constructs can include viral sequences for transfection, if desired.
  • the construct can be incorporated into vectors capable of episomal replication, e.g., EPV and EBV vectors. See, e.g., U.S. Patent Nos.6,534,261; 6,607,882; 6,824,978; 6,933,113; 6,979,539; 7,013,219; and 7,163,824, the entire contents of each of which is incorporated by reference herein.
  • Vectors including those derived from retroviruses such as lentivirus, are FH12877464.1 Attorney Docket: OGY-02925 suitable tools to achieve long- term gene transfer since they allow long-term, stable integration of a transgene and its propagation in daughter cells.
  • vectors include expression vectors, replication vectors, probe generation vectors, and sequencing vectors.
  • an expression vector may be provided to a cell in the form of a viral vector.
  • Viral vector technology is known in the art and described in a variety of virology and molecular biology manuals.
  • a suitable viral vector for use in the present invention is an adeno-associated viral vector, such as a recombinant adeno-associated viral vector.
  • Recombinant adeno-associated virus vectors are gene delivery systems based on the defective and nonpathogenic parvovirus adeno-associated type 2 virus.
  • the vectors are derived from a plasmid that retains only the AAV 145 bp inverted terminal repeats flanking the transgene expression cassette. Efficient gene transfer and stable transgene delivery due to integration into the genomes of the transduced cell are key features for this vector system. (Wagner et al., Lancet 351:91171702-3 (1998), Kearns et al., Gene Ther.9:748-55 (1996)).
  • AAV serotypes including AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8 and AAV9, can be used in accordance with the present invention.
  • Replication-deficient recombinant adenoviral vectors can be produced at high titer and readily infect a number of different cell types.
  • Most adenovirus vectors are engineered such that a transgene replaces the Ad E1a, E1b, and/or E3 genes; subsequently, the replication defective vector is propagated in a suitable cell system, e.g., HEK293 and variants thereof, that supply deleted gene function in trans.
  • Ad vectors can transduce multiple types of tissues in vivo, including nondividing, differentiated cells, such as those found in liver, kidney, and muscle. Conventional Ad vectors have a large carrying capacity.
  • An example of the use of an Ad vector in a clinical trial involved polynucleotide therapy for antitumor immunization with intramuscular injection (Sterman et al., Hum. Gene Ther.7:1083-9 (1998)). Additional examples of the use of adenovirus vectors for gene transfer in clinical trials include Rosenecker et al., Infection 24:15-10 (1996); Sterman et al., Hum. Gene Ther.9:71083-1089 (1998); Welsh et al., Hum.
  • Packaging cells are used to form virus particles that are capable of infecting a host cell. Such cells include, for example and without limitation, HEK293 cells, and variants thereof, ⁇ 2 cells, and PA317 cells.
  • Viral vectors used in gene therapy are usually generated by a producer cell line that packages a nucleic acid vector into a viral particle.
  • the vectors FH12877464.1 Attorney Docket: OGY-02925 typically contain the minimal viral sequences required for packaging and subsequent integration into a host (if applicable), other viral sequences being replaced by an expression cassette encoding the protein to be expressed.
  • the missing viral functions are supplied in trans by the packaging cell line.
  • AAV vectors used in gene therapy typically only possess inverted terminal repeat (ITR) sequences from the AAV genome which are required for packaging and integration into the host genome.
  • viral DNA is packaged in a cell line, which contains a helper plasmid encoding the other AAV genes, namely rep and cap, but lacking ITR sequences.
  • the cell line is also infected with adenovirus as a helper.
  • the helper virus promotes replication of the AAV vector and expression of AAV genes from the helper plasmid.
  • the helper plasmid is not packaged in significant amounts due to a lack of ITR sequences.
  • contamination with adenovirus can be reduced by, e.g., heat treatment to which adenovirus is more sensitive than AAV.
  • the present disclosure provides a method for introducing one or more epigenetic modifications (e.g., DNA demethylation and/or histone modification) to a site in a region of the genome comprising a target gene in a cell or a population of cells, comprising contacting the cell or the population of cells with a dose of an expression activator described herein or a nucleic acid encoding the expression activator (e.g., an LNP-formulated expression activator or an LNP-formulated nucleic acid encoding the expression activator, wherein the expression activator comprises (i) a DNA-targeting moiety (e.g., a TALE, a ZFN, a dCas9/gRNA) that binds a target sequence described herein, and (ii) an effector domain described herein.
  • a DNA-targeting moiety e.g., a TALE, a ZFN, a dCas9/gRNA
  • the present disclosure provides a method for introducing one or more epigenetic modifications (e.g., DNA demethylation and/or histone modification) to a site in a region of the genome comprising a target gene in a cell or a population of cells, comprising contacting the cell or the population of cells with a dose of an expression activation system described herein comprising at least one expression activator (e.g., 1, 2, 3, 4, 5 or more expression activators described herein) or a nucleic acid encoding the at least one expression activator (e.g., an expression activation system comprising at least one LNP-formulated expression activator or an LNP-formulated nucleic acid encoding the at least one expression activator), wherein the at least one expression activator comprises (i) a DNA-targeting moiety FH12877464.1 Attorney Docket: OGY-02925 (e.g., a TALE, a ZFN, a dCas9/gRNA) that binds a target sequence described here
  • the present disclosure provides a method for introducing one or more epigenetic modifications to a site in a region of the genome comprising the target gene in a subject, comprising administering to the subject a dose of an expression activator described herein or a nucleic acid encoding the expression activator (e.g., an LNP-formulated expression activator or an LNP-formulated nucleic acid encoding the expression activator), wherein the expression activator comprises (i) a DNA-targeting moiety (e.g., a TALE, a ZFN, a dCas9/gRNA) that binds a target sequence described herein, and (ii) an effector domain described herein.
  • a DNA-targeting moiety e.g., a TALE, a ZFN, a dCas9/gRNA
  • the present disclosure provides a method of introducing one or more epigenetic modifications to a site in a region of the genome comprising the target gene in a subject, comprising administering to the subject a dose of an expression activation system described herein comprising at least one expression activator (e.g., 1, 2, 3, 4, 5 or more expression activators described herein) or a nucleic acid encoding the at least one expression activator (e.g., an expression activation system comprising at least one LNP-formulated expression r activator or an LNP-formulated nucleic acid encoding the at least one expression activator), wherein the at least one expression activator comprises (i) a DNA-targeting moiety (e.g., a TALE, a ZFN, a dCas9/gRNA) that binds a target sequence described herein, and (ii) an effector domain described herein.
  • a DNA-targeting moiety e.g., a TALE, a ZFN, a dCa
  • epigenetic modification at the site is increased as compared to prior to the contacting or as compared to a control cell or control population of cells not contacted with the expression activator or expression activation system. In some embodiments, epigenetic modification at the site is decreased as compared to prior to the contacting or as compared to a control cell or control population of cells not contacted with the expression activator or expression activation system. In some embodiments, the increase or decrease of the epigenetic modification at the site is associated with increased expression of the target gene in the cell or the population of cells. In some embodiments, the epigenetic modification comprises DNA demethylation, wherein an increase in DNA demethylation at the site is associated with increased expression of the target gene in the cell or the population of cells.
  • the epigenetic modification comprises a histone modification, wherein a decrease in the histone modification at the site is associated with increased expression of the target gene in the cell or the population of cells.
  • the epigenetic modification comprises a histone modification, wherein an increase in the histone FH12877464.1 Attorney Docket: OGY-02925 modification at the site is associated with increased expression of the target gene in the cell or the population of cells.
  • the histone modification comprises histone acetylation, wherein an increase in histone acetylation is associated with increased expression of the target gene in the cell or the population of cells.
  • the histone modification comprises histone methylation, wherein an increase in histone methylation is associated with increased expression of the target gene in the cell or the population of cells.
  • Methods to measure histone modification are known in the art.
  • methods to detect histone modification of genomic DNA include, but are not limited to, mass spectrometry and genomic approaches based upon chromatin immunoprecipitation (ChIP) in combination with DNA microarray (i.e., ChIP-chip), high- throughput sequencing (i.e., ChIP-seq), or serial analysis of gene expression (ChIP-SAGE).
  • ChIP chromatin immunoprecipitation
  • ChIP-chip DNA microarray
  • ChIP-seq high- throughput sequencing
  • serial analysis of gene expression ChIP-SAGE
  • Methods to measure DNA methylation are known in the art, including, but not limited to, mass spectrometry, methylation-specific PCR, sequencing based-assay such as bisulfite sequencing, the Hpall tiny fragment Enrichment by Ligation-mediated PCR (HELP) assay, GLAD-PCR assay, ChIP-on-chip assay, restriction landmark genomic scanning, methylated DNA immunoprecipitation, methyl sensitive southern blotting, high resolution Melt analysis, and methylation sensitive single nucleotide primer extension assay.
  • the method to measure DNA methylation of a target gene comprises use of a DNA methylation microarray (e.g., an Illumina Methylation Array).
  • the method comprises a sequencing-based assay, wherein genomic DNA is treated with an agent prior to sequencing that converts cytosine residues to uracil (or another base having distinct hybridization properties from cytosine), but does not affect 5-methylcytosine residues.
  • agents are known in the art and include bisulfite, hydrogen sulfite, disulfite, and combinations thereof.
  • DNA treated with bisulfite retains the methylated cytosines, but not unmethylated cytosines.
  • the treated DNA is then subjected to sequencing analysis (see, e.g., Campan et al (2009) Methods Mol Biol 507:325-37; Adusumalli, et al (2015) Brief Bioinform 16:369-79).
  • Exemplary methods for sequencing analysis are known in the art and include use of next generation sequencing platforms based on sequencing-by-synthesis or sequencing-by- ligation as employed by Illumina, Life Technologies, and Roche; or based on nanopore sequencing or electronic-detection as employed by Ion Torrent technology.
  • the method to measure DNA methylation comprises enzymatic methyl-seq (EM-seq) (see, e.g., Vaisvila et al (2021) Genome Res 31:1280).
  • enzymatic reactions are used to convert 5-methylcytosine (5mC) and 5-hydroxymethylcytosine (i.e., the oxidation product of 5mC; also referred to as 5hmC) into products resistant to an enzymatic reaction that deaminates unmodified cytosines by converting them to uracils (e.g., performed using APOBEC3A).
  • the enzymatically processed DNA is then amplified by PCR using EM-seq adaptor primers and subjected to sequencing analysis, e.g., using Illumina sequencing.
  • the present disclosure provides a method of modulating, e.g., increasing, expression of a target gene.
  • the method comprises providing an expression activator (or a nucleic acid encoding the same, or a pharmaceutical composition comprising said expression activator nucleic acid) or an expression activation system described herein (or a nucleic acid encoding the same, or pharmaceutical composition comprising said expression activation system or nucleic acid), and contacting the target gene, and/or operably linked transcription control element(s) with the expression activator or the expression activation system.
  • modulating, e.g., increasing expression of a target gene comprises modulation of transcription of a target gene as compared with a reference value, e.g., transcription of a target gene in absence of the expression activator or the expression activation.
  • the method of modulating, e.g., increasing, expression of a target gene are used ex vivo, e.g., on a cell from a subject, e.g., a mammalian subject, e.g., a human subject.
  • the methods of modulating, e.g., increasing, expression of a target gene are used in vivo, e.g., on a mammalian subject, e.g., a human subject. In some embodiments, the method of modulating, e.g., increasing, expression of a target gene are used in vitro, e.g., on a cell or cell line as described herein.
  • the present disclosure provides a method of modulating (e.g., increasing) expression of a target gene in a cell or a population of cells, comprising contacting the cell or the population of cells with a dose of an expression activator described herein or a nucleic acid encoding the expression activator (e.g., an LNP-formulated expression activator or an LNP-formulated nucleic acid encoding the expression activator), wherein the expression activator comprises (i) a DNA-targeting moiety (e.g., a TALE, a ZFN, a dCas9/gRNA) that binds a target sequence described herein, and (ii) an effector domain described herein.
  • a DNA-targeting moiety e.g., a TALE, a ZFN, a dCas9/gRNA
  • the present disclosure provides a method of modulating (e.g., increasing) expression of a target gene in a cell or a population of cells, comprising contacting the cell or the population of cells with a dose of an expression activation system described herein comprising at least one expression activator (e.g., 1, 2, 3, 4, 5 or more expression activators described herein) or a nucleic acid encoding the at least one expression activator (e.g., an expression activation system comprising at least one LNP-formulated expression activator or an LNP-formulated nucleic acid encoding the at least one expression activator), wherein the at least one expression activator comprises (i) a DNA-targeting moiety (e.g., a TALE, a ZFN, a dCas9/gRNA) that binds a target sequence described herein, and (ii) an effector domain described herein.
  • a DNA-targeting moiety e.g., a TALE, a ZFN, a dCa
  • expression of a target gene is increased as compared to prior to the contacting or as compared to a control cell or control population of cells not contacted with the expression activator or the expression activation system.
  • the contacting is ex vivo.
  • the contacting is in vivo.
  • the expression of the target gene is measured by harvesting the cell or population of cells at a time point following the contacting, obtaining the transcriptional or translational product of the target gene in the cell or the population of cells, and quantifying a level of the transcriptional or translation product as compared to a control cell or control population of cells (e.g., a cell or population of cells not contacted with the expression activator or the expression activation system).
  • expression of the target gene is measured by quantifying the level of mRNA as compared to the control cell or control population of cells.
  • the level of mRNA is increased by at least about 5%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, or about 50% as compared to the control cell or control population of cells.
  • the level of mRNA is increased by about 1.1-fold to about 10- fold, by about 1.2-fold to about 10-fold, by about 1.3-fold to about 10-fold, by about 1.4-fold to about 10-fold, by about 1.5-fold to about 10-fold, by about 2-fold to about 10-fold, by about 3-fold to about 10-fold, by about 4-fold to about 10-fold, or by about 5-fold to about 10-fold as compared to the control cell or control population of cells.
  • expression of the target gene is measured by quantifying the level of polypeptide expressed by the gene as compared to the control cell or control population of cells.
  • the level of polypeptide is increased by at least about 5%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, or about 50% as compared to the control tissue.
  • the level of polypeptide is increased FH12877464.1 Attorney Docket: OGY-02925 by about 1.1-fold to about 10-fold, by about 1.2-fold to about 10-fold, by about 1.3-fold to about 10-fold, by about 1.4-fold to about 10-fold, by about 1.5-fold to about 10-fold, by about 2-fold to about 10-fold, by about 3-fold to about 10-fold, by about 4-fold to about 10-fold, or by about 5-fold to about 10-fold as compared to the control cell or control population of cells.
  • the present disclosure provides a method of modulating (e.g., increasing) expression of a target gene in a target tissue in vivo, comprising administering to a subject a dose of an expression activator described herein or a nucleic acid encoding the expression activator(e.g., an LNP-formulated expression activator or an LNP-formulated nucleic acid encoding the expression activator, wherein the expression activator comprises (i) a DNA-targeting moiety (e.g., a TALE, a ZFN, a dCas9/gRNA) that binds a target sequence described herein, and (ii) an effector domain described herein.
  • a DNA-targeting moiety e.g., a TALE, a ZFN, a dCas9/gRNA
  • the present disclosure provides a method of modulating (e.g., increasing) expression of a target gene in a target tissue in vivo, comprising administering to a subject a dose of an expression activation system described herein comprising at least one expression activator (e.g., 1, 2, 3, 4, 5 or more expression activators described herein) or a nucleic acid encoding the at least one expression activator (e.g., an expression activation system comprising at least one LNP-formulated expression activator or an LNP-formulated nucleic acid encoding the at least one expression activator), wherein the at least one expression activator comprises (i) a DNA-targeting moiety (e.g., a TALE, a ZFN, a dCas9/gRNA) that binds a target sequence described herein, and (ii) an effector domain described herein.
  • a DNA-targeting moiety e.g., a TALE, a ZFN, a dCas9/gRNA
  • expression of a target gene is measured by harvesting the target tissue at a time point following the administering, obtaining the transcriptional or translational product of the target gene in the target tissue, and quantifying a level of the transcriptional or translation product as compared to a control tissue (e.g., the same tissue obtained from a subject not administered the expression activator or the expression activation system).
  • expression of the target gene is measured by quantifying the level of mRNA as compared to the control tissue.
  • the level of mRNA is increased by at least about 5%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, or about 50% as compared to the control tissue.
  • the level of mRNA is increased by about 1.1-fold to about 10-fold, by about 1.2-fold to about 10-fold, by about 1.3-fold to about 10-fold, by about 1.4-fold to about 10-fold, by about 1.5-fold to about 10-fold, by about 2-fold to about 10-fold, by about 3-fold to about 10-fold, by about 4-fold to about 10-fold, or by about 5-fold to about FH12877464.1 Attorney Docket: OGY-02925 10-fold as compared to the control tissue.
  • expression of the target gene is measured by quantifying the level of polypeptide encoded by the gene as compared to the control tissue.
  • the level of polypeptide is increased by at least about 5%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, or about 50% as compared to the control tissue. In some embodiments, the level of polypeptide is increased by about 1.1-fold to about 10-fold, by about 1.2-fold to about 10-fold, by about 1.3-fold to about 10-fold, by about 1.4-fold to about 10-fold, by about 1.5-fold to about 10-fold, by about 2-fold to about 10-fold, by about 3-fold to about 10-fold, by about 4-fold to about 10-fold, or by about 5-fold to about 10-fold as compared to the control tissue.
  • Methods to measure a level of a gene transcriptional or translational product are known in the art.
  • the method comprises measuring a level of transcriptional product (e.g., mRNA) using any technique known in the art for measuring or quantifying target RNAs in a cell culture or primary cells harvested from a subject, e.g., RNAseq, transcriptome microarrays, and RT-qPCR.
  • the method comprises measuring a level of polypeptide using any technique known in the art for measuring or quantifying polypeptides in a cell culture or primary cells harvested from a subject, e.g., ELISA, immunoassays (e.g., western blot), and mass spectrometry.
  • the present disclosure provides a method of modulating (e.g., increasing) expression of a target gene in a subject, comprising administering to the subject a dose of an expression activator described herein or a nucleic acid encoding the expression activator (e.g., an LNP- formulated expression activator or an LNP-formulated nucleic acid encoding the expression activator), wherein the expression activator comprises (i) a DNA-targeting moiety (e.g., a TALE, a ZFN, a dCas9/gRNA) that binds a target sequence described herein, and (ii) an effector domain described herein.
  • a DNA-targeting moiety e.g., a TALE, a ZFN, a dCas9/gRNA
  • the present disclosure provides a method of modulating (e.g., increasing) expression of a target gene in a subject, comprising administering to the subject a dose of an expression activation system described herein comprising at least one expression activator (e.g., 1, 2, 3, 4, 5 or more expression activators described herein) or a nucleic acid encoding the at least one expression activator (e.g., an expression activation system comprising at least one LNP-formulated expression activator or an LNP-formulated nucleic acid encoding the at least one expression activator), wherein the at least one expression activator comprises (i) a DNA-targeting moiety (e.g., a TALE, a ZFN, a dCas9/gRNA) that binds a target sequence described herein, and (ii) an effector domain described herein.
  • a DNA-targeting moiety e.g., a TALE, a ZFN, a dCas9/gRNA
  • expression of a target gene is increased in the subject (e.g., as measured in a tissue sample or serum obtained from the subject) as compared to prior to the administering or as compared to a control subject who has not received a dose of the expression activator or the expression activation system.
  • a level of a transcriptional or translation product of the target gene is increased in the subject (e.g., as measured in a tissue sample or serum obtained from the subject) as compared to prior to the administering or as compared to a control subject who has not received a dose of the expression activator or the expression activation system.
  • expression of the target gene or the level of a transcriptional or translation product thereof is measured in a tissue sample obtained from the subject following administering of the dose of the expression activator or the expression activation system.
  • the tissue sample is a fresh, frozen, and/or preserved organ, biopsy, and/or aspirate obtained from the subject.
  • the tissue sample is blood or any blood constituent (e.g., plasma) collected from the subject.
  • expression of the target gene or the level of a transcriptional or translation product thereof as measured in the tissue sample is compared to expression or a level in a reference tissue sample obtained from the subject prior to the administering or from a control subject.
  • Methods to measure expression of the target gene or the level of a transcriptional or translation product thereof include assays for measuring genomic DNA, mRNA, or cDNA (e.g., RNAseq, RT-PCR, real- time RT-PCR, competitive RT-PCR, northern blotting, nucleic acid microarray) and assays for measuring protein expression (e.g., quantitative immunofluorescence, flow cytometry, western blotting, ELISA, tissue immunostaining, immunoprecipitation, mass spectrometry, immunohistochemistry).
  • expression of the target gene is measured by harvesting a tissue sample or serum from the subject, obtaining the transcriptional or translational product of the gene in the tissue or serum, and quantifying a level of the transcriptional or translation product as compared to a level in a reference tissue sample obtained from the subject prior to the administering or from a control subject.
  • expression of the target gene is measured by quantifying the level of mRNA as compared to the reference tissue sample.
  • the level of mRNA is increased by at least about 5%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, or about 50% as compared to the reference tissue sample.
  • the level of mRNA is increased by about 1.1-fold to about 10-fold, by about 1.2-fold to about 10-fold, by about 1.3-fold to about 10-fold, by about 1.4-fold to about 10-fold, by about 1.5-fold to about FH12877464.1
  • expression of the target gene is measured by quantifying the level of polypeptide encoded by the gene as compared to the reference tissue sample.
  • the level of polypeptide is increased by at least about 5%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, or about 50% as compared to the reference tissue sample. In some embodiments, the level of polypeptide is increased by about 1.1-fold to about 10-fold, by about 1.2-fold to about 10-fold, by about 1.3- fold to about 10-fold, by about 1.4-fold to about 10-fold, by about 1.5-fold to about 10-fold, by about 2-fold to about 10-fold, by about 3-fold to about 10-fold, by about 4-fold to about 10-fold, or by about 5-fold to about 10-fold as compared to the reference tissue sample.
  • the disclosure provides a method of increasing expression of a target gene in a cell or a population of cells, comprising contacting the cell or the population with a dose of an expression activator described herein or a nucleic acid encoding the expression activator (e.g., an LNP-formulated expression activator or an LNP-formulated nucleic acid encoding the expression activator), wherein the expression activator comprises (i) a DNA- targeting moiety (e.g., a TALE, a ZFN, a dCas9/gRNA) that binds a target sequence described herein, and (ii) an effector domain described herein.
  • a DNA- targeting moiety e.g., a TALE, a ZFN, a dCas9/gRNA
  • the disclosure provides a method of increasing expression of a target gene in a cell or a population of cells, comprising contacting the cell or the population of cells with a dose of an expression activation system described herein comprising at least one expression activator (e.g., 1, 2, 3, 4, 5 or more expression activators described herein) or a nucleic acid encoding the at least one expression activator (e.g., an expression activation system comprising at least one LNP-formulated expression activator or an LNP-formulated nucleic acid encoding the at least one expression activator), wherein the at least one expression activator comprises (i) a DNA-targeting moiety (e.g., a TALE, a ZFN, a dCas9/gRNA) that binds a target sequence described herein, and (ii) an effector domain described herein.
  • a DNA-targeting moiety e.g., a TALE, a ZFN, a dCas9/gRNA
  • expression of the target gene is increased as compared to prior to the contacting or as compared to a control cell or control population of cells not contacted with the expression activator or the expression activation system.
  • the present disclosure is further directed, in another aspect, to a cell made by a method or process described herein.
  • the disclosure provides a cell produced by: providing an expression activator or an expression activation system described FH12877464.1 Attorney Docket: OGY-02925 herein, providing the cell, and contacting the cell with the expression activator (or a nucleic acid encoding the expression activator, or a composition comprising said expression activator or nucleic acid) or the expression activation system (or a nucleic acid encoding the expression activation system, or a composition comprising said expression activation system or nucleic acid).
  • contacting a cell with an expression activator comprises contacting the cell with a nucleic acid encoding the expression activator under conditions that allow the cell to produce the expression activator.
  • contacting a cell with an expression activator comprises contacting an organism that comprises the cell with the expression activator or a nucleic acid encoding the expression activator under conditions that allow the cell to produce the expression activator.
  • Methods and compositions as provided herein may treat a condition associated with mis-regulation of a target gene by stably or transiently altering (e.g., increasing) transcription of a target gene.
  • a method or composition provided herein may increase expression of a target gene in a cell by at least 10, 20, 30, 40, 50, 60, 70, 80, 90, or 100% (and optionally up to 100%) relative to expression of the target gene in a cell not contacted by the composition or treated with the method.
  • the disclosure provides a method of modulating (e.g., increasing) expression of a target gene in a subject having a disorder associated with dysregulation (e.g., mutation) of the target gene in a subject, comprising administering to the subject an expression activator described herein or a nucleic acid encoding the expression activator (e.g., an LNP-formulated expression activator or an LNP-formulated nucleic acid encoding the expression activator), wherein the expression activator comprises (i) a DNA- targeting moiety (e.g., a TALE, a ZFN, a dCas9/gRNA) that binds a target sequence described herein, and (ii) an effector domain described herein.
  • a DNA- targeting moiety e.g., a TALE, a ZFN, a dCas9/gRNA
  • the disclosure provides a method of modulating (e.g., increasing) expression of a target gene in a subject having a disorder associated with dysregulation (e.g., mutation) of the target gene in a subject an expression activation system described herein comprising at least one expression activator (e.g., 1, 2, 3, 4, 5 or more expression activators described herein) or a nucleic acid encoding the at least one expression activator (e.g., an expression activation system comprising at least one LNP-formulated expression activator or an LNP-formulated nucleic acid encoding the at least one expression activator), wherein the at least one expression activator comprises (i) a DNA-targeting moiety (e.g., a TALE, a ZFN, a dCas9/gRNA) that binds a target sequence described herein, and (ii) an effector domain described herein.
  • a DNA-targeting moiety e.g., a TALE, a ZFN, a dCa
  • FH12877464.1 Attorney Docket: OGY-02925 Methods are described herein to deliver agents, or a composition as disclosed herein, to a subject for treatment of a disorder such that the subject suffers minimal side effects or systemic toxicity in comparison to an alternative, e.g., standard of care, treatment.
  • the subject does not experience any significant side effects typically associated with standard of care, when treated with the agents and/or compositions described herein.
  • the subject does not experience a significant side effect including but not limited to alopecia, nausea, vomiting, poor appetite, soreness, neutropenia, anemia, thrombocytopenia, dizziness, fatigue, constipation, oral ulcers, itchy skin, peeling, nerve and muscle damage, auditory changes, weight loss, diarrhea, immunosuppression, bruising, heart damage, bleeding, liver damage, kidney damage, edema, mouth and throat sores, infertility, fibrosis, epilation, moist desquamation, mucosal dryness, vertigo and encephalopathy when treated with the agents and/or compositions described herein.
  • alopecia nausea, vomiting, poor appetite, soreness, neutropenia, anemia, thrombocytopenia, dizziness, fatigue, constipation, oral ulcers, itchy skin, peeling, nerve and muscle damage
  • auditory changes weight loss, diarrhea, immunosuppression, bruising, heart damage, bleeding, liver damage, kidney damage, edema,
  • the subject does not show a significant loss of body weight when treated with the agents and/or compositions described herein.
  • the agents and compositions described herein can be administered to a subject, e.g., a mammal, e.g., in vivo, to treat or prevent a variety of disorders as described herein.
  • compositions comprising an expression activator or an expression activation system, e.g., expression activator(s), described herein, to pharmaceutical compositions comprising nucleic acids encoding the expression activator or expression activation system, e.g., expression activator(s), described herein, and/or to and/or compositions that deliver an expression activator or expression activation system, e.g., expression activator(s), described herein to a cell, tissue, organ, and/or subject.
  • an expression activator or an expression activation system e.g., expression activator(s), described herein
  • the term “pharmaceutical composition” refers to an active agent (e.g., an expression activator or nucleic acids of the expression activator, e.g., an expression activation system, e.g., expression activator(s) of an expression activation system, or a nucleic acid encoding the same), formulated together with one or more pharmaceutically acceptable carriers (e.g., pharmaceutically acceptable carriers known to those of skill in the art).
  • an active agent is present in unit dose amount appropriate for administration in a therapeutic regimen that shows a statistically significant probability of achieving a predetermined therapeutic effect when administered to a relevant population.
  • a pharmaceutical composition comprising an expression activator of the FH12877464.1 Attorney Docket: OGY-02925 present disclosure comprises an expression activator or nucleic acid(s) encoding the same.
  • a pharmaceutical composition comprising an expression activation system of the present disclosure comprises each of the expression activators of the expression activation system or nucleic acid(s) encoding the same (e.g., if an expression activation system comprises a first expression activator and a second expression activator, the pharmaceutical composition comprises the first and second expression activator).
  • a pharmaceutical composition comprises less than all of the expression activators of an expression activation system comprising a plurality of expression activators.
  • an expression activation system may comprise a first expression activator and a second expression activator, and a first pharmaceutical composition may comprise the first expression activator or nucleic acid encoding the same and a second pharmaceutical composition may comprise the second expression activator or nucleic acid encoding the same.
  • a pharmaceutical composition may comprise coformulation of one or more expression activators, or nucleic acid(s) encoding the same.
  • compositions may be specially formulated for administration in solid or liquid form, including those adapted for the following: oral administration, for example, drenches (aqueous or non-aqueous solutions or suspensions), tablets, e.g., those targeted for buccal, sublingual, and systemic absorption, boluses, powders, granules, pastes for application to the tongue; parenteral administration, for example, by subcutaneous, intramuscular, intravenous or epidural injection as, for example, a sterile solution or suspension, or sustained-release formulation; topical application, for example, as a cream, ointment, or a controlled-release patch or spray applied to the skin, lungs, or oral cavity; intravaginally or intrarectally, for example, as a pessary, cream, or foam; sublingually; ocularly; trans-dermally; or nasally, pulmonary, and/or to other mucosal surfaces.
  • oral administration for example, drenches (aqueous or non-aqueous
  • the term “pharmaceutically acceptable” refers to those compounds, materials, compositions, and/or dosage forms which are, within the scope of sound medical judgment, suitable for use in contact with the tissues of human beings and animals without excessive toxicity, irritation, allergic response, or other problem or complication, commensurate with a reasonable benefit/risk ratio.
  • pharmaceutically acceptable carrier means a pharmaceutically-acceptable material, composition, or vehicle, such as a liquid or solid filler, diluent, excipient, or solvent encapsulating material, involved in carrying or transporting the subject compound from one organ, or portion of the body, to another organ, or portion of the body.
  • materials which can serve as pharmaceutically-acceptable carriers include: sugars, such as lactose, glucose and sucrose; starches, such as com starch and potato starch; cellulose, and its derivatives, such as sodium carboxymethyl cellulose, ethyl cellulose and cellulose acetate; powdered tragacanth; malt; gelatin; talc; excipients, such as cocoa butter and suppository waxes; oils, such as peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, corn oil and soybean oil; glycols, such as propylene glycol; polyols, such as glycerin, sorbitol, mannitol and polyethylene glycol; esters, such as ethyl oleate
  • the term “pharmaceutically acceptable salt” refers to salts of such compounds that are appropriate for use in pharmaceutical contexts, i.e., salts which are, within the scope of sound medical judgment, suitable for use in contact with the tissues of humans and lower animals without undue toxicity, irritation, allergic response and the like, and are commensurate with a reasonable benefit/risk ratio.
  • Pharmaceutically acceptable salts are well known in the art. For example, S. M. Berge, et al. describes pharmaceutically acceptable salts in detail in J. Pharmaceutical Sciences, 66: 1-19 (1977).
  • pharmaceutically acceptable salts include, but are not limited to, nontoxic acid addition salts, which are salts of an amino group formed with inorganic acids such as hydrochloric acid, hydrobromic acid, phosphoric acid, sulfuric acid and perchloric acid or with organic acids such as acetic acid, maleic acid, tartaric acid, citric acid, succinic acid, or malonic acid or by using other methods used in the art such as ion exchange.
  • nontoxic acid addition salts which are salts of an amino group formed with inorganic acids such as hydrochloric acid, hydrobromic acid, phosphoric acid, sulfuric acid and perchloric acid or with organic acids such as acetic acid, maleic acid, tartaric acid, citric acid, succinic acid, or malonic acid or by using other methods used in the art such as ion exchange.
  • pharmaceutically acceptable salts include, but are not limited to, adipate, alginate, ascorbate, aspartate, benzenesulfonate, benzoate, bisulfate, borate, butyrate, camphorate, camphorsulfonate, citrate, cyclopentanepropionate, digluconate, dodecylsulfate, ethanesulfonate, formate, fumarate, glucoheptonate, glycerophosphate, gluconate, hemisulfate, heptanoate, hexanoate, hydroiodide, 2-hydroxy-ethanesulfonate, lactobionate, lactate, laurate, lauryl sulfate, malate, maleate, malonate, methanesulfonate, 2- naphthalenesulfonate, nicotinate, nitrate, oleate, oxalate, palm
  • Representative alkali or alkaline earth metal salts include sodium, lithium, potassium, FH12877464.1 Attorney Docket: OGY-02925 calcium, magnesium, and the like.
  • pharmaceutically acceptable salts include, when appropriate, nontoxic ammonium, quaternary ammonium, and amine cations formed using counterions such as halide, hydroxide, carboxylate, sulfate, phosphate, nitrate, alkyl having from 1 to 6 carbon atoms, sulfonate, and aryl sulfonate.
  • the present disclosure provides pharmaceutical compositions described herein with a pharmaceutically acceptable excipient.
  • compositions that are generally safe, non- toxic, and desirable, and includes excipients that are acceptable for veterinary use as well as for human pharmaceutical use.
  • excipients may be solid, liquid, semisolid, or, in the case of an aerosol composition, gaseous.
  • Pharmaceutical preparations may be made following conventional techniques of pharmacy involving milling, mixing, granulation, and compressing, when necessary, for tablet forms; or milling, mixing, and filling for hard gelatin capsule forms.
  • a preparation can be in the form of a syrup, elixir, emulsion or an aqueous or non-aqueous solution or suspension.
  • compositions may be formulated for delivery to a cell and/or to a subject via any route of administration.
  • Modes of administration to a subject may include injection, infusion, inhalation, intranasal, intraocular, topical delivery, inter-cannular delivery, or ingestion.
  • Injection includes, without limitation, intravenous, intramuscular, intra-arterial, intrathecal, intraventricular, intracapsular, intra-orbital, intracardiac, intradermal, intraperitoneal, transtracheal, subcutaneous, subcuticular, intraarticular, sub capsular, subarachnoid, intraspinal, intra-cerebrospinal, and intra-stemal injection and infusion.
  • administration includes aerosol inhalation, e.g., with nebulization.
  • administration is systemic (e.g., oral, rectal, nasal, sublingual, buccal, or parenteral), enteral (e.g., system-wide effect, but delivered through the gastrointestinal tract), or local (e.g., local application on the skin, or intravitreal injection).
  • one or more compositions is administered systemically.
  • administration is non-parenteral and a therapeutic is a parenteral therapeutic.
  • administration may be bronchial (e.g., by bronchial instillation), buccal, dermal (which may be or comprise, for example, one or more of topical to the dermis, intradermal, inter-dermal, transdermal, etc.), enteral, intra-arterial, intradermal, intragastric, intramedullary, intramuscular, intranasal, intraperitoneal, intrathecal, intravenous, intraventricular, within a specific organ (e. g.
  • administration may be a single dose.
  • administration may involve dosing that is intermittent (e.g., a plurality of doses separated in time) and/or periodic (e.g., individual doses separated by a common period of time) dosing.
  • administration may involve continuous dosing (e.g., perfusion) for at least a selected period of time.
  • administering may be given to the subject during one treatment or over a period of time as a treatment regimen.
  • administrations may be given as needed, e.g., for as long as symptoms associated with the disease, disorder or condition persist.
  • repeated administrations may be indicated for the remainder of the subject’s life.
  • Treatment periods may vary and could be, e.g., one day, two days, three days, one week, two weeks, one month, two months, three months, six months, a year, or longer.
  • the dosage of the administered agent or composition can vary based on, e.g., the condition being treated, the severity of the disease, the subject’s individual parameters, including age, physiological condition, size and weight, duration of treatment, the type of treatment to be performed (if any), the particular route of administration and similar factors. Thus, the dose administered of the agents described herein can depend on such various parameters.
  • the dosage of an administered composition may also vary depending upon other factors as the subject’s sex, general medical condition, and severity of the disorder to be treated.
  • a dosage of a modulatory agent or combination of modulatory agents disclosed herein that is in the range of from about 1 mg/kg to 6 mg/kg as a single intravenous infusion, although a lower or higher dosage also may be administered as circumstances dictate.
  • the dosage may be repeated as needed, for example, once every day (e.g., for 1-30 days), once every 3 days (e.g., for 1-30 days) once every 5 days (e.g., for 1-30 days), once per week (e.g., for 1-6 weeks or for 2-5 weeks).
  • dosages may include, but are not limited to, 1.0 mg/kg - 6 mg/kg, 1.0 mg/kg - 5 mg/kg, 1.0 mg/kg - 4 mg/kg, 1.0 mg/kg - 3.0 mg/kg, 1.5 mg/kg - 3.0 mg/kg, 1.0 mg/kg - 1.5 mg/kg, 1.5 mg/kg - 3 mg/kg, 3 mg/kg - 4 mg/kg, 4 mg/kg - 5 mg/kg, or 5 mg/kg - 6 mg/kg.
  • the dosage may be administered multiple times, e.g., once, or twice a week, once every 1, or once every 2 weeks.
  • the subject is provided with a dosage of a modulatory agent or combination of modulatory agents disclosed herein that is in the range of FH12877464.1 Attorney Docket: OGY-02925 from about 1 mg/kg to 6 mg/kg as multiple intravenous infusions although a lower or higher dosage also may be administered as circumstances dictate.
  • a modulatory agent or a combination of modulatory agents as disclosed herein may be administered as one dosage every 3-5 days, repeated for a total of at least 3 dosages.
  • a modulatory agent or a combination of modulatory agents as disclosed herein may be administered at 3 mg/kg every 5 days for 25 days.
  • a modulatory agent or a combination of modulatory agents as disclosed herein may be administered at 1.0-5.0 mg/kg every 3-5 days for 1-10 doses.
  • a modulatory agent or a combination of modulatory agents as disclosed herein may be administered at 1.0- 3.0 mg/kg every 5 days for 3 doses then every 3 days for 3 doses.
  • a modulatory agent or a combination of modulatory agents as disclosed herein may be administered at 1.0-3.0 mg/kg every 5 days for 4 doses then every 3 days for 3 doses.
  • a modulatory agent or a combination of modulatory agents as disclosed herein may be administered at 6 mg/kg every 5 days for 1- 10 doses.
  • a modulatory agent or a combination of modulatory agents as disclosed herein may be administered at 3 mg/kg every 5 days for 1-10 doses.
  • a modulatory agent or a combination of modulatory agents as disclosed herein may be administered at 1.5 mg/kg every 5 days for 2 doses, 3 mg/kg every 5 days for 3 doses, 3 mg/kg every 3 days for 1 dose.
  • a modulatory agent or a combination of modulatory agents as disclosed herein may be administered at 6 mg/kg at every 5 days or at 1 .5 mg/kg once a day for 5 days with 2 days off.
  • the dosing schedule can optionally be repeated at other intervals and dosage may be given through various parenteral routes, with appropriate adjustment of the dose and schedule.
  • the dosing of modulatory agents or a combination of modulatory agents may include a dosage of between 1.0 mg/kg to 6.0 mg/kg, optionally given either weekly, twice per week, or every other week.
  • a dosage of a modulatory agent or a combination of modulatory agents as disclosed herein, and that the dosage and/or frequency of administration may be increased or decreased during the course of therapy.
  • the dosage may be repeated as needed, with evidence of reduction of tumor volume observed after as few as 2 to 8 doses.
  • the dosages and schedules of administration disclosed herein show minimal effect on overall weight of the subject compared to cisplatin, sorafenib, or a small molecule comparator.
  • the subject methods may include use of CT and/or PET/CT, or MRI, to measure tumor response at regular intervals. Blood levels of tumor markers may also FH12877464.1 Attorney Docket: OGY-02925 be monitored. Dosages and/or administration schedules may be adjusted as needed, according to the results of imaging and/or marker blood levels.
  • Pharmaceutical compositions according to the present disclosure may be delivered in a therapeutically effective amount.
  • a precise therapeutically effective amount is an amount of a composition that will yield the most effective results in terms of efficacy of treatment in a given subject.
  • the present disclosure provides methods of delivering a therapeutic comprising administering a composition as described herein to a subject, wherein a modulating agent is a therapeutic and/or wherein delivery of a therapeutic causes changes in gene expression relative to gene expression in absence of a therapeutic.
  • compositions e.g., comprising an expression activator or an expression activation system described herein
  • one or more compositions is/are targeted to specific cells, or one or more specific tissues.
  • one or more compositions e.g., comprising an expression activator or an expression activation system described herein, is/are targeted to hepatic, epithelial, connective, muscular, reproductive, and/or nervous tissue or cells.
  • a composition is targeted to a cell or tissue of a particular organ system, e.g., cardiovascular system (heart, vasculature); digestive system (esophagus, stomach, liver, gallbladder, pancreas, intestines, colon, rectum and anus); endocrine system (hypothalamus, pituitary gland, pineal body or pineal gland, thyroid, parathyroids, adrenal glands); excretory system (kidneys, ureters, bladder); lymphatic system (lymph, lymph nodes, lymph vessels, tonsils, adenoids, thymus, spleen); integumentary system (skin, hair, nails); muscular system (e.g., skeletal muscle); nervous system (brain, spinal cord, nerves); reproductive system (ovaries, uterus, mammary glands, testes, vas deferens, seminal vesicles, prostate); respiratory system (pharynx, larynx, trachea
  • an expression FH12877464.1 Attorney Docket: OGY-02925 activator or an expression activation system described herein is targeted to the liver or liver cells.
  • a composition of the present disclosure crosses a blood-brain- barrier, a placental membrane, or a blood-testis barrier.
  • a pharmaceutical composition as provided herein is administered systemically.
  • administration is non-parenteral and a therapeutic is a parenteral therapeutic.
  • Methods and compositions provided herein, e.g., comprising an expression activator or an expression activation system described herein may comprise a pharmaceutical composition administered by a regimen sufficient to alleviate a symptom of a disease, disorder, and/or condition.
  • the present disclosure provides methods of delivering a therapeutic by administering compositions as described herein.
  • Pharmaceutical uses of the present disclosure may include compositions (e.g., modulating agents, e.g., disrupting agents) as described herein.
  • a pharmaceutical composition of the present disclosure has improved PK/PD, e.g., increased pharmacokinetics or pharmacodynamics, such as improved targeting, absorption, or transport (e.g., at least 5%, 10%, 15%, 20%, 30%, 40%, 50%, 60%, 75%, 80%, 90% improved or more) as compared to an active agent alone.
  • a pharmaceutical composition has reduced undesirable effects, such as reduced diffusion to a nontarget location, off-target activity, or toxic metabolism, as compared to a therapeutic alone (e.g., at least 5%, 10%, 15%, 20%, 30%, 40%, 50%, 60%, 75%, 80%, 90% or more reduced) as compared to an active agent alone.
  • a composition increases efficacy and/or decreases toxicity of a therapeutic (e.g., at least 5%, 10%, 15%, 20%, 30%, 40%, 50%, 60%, 75%, 80%, 90% or more) as compared to an active agent alone.
  • the composition can be administered by any means known in the art including, but not limited to oral, intraperitoneal, or parenteral routes, including intracranial (e.g., intraventricular, intraparenchymal, and intrathecal), intravenous, intramuscular, subcutaneous, transdermal, airway (aerosol), nasal, rectal, and topical (including buccal and sublingual) administration.
  • intracranial e.g., intraventricular, intraparenchymal, and intrathecal
  • intravenous intramuscular
  • subcutaneous transdermal
  • nasal rectal
  • topical including buccal and sublingual
  • the compositions are administered by intravenous infusion or injection.
  • kits comprises an expression activator or an expression activation system (e.g., the expression activator(s) of the expression activation system) and instructions for the use of said expression activator or expression activation system.
  • a kit comprises a nucleic acid encoding the expression activator or a nucleic acid encoding the expression activation system or a component thereof (e.g., the expression activator(s) of the expression activation system) and instructions for the use of said expression activator (and/or said nucleic acid) and/or said expression activation system (and/or said nucleic acid).
  • a kit comprises a cell comprising a nucleic acid encoding the expression activator or a nucleic acid encoding the expression activation system or a component thereof (e.g., the expression activator(s) of the expression activation system) and instructions for the use of said cell, nucleic acid, and/or said expression activator or expression activation system.
  • the kit further comprises a set of instructions comprising at least one method for treating a disease or modulating, e.g., increasing the expression of target gene within a cell with said composition.
  • the kits can optionally include a delivery vehicle for said composition (e.g., a lipid nanoparticle).
  • kits may optionally contain additional therapeutics to be co- administered with the compositions to affect the desired target gene expression.
  • instructional materials typically comprise written or printed materials, they are not limited to such. Any medium capable of storing such instructions and communicating them to an end user is contemplated. Such media include but are not limited to electronic storage media (e.g., magnetic discs, tapes, cartridges, chips), optical media (e.g., CD ROM), and the like. Such media may include addresses to internet sites that provide such instructional materials.
  • a kit comprises a unit dosage of an expression activator or an expression activation system, e.g., expression activator(s), described herein, or a unit dosage of a nucleic acid, e.g., a vector, encoding an expression activation system, e.g., expression activator(s), described herein.
  • the term “agent”, may be used to refer to a compound or entity of any chemical class including, for example, a polypeptide, nucleic acid, saccharide, lipid, small FH12877464.1 Attorney Docket: OGY-02925 molecule, metal, or combination or complex thereof.
  • the term may be utilized to refer to an entity that is or comprises a cell or organism, or a fraction, extract, or component thereof.
  • the term may be used to refer to a natural product in that it is found in and/or is obtained from nature.
  • the term may be used to refer to one or more entities that is man- made in that it is designed, engineered, and/or produced through action of the hand of man and/or is not found in nature.
  • an agent may be utilized in isolated or pure form; in some embodiments, an agent may be utilized in crude form.
  • potential agents may be provided as collections or libraries, for example that may be screened to identify or characterize active agents within them.
  • the term “agent” may refer to a compound or entity that is or comprises a polymer; in some embodiments, the term may refer to a compound or entity that comprises one or more polymeric moieties.
  • the term “agent” may refer to a compound or entity that is not a polymer and/or is substantially free of any polymer and/or of one or more particular polymeric moieties. In some embodiments, the term may refer to a compound or entity that lacks or is substantially free of any polymeric moiety.
  • anchor sequence refers to a nucleic acid sequence recognized by a nucleating agent that binds sufficiently to form an anchor sequence-mediated conjunction, e.g., a complex. In some embodiments, an anchor sequence comprises one or more CTCF binding motifs. In some embodiments, an anchor sequence is not located within a gene coding region. In some embodiments, an anchor sequence is located within an intergenic region.
  • an anchor sequence is not located within either of an enhancer or a promoter. In some embodiments, an anchor sequence is located at least 400 bp, at least 450 bp, at least 500 bp, at least 550 bp, at least 600 bp, at least 650 bp, at least 700 bp, at least 750 bp, at least 800 bp, at least 850 bp, at least 900 bp, at least 950 bp, or at least 1 kb away from any transcription start site. In some embodiments, an anchor sequence is located within a region that is not associated with genomic imprinting, monoallelic expression, and/or monoallelic epigenetic marks.
  • the anchor sequence has one or more functions selected from binding an endogenous nucleating polypeptide (e.g., CTCF), interacting with a second anchor sequence to form an anchor sequence mediated conjunction, or insulating against an enhancer that is outside the anchor sequence mediated conjunction.
  • an endogenous nucleating polypeptide e.g., CTCF
  • technologies are provided that FH12877464.1 Attorney Docket: OGY-02925 may specifically target a particular anchor sequence or anchor sequences, without targeting other anchor sequences (e.g., sequences that may contain a nucleating agent (e.g., CTCF) binding motif in a different context); such targeted anchor sequences may be referred to as the “target anchor sequence”.
  • sequence and/or activity of a target anchor sequence is modulated while sequence and/or activity of one or more other anchor sequences that may be present in the same system (e.g., in the same cell and/or in some embodiments on the same nucleic acid molecule – e.g., the same chromosome) as the targeted anchor sequence is not modulated.
  • the anchor sequence comprises or is a nucleating polypeptide binding motif. In some embodiments, the anchor sequence is adjacent to a nucleating polypeptide binding motif.
  • anchor sequence-mediated conjunction refers to a DNA structure, in some cases, a complex, that occurs and/or is maintained via physical interaction or binding of at least two anchor sequences in the DNA by one or more polypeptides, such as nucleating polypeptides, or one or more proteins and/or a nucleic acid entity (such as RNA or DNA), that bind the anchor sequences to enable spatial proximity and functional linkage between the anchor sequence.
  • polypeptides such as nucleating polypeptides, or one or more proteins and/or a nucleic acid entity (such as RNA or DNA)
  • Two events or entities are “associated” with one another, as that term is used herein, if presence, level, form and/or function of one is correlated with that of the other.
  • a particular entity e.g., polypeptide, genetic signature, metabolite, microbe, etc.
  • a particular entity e.g., polypeptide, genetic signature, metabolite, microbe, etc.
  • two or more entities are physically “associated” with one another if they interact, directly or indirectly, so that they are and/or remain in physical proximity with one another.
  • two or more entities that are physically associated with one another are covalently linked to one another; in some embodiments, two or more entities that are physically associated with one another are not covalently linked to one another but are non- covalently associated, for example by means of hydrogen bonds, van der Waals interaction, hydrophobic interactions, magnetism, and combinations thereof.
  • a DNA sequence is “associated with” a target genomic or transcription complex when the nucleic acid is at least partially within the target genomic or transcription complex, and expression of a gene in the DNA sequence is affected by formation or disruption of the target genomic or transcription complex.
  • FH12877464.1 Attorney Docket: OGY-02925
  • domain refers to a section or portion of an entity.
  • a “domain” is associated with a particular structural and/or functional feature of the entity so that, when the domain is physically separated from the rest of its parent entity, it substantially or entirely retains the particular structural and/or functional feature.
  • a domain may be or include a portion of an entity that, when separated from that (parent) entity and linked with a different (recipient) entity, substantially retains and/or imparts on the recipient entity one or more structural and/or functional features that characterized it in the parent entity.
  • a domain is or comprises a section or portion of a molecule (e.g., a small molecule, carbohydrate, lipid, nucleic acid, polypeptide, etc.).
  • a domain is or comprises a section of a polypeptide.
  • a domain is characterized by a particular structural element (e.g., a particular amino acid sequence or sequence motif, alpha-helix character, beta-sheet character, coiled-coil character, random coil character, etc.), and/or by a particular functional feature (e.g., binding activity, enzymatic activity, folding activity, signaling activity, etc.).
  • CpG sequence also called “CpG site” or “CpG dyad,” are regions of DNA having 5 ⁇ to 3 ⁇ a cytosine nucleoside linked to a guanine nucleoside by a phosphate group (i.e., 5 ⁇ -C-phosphate linkage-G-3 ⁇ ).
  • CpG islands also called “CG islands,” are regions of the genome comprising a high frequency of CpG sequences. GpG islands and criteria for identifying CpG islands are known in the art and described in, for example, Bird et al, (1985) Cell 40:91-99).
  • CpG island is a region of (1) at least 200 bp in length, (2) a GC percentage greater than 50%, and (3) an observed-to-expected CpG ratio greater than 60%.
  • the observed-to-expected CpG ratio may be calculated in multiple ways. Two methods of calculating the observed-to-expected CpG ratio are as follows: (a) (number of C * number of G) / length of sequence (b) ((number of C + number of G) / length of sequence) 2 See, e.g., Gardiner-Garden M, Frommer M (1987). "CpG islands in vertebrate genomes". Journal of Molecular Biology.196 (2): 261–282. doi:10.1016/0022- 2836(87)90689-9.
  • CpG islands often occur near transcription start sites and promote regions. Indeed, many gene promoters reside within or near CpG islands (see, e.g., Saxonov et al (2006) PNAS 103:1412- 17).
  • DNA-targeting moiety refers to an agent or entity that specifically targets, e.g., binds, a target sequence in genomic DNA (e.g., a transcriptional control element or an anchor sequence).
  • effector domain refers to a domain capable of altering the expression of a target gene when localized to an appropriate site in the nucleus of a cell.
  • expression activator refers to an agent or entity with one or more functionalities that increases expression of a target gene in a cell and that specifically binds to a DNA sequence (e.g., a DNA sequence associated with a target gene or a transcription control element operably linked to a target gene).
  • An expression activator comprises at least one targeting moiety and optionally one effector domain.
  • genomic complex is a complex that brings together two genomic sequence elements that are spaced apart from one another on one or more chromosomes, via interactions between and among a plurality of protein and/or other components (potentially including, the genomic sequence elements).
  • a genomic sequence element may be or comprise a CTCF binding motif, a promoter and/or an enhancer.
  • a genomic sequence element includes at least one or both of a promoter and/or regulatory site (e.g., an enhancer).
  • complex formation is nucleated at the genomic sequence element(s) and/or by binding of one or more of the protein component(s) to the genomic sequence element(s).
  • co-localization e.g., conjunction
  • a genomic complex comprises an anchor sequence-mediated conjunction, which comprises one or more loops.
  • a genomic complex as described herein is nucleated by a nucleating polypeptide such as, for example, CTCF and/or Cohesin.
  • a genomic complex as described herein may include, for example, one or more of CTCF, Cohesin, non-coding RNA (e.g., eRNA), transcriptional machinery proteins (e.g., RNA polymerase, one or more transcription factors, for example selected from the group consisting of TFIIA, TFIIB, TFIID, TFIIE, TFIIF, TFIIH, etc.), transcriptional regulators FH12877464.1 Attorney Docket: OGY-02925 (e.g., Mediator, P300, enhancer-binding proteins, repressor-binding proteins, histone modifiers, etc.), etc.
  • OGY-02925 e.g., Mediator, P300, enhancer-binding proteins, repressor-binding proteins, histone modifiers, etc.
  • a genomic complex as described herein includes one or more polypeptide components and/or one or more nucleic acid components (e.g., one or more RNA components), which may, in some embodiments, be interacting with one another and/or with one or more genomic sequence elements (e.g., anchor sequences, promoter sequences, regulatory sequences (e.g., enhancer sequences)) so as to constrain a stretch of genomic DNA into a topological configuration (e.g., a loop) that it does not adopt when the complex is not formed.
  • genomic sequence elements e.g., anchor sequences, promoter sequences, regulatory sequences (e.g., enhancer sequences)
  • topological configuration e.g., a loop
  • the term “moiety” refers to a defined chemical group or entity with a particular structure and/or or activity, as described herein.
  • the term “modulating agent” refers to an agent comprising one or more targeting moieties and one or more effector moieties that is capable of altering (e.g., increasing or decreasing) expression of a target gene.
  • the term “nucleic acid” refers to any compound and/or substance that is or can be incorporated into an oligonucleotide chain.
  • a nucleic acid is a compound and/or substance that is or can be incorporated into an oligonucleotide chain via a phosphodiester linkage.
  • nucleic acid refers to an individual nucleic acid residue (e.g., a nucleotide and/or nucleoside); in some embodiments, “nucleic acid” refers to an oligonucleotide chain comprising individual nucleic acid residues.
  • a "nucleic acid” is or comprises RNA; in some embodiments, a “nucleic acid” is or comprises DNA.
  • a nucleic acid is, comprises, or consists of one or more natural nucleic acid residues.
  • a nucleic acid is, comprises, or consists of one or more nucleic acid analogs.
  • a nucleic acid analog differs from a nucleic acid in that it does not utilize a phosphodiester backbone.
  • a nucleic acid is, comprises, or consists of one or more "peptide nucleic acids", which are known in the art and have peptide bonds instead of phosphodiester bonds in the backbone, are considered within the scope of the present disclosure.
  • a nucleic acid has one or more phosphorothioate and/or 5'-N-phosphoramidite linkages rather than phosphodiester bonds.
  • a nucleic acid is, comprises, or consists of one or more natural nucleosides (e.g., adenosine, thymidine, guanosine, cytidine, uridine, deoxyadenosine, deoxythymidine, deoxy guanosine, and deoxycytidine).
  • adenosine thymidine, guanosine, cytidine
  • uridine deoxyadenosine
  • deoxythymidine deoxy guanosine
  • deoxycytidine deoxycytidine
  • a nucleic acid is, comprises, or consists of one or more nucleoside analogs (e.g., 2-aminoadenosine, 2-thiothymidine, inosine, pyrrolo- FH12877464.1 Attorney Docket: OGY-02925 pyrimidine, 3 -methyl adenosine, 5-methylcytidine, C-5 propynyl-cytidine, C-5 propynyl- uridine, 2-aminoadenosine, C5-bromouridine, C5-fluorouridine, C5-iodouridine, C5- propynyl-uridine, C5 -propynyl-cytidine, C5-methylcytidine, 2-aminoadenosine, 7- deazaadenosine, 7-deazaguanosine, 8-oxoadenosine, 8-oxoguanosine, 0(6)-methylguan
  • a nucleic acid comprises one or more modified sugars (e.g., 2'-fluororibose, ribose, 2'-deoxyribose, arabinose, and hexose) as compared with those in natural nucleic acids.
  • a nucleic acid has a nucleotide sequence that encodes a functional gene product such as an RNA or protein.
  • a nucleic acid includes one or more introns.
  • nucleic acids are prepared by one or more of isolation from a natural source, enzymatic synthesis by polymerization based on a complementary template (in vivo or in vitro), reproduction in a recombinant cell or system, and chemical synthesis.
  • a nucleic acid is at least 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 20, 225, 250, 275, 300, 325, 350, 375, 400, 425, 450, 475, 500, 600, 700, 800, 900, 1000, 1500, 2000, 2500, 3000, 3500, 4000, 4500, 5000 or more residues long.
  • a nucleic acid is partly or wholly single stranded; in some embodiments, a nucleic acid is partly or wholly double stranded.
  • nucleic acid has a nucleotide sequence comprising at least one element that encodes, or is the complement of a sequence that encodes, a polypeptide. In some embodiments, a nucleic acid has enzymatic activity.
  • nucleating polypeptide or “conjunction nucleating polypeptide” as used herein, refers to a protein that associates with an anchor sequence directly or indirectly and may interact with one or more conjunction nucleating polypeptides (that may interact with an anchor sequence or other nucleic acids) to form a dimer (or higher order structure) comprised of two or more such conjunction nucleating polypeptides, which may or may not be identical to one another.
  • nucleating polypeptides associated with different anchor sequences associate with each other so that the different anchor sequences are maintained in physical proximity with one another
  • the structure generated thereby is an anchor-sequence-mediated conjunction. That is, the close physical proximity of a nucleating polypeptide-anchor sequence interacting with another nucleating polypeptide-anchor sequence generates an anchor sequence-mediated conjunction (e.g., in some cases, a DNA loop), that begins and ends at the anchor sequence.
  • operably linked refers to a juxtaposition wherein the components described are in a relationship permitting them to function in their intended manner.
  • a transcription control element "operably linked" to a functional element, e.g., gene is associated in such a way that expression and/or activity of the functional element, e.g., gene, is achieved under conditions compatible with the transcription control element.
  • "operably linked" transcription control elements are contiguous (e.g., covalently linked) with coding elements, e.g., genes, of interest; in some embodiments, operably linked transcription control elements act in trans to or otherwise at a distance from the functional element, e.g., gene, of interest.
  • operably linked means two nucleic acid sequences are comprised on the same nucleic acid molecule. In a further embodiment, operably linked may further mean that the two nucleic acid sequences are proximal to one another on the same nucleic acid molecule, e.g., within 1,000, 500, 100, 50, or 10 base pairs of each other or directly adjacent to each other.
  • the terms “peptide,” “polypeptide,” and “protein” refer to a compound comprised of amino acid residues covalently linked by peptide bonds, or by means other than peptide bonds.
  • a protein or peptide must contain at least two amino acids, and no limitation is placed on the maximum number of amino acids that can comprise a protein’s or peptide’s sequence.
  • Polypeptides include any peptide or protein comprising two or more amino acids joined to each other by peptide bonds or by means other than peptide bonds.
  • the term refers to both short chains, which also commonly are referred to in the art as peptides, oligopeptides and oligomers, for example, and to longer chains, which generally are referred to in the art as proteins, of which there are many types.
  • proximal refers to the location of a first site and a second site in the genome that occur sufficiently close (e.g., occurring within a span of bases of up to 2,000 bases) for a function directed to the first site results in a desired functional outcome at the second site or vice versa.
  • the first site is a target sequence described herein and the second site is a site for epigenetic modulation (e.g., a CpG FH12877464.1 Attorney Docket: OGY-02925 island), wherein the first site and the second site are sufficiently close that an expression activator targeting the first site via its DNA-targeting moiety results in a desired epigenetic modulation at the second site via its effector domain.
  • a site for epigenetic modulation e.g., a CpG FH12877464.1 Attorney Docket: OGY-02925 island
  • the first site is a site for epigenetic modulation (e.g., a CpG island) and the second site is a transcriptional control element (e.g., a promoter) operably linked to a target gene, wherein the first site and the second site are sufficiently close that an expression activator that introduces an epigenetic modulation at the first site via its effector domain results in altered transcriptional regulation at the second site (e.g., transcriptional regulation resulting in increased expression of the target gene).
  • the location of the first site and the location of the second site occur within or overlapping a span of about 300 bases to about 2,000 bases.
  • the location of the first site and the location of the second site occur within or overlapping a span of about 500 bases to about 1,500 bases. In some embodiments, the location of the first site and the location of the second site occur within or overlapping a span of about 500 bases to about 1,000 bases.
  • the term “pharmaceutical composition” refers to an active agent (e.g., a modulating agent, e.g., an expression activator or expression activation system of the present disclosure), formulated together with one or more pharmaceutically acceptable carriers.
  • an active agent is present in unit dose amount appropriate for administration in a therapeutic regimen that shows a statistically significant probability of achieving a predetermined therapeutic effect when administered to a relevant population.
  • compositions may be specially formulated for administration in solid or liquid form, including those adapted for the following: oral administration, for example, drenches (aqueous or non-aqueous solutions or suspensions), tablets, e.g., those targeted for buccal, sublingual, and systemic absorption, boluses, powders, granules, pastes for application to the tongue; parenteral administration, for example, by subcutaneous, intramuscular, intravenous or epidural injection as, for example, a sterile solution or suspension, or sustained-release formulation; topical application, for example, as a cream, ointment, or a controlled-release patch or spray applied to the skin, lungs, or oral cavity; intravaginally or intrarectally, for example, as a pessary, cream, or foam; sublingually; ocularly; trans-dermally; or nasally, pulmonary, and/or to other mucosal surfaces.
  • oral administration for example, drenches (aqueous or non-aqueous
  • proximal refers to a closeness of two sites, e.g., nucleic acid sites, such that binding of an expression activator at the first site and/or modification of the first site by an expression activator will produce the same or substantially the same effect as binding and/or modification of the other site.
  • a targeting moiety may bind to a first site FH12877464.1 Attorney Docket: OGY-02925 that is proximal to an enhancer (the second site), and the effector moiety associated with said targeting moiety may epigenetically modify the first site such that the enhancer’s effect on expression of a target gene is modified, substantially the same as if the second site (the enhancer sequence) had been bound and/or modified.
  • a site proximal to a target gene e.g., an exon, intron, or splice site within the target gene
  • proximal to a transcription control element operably linked to the target gene, or proximal to an anchor sequence is less than 5,000, 4,000, 3,000, 2,000, 1,000, 900, 800, 700, 600, 500, 400, 300, 200, 100, 50, or 25 base pairs from the target gene (e.g., an exon, intron, or splice site within the target gene), transcription control element, or anchor sequence (and optionally at least 20, 25, 50, 100, 200, or 300 base pairs from the target gene (e.g., an exon, intron, or splice site within the target gene), transcription control element, or anchor sequence).
  • the term “specific”, referring to an agent having an activity, is understood by those skilled in the art to mean that the agent discriminates between potential target entities or states.
  • an agent is said to bind “specifically” to its target if it binds preferentially with that target in the presence of one or more competing alternative targets.
  • specific interaction is dependent upon the presence of a particular structural feature of the target entity (e.g., an epitope, a cleft, a binding site). It is to be understood that specificity need not be absolute. In some embodiments, specificity may be evaluated relative to that of the binding agent for one or more other potential target entities (e.g., competitors).
  • specificity is evaluated relative to that of a reference specific binding agent. In some embodiments, specificity is evaluated relative to that of a reference non-specific binding agent. In some embodiments, the agent or entity does not detectably bind to the competing alternative target under conditions of binding to its target entity. In some embodiments, binding agent binds with higher on-rate, lower off-rate, increased affinity, decreased dissociation, and/or increased stability to its target entity as compared with the competing alternative target(s). As used herein, the term “specific binding” refers to an ability to discriminate between possible binding partners in the environment in which binding is to occur. In some embodiments, a binding agent that interacts with one particular target when other potential targets are present is said to "bind specifically" to the target with which it interacts.
  • specific binding is assessed by detecting or determining degree of association between the binding agent and its partner; in some embodiments, specific binding is assessed by detecting or determining degree of dissociation of a binding agent-partner complex. In some embodiments, specific binding is assessed by detecting or determining ability of the FH12877464.1 Attorney Docket: OGY-02925 binding agent to compete with an alternative interaction between its partner and another entity. In some embodiments, specific binding is assessed by performing such detections or determinations across a range of concentrations. As used herein, the term “substantially” refers to the qualitative condition of exhibiting total or near-total extent or degree of a characteristic or property of interest.
  • an agent or entity is considered to “target” another agent or entity, in accordance with the present disclosure, if it binds specifically to the targeted agent or entity under conditions in which they come into contact with one another.
  • an antibody or antigen-binding fragment thereof targets its cognate epitope or antigen.
  • a nucleic acid having a particular sequence targets a nucleic acid of substantially complementary sequence.
  • target gene means a gene that is targeted for modulation, e.g., of expression.
  • a target gene is part of a targeted genomic complex (e.g., a gene that has at least part of its genomic sequence as part of a target genomic complex, e.g., inside an anchor sequence-mediated conjunction), which genomic complex is targeted by one or more modulating agents as described herein.
  • modulation comprises inhibition of expression of the target gene.
  • a target gene is modulated by contacting the target gene or a transcription control element operably linked to the target gene with an expression activation system, e.g., expression activator(s), described herein.
  • a target gene is aberrantly expressed (e.g., over-expressed) in a cell, e.g., a cell in a subject (e.g., patient).
  • targeting moiety means an agent or entity that specifically targets, e.g., binds, a genomic sequence element (e.g., an expression control sequence or anchor sequence).
  • the genomic sequence element is proximal to and/or operably linked to a target gene.
  • therapeutic agent refers to an agent that, when administered to a subject, has a therapeutic effect and/or elicits a desired biological and/or pharmacological effect.
  • a therapeutic agent is any substance that can be used to alleviate, ameliorate, relieve, inhibit, prevent, delay onset of, reduce severity of, and/or reduce incidence of one or more symptoms or features of a disease, disorder, and/or FH12877464.1 Attorney Docket: OGY-02925 condition.
  • a therapeutic agent comprises an expression activation system, e.g., an expression activator, described herein.
  • a therapeutic agent comprises a nucleic acid encoding an expression activation system, e.g., an expression activator, described herein.
  • a therapeutic agent comprises a pharmaceutical composition described herein.
  • a therapeutically effective amount means an amount of a substance (e.g., a therapeutic agent, composition, and/or formulation) that elicits a desired biological response when administered as part of a therapeutic regimen.
  • a therapeutically effective amount of a substance is an amount that is sufficient, when administered to a subject suffering from or susceptible to a disease, disorder, and/or condition, to treat, diagnose, prevent, and/or delay the onset of the disease, disorder, and/or condition.
  • an effective amount of a substance may vary depending on such factors as desired biological endpoint(s), substance to be delivered, target cell(s) or tissue(s), etc.
  • an effective amount of compound in a formulation to treat a disease, disorder, and/or condition is an amount that alleviates, ameliorates, relieves, inhibits, prevents, delays onset of, reduces severity of and/or reduces incidence of one or more symptoms or features of the disease, disorder, and/or condition.
  • the disclosure relates to the following embodiments. Throughout this section, the term “embodiment” is abbreviated as “E” followed by an ordinal. For example, E1 is equivalent to Embodiment 1. E1.
  • An expression activator for increasing expression of a target gene comprising: (i) a DNA-targeting moiety that binds to a target sequence of 15-20 nucleotides in or near the target gene; and (ii) an effector domain comprising an epigenetic activating domain encoded by a fragment of a gene selected from MYBL1, DUX4, MYB, CEPBPD, ATF4, NCOA2, TP53, FOXO1, NFE2, E2F2, UBN1, CREB3L1, FOXO3, KLF15, MLXIPL, LEUTX, INO80D, CR3L1, ATF6A, CSN9, FOXO6, CACTIN, C1QBP, KLF4, MYC, NUTM1, VP16, VP64, YLR278C, PIP2 and STB3.
  • the expression activator of Embodiment 1, wherein the DNA-targeting moiety comprises a zinc finger (ZF) domain.
  • FH12877464.1 Attorney Docket: OGY-02925 E3.
  • the expression activator of Embodiment 1, wherein the DNA-targeting moiety comprises a transcription activator-like effector (TALE) domain.
  • TALE transcription activator-like effector
  • the expression activator of Embodiment 1, wherein the DNA-targeting moiety comprises a nuclease inactive polypeptide and a gRNA comprising a sequence complementary to the target sequence.
  • E5. The expression activator of Embodiment 4, wherein the nuclease inactive polypeptide is a nuclease inactive Cas (dCas) polypeptide.
  • dCas nuclease inactive Cas
  • the expression activator of any one of Embodiments 1-7, wherein the effector domain comprises an amino acid sequence having at least 90% identity to an amino acid sequence selected from SEQ ID Nos: 1-35.
  • FH12877464.1 Attorney Docket: OGY-02925 E16. A method of increasing expression of a target gene in a cell, comprising contacting the cell with the expression activator of any one of Embodiments 1-15.
  • E17 A nucleic acid encoding the expression activator of any one of Embodiments 1-15.
  • E18. A recombinant expression vector comprising the nucleic acid of Embodiment 17.
  • E19. A messenger RNA (mRNA) encoding the expression activator of any one of Embodiments 1-15.
  • E20. A lipid nanoparticle (LNP) comprising the expression activator of any one of Embodiments 1-15, the nucleic acid of Embodiment 17, the recombinant expression vector of Embodiment 18, or the mRNA of Embodiments 19. E21.
  • a pharmaceutical composition comprising the expression activator of any one of Embodiments 1-15, the nucleic acid of Embodiment 17, the recombinant expression vector of Embodiment 18, the mRNA of Embodiment 19, or the LNP of Embodiment 20, and a pharmaceutically acceptable carrier.
  • E22. A method of altering expression of a target gene in a cell, comprising contacting the cell with the expression activator of any one of Embodiment 1-15, the nucleic acid of Embodiment 17, the recombinant expression vector of Embodiment 18, the mRNA of Embodiment 19, the LNP of Embodiment 20, or the pharmaceutical composition of Embodiment 21.
  • Example 1 Development and Validation of Epigenetic Activators for Upregulation of Target Genes Described in this Example are methods to generate and validate fusion proteins composed of an epigenetic activator to upregulate expression of target genes, which may occur by demethylation of CpG sequences in a transcriptional regulatory element (e.g., promoter).
  • a transcriptional regulatory element e.g., promoter
  • Epigenetic regulators having activating domains encoded by fragments of a selected gene are generated and utilized in a three component system.
  • the system includes i) a dCas9 fused to 5 GCN4 peptides (e.g., dCas9-SunTag), ii) a fusion protein having an scFv fused to 104 FH12877464.1 Attorney Docket: OGY-02925 the activating domain, where the scFv is specific for a GCN4 peptide of the dCas9-SunTag, and iii) a guide RNA (gRNA) designed to direct the dCas9 and fusion protein to a target DNA sequence.
  • gRNA guide RNA
  • a target gene is scanned using a bioinformatics tool to identify regions predicted to increase gene expression, for example, upon decreased methylation of CpG residues within the region.
  • a database is used that contains the human genome annotated with potential regions that are CpG islands.
  • UCSC Genome Browser is an exemplary database that provides the human genome annotated with potential CpG islands characterized by a minimum length of about 200 bases, a GC content of 50% or greater, and a ratio greater than 0.6 of observed number of CpG residues to the expected number based on the number of guanine and cytosine nucleotides in the segment.
  • the promoter region of a target gene is evaluated to identify potential CpG islands using this approach.
  • Target sequences within this region are selected for experimental evaluation based upon criteria such as low likelihood of off-target binding.
  • a gRNA comprising a nucleic acid sequence complementary to the selected target sequence is designed to be used with an mRNA encoding a dCas9 linked to 5 GCN4 peptides, e.g., by linker, and an mRNA encoding an scFv specific for a GCN4 peptide and fused to an epigenetic activator, e.g., by a linker.
  • mRNA encoding a a TALE domain or a zinc-finger (ZF) domain fused to an epigenetic activator is designed.
  • a TALE domain that binds the selected target sequence is designed by modifying the repeat array in a Xanthomonas TALE to have RVDs that correspond to each nucleotide in the target sequence (e.g., NI for A; HD for C; NN or NK for G; and NG for T).
  • a ZF domain that binds the selected target sequence is designed by modifying the one or more Zn fingers or fragments thereof.
  • the mRNA molecules are constructed with a 5 ⁇ Cap 1 structure, a polyA tail, and fully-modified with N1-methyl-pseudouridine.
  • the effect on target gene expression is measured in vitro.
  • cells e.g., Hepa1-6 cells, HEP3B cells, or K562 cells
  • LNP-formulated mRNA encoding the dCas9-SunTag, and scFv fusion protein. Negative control cells are untreated.
  • RNA is reverse-transcribed to cDNA and analyzed by multiplexed qPCR using FH12877464.1 Attorney Docket: OGY-02925 TaqMan ® primer probes specific to either ACTB or GAPDH (housekeeper control) and a target gene. Relative target gene mRNA expression is determined through the comparative ⁇ Ct method. An increase in target gene mRNA expressed relative to untreated cells indicates the LNP-formulated mRNA functioned for upregulation of the target gene.
  • genomic DNA is isolated from cells samples at the indicated time points following treatment with LNP- formulated mRNA encoding dCas9-SunTag and mRNA encoding an scFv-effector fusion protein. Negative control cells are untreated. Genomic DNA is sheared, e.g., by sonication.
  • Fragmented DNA was purified using SPRI beads (1 ⁇ SPRISelect, Beckman-Coulter ® Cat #B23319) and subjected to EM-conversion using a NEB ® EM-seq Conversion Kit (Cat #E7125) according to the manufacturer’s instructions.
  • Purified, converted DNA is PCR amplified at the target gene locus.
  • the amplicon is transposase-labeled with Illumina ® sequencing adapters. Libraries are dual-indexed (combinatorial) via PCR (see, e.g., Mezger A, et al. Nature Comm.2018 (PMID: 30194434)).
  • Final libraries are purified using SPRI beads and sequenced on a MiSeq (Illumina ® ).
  • the EM-Seq data is assessed for alignment quality using FastQC, adapter-trimmed and quality filtered using TrimGalore, and aligned to the mm10 reference genome using Bismark.
  • Fragment-level methylation calls are made using the "Bismark Methylation Extractor" tool for each sample and aggregated into strand-specific CpG, CHG, and CHH context files.
  • CpG context is the measure of interest while CHG and CHG files are used to assess conversion efficiency. Fragments are flagged if there is any sign of incomplete conversion in CHG and CHH contexts, and these fragment IDs are used to filter the CpG context files prior to quantifying methylation levels.
  • CpG methylation is determined by the ratio of methylated read coverage to total read coverage for each CpG. Amplicon-wide and CpG-specific mean methylation values are calculated at the level of technical replicates, biological replicates, and experimental groups and summarized in box / dot plots. A decrease in amplicon-wide CpG methylation relative to untreated cells indicates the LNP-formulated mRNA functioned for epigenetic regulation at the targeted region.
  • the effect on target gene expression and/or DNA methylation with a dCas9-SunTag and scFv-effector fusion protein as described is not appreciably altered relative to a negative control (e.g., target gene expression is not appreciably increased and/or DNA methylation is not appreciably decreased, each relative to untreated control cells), attributable, at least in part and without being bound by theory, to factors such as insufficient binding affinity of the particular DNA binding domain to the target sequence, insufficient FH12877464.1 Attorney Docket: OGY-02925 loading of mRNA encoding the construct into the LNP (e.g., as a result of the particular mRNA sequence tested), and/or insufficient expression of the mRNA upon introducing the LNP-formulated mRNA encoding the construct to cells.
  • an scFv-effector fusion protein directed to a target sequence e.g., by codon optimization of the mRNA sequence, selection of RVDs in the TALE approach that enhance binding affinity to the target sequence, and selection of alternate lipid formulations to improve LNP packaging.
  • Example 2 Targeted Modification Results in Upregulation of Target Gene mRNA
  • This example describes editing target genes with (i) a fusion of a catalytically inactive Cas9 (dCas9) to 5 GCN4 peptides, (ii) an scFv specific for GCN4 peptide fused to an epigenetic activating domain encoded by a fragment of a selected gene, and (iii) gRNA to target sequences within a target gene (e.g., Target Gene 1, Target Gene 2, Target Gene 3, Target Gene 4, Target Gene 5, Target Gene 6, Target Gene 7, Target Gene 8, or Target Gene 9) to upregulate target gene expression.
  • a target gene e.g., Target Gene 1, Target Gene 2, Target Gene 3, Target Gene 4, Target Gene 5, Target Gene 6, Target Gene 7, Target Gene 8, or Target Gene
  • gRNAs were designed to target sequences near or within the promoter or enhancer region CpG-island of a target gene (i.e., Target Gene 1, Target Gene 2, Target Gene 3, Target Gene 4, Target Gene 5, Target Gene 6, Target Gene 7, Target Gene 8, or Target Gene 9) and were assessed for target gene upregulation. Effectors designed and generated are provided in Table 3.
  • the mRNA sequence of dCas9-SunTag is provided in SEQ ID NO: 36
  • the DNA sequence of dCas9-SunTag is provided in SEQ ID NO: 72. Table 3.
  • expression activators having epigenetic activating domains encoded by a fragment of gene VPR, VP64, NC0A1, LEUTX, E2F2, KLF15, FH12877464.1 Attorney Docket: OGY-02925 INO80D, FOXO6, or TP53 and used in combination with gRNAs targeting the distal or proximal promoter resulted in significantly increased Target Gene 1 expression compared to untreated control cells.
  • expression activators having epigenetic activating domains encoded by a fragment of gene VPR, VP16, VP64, NCOA2, MYBL1, LEUTX, CREB3L1, E2F2, or KLF15 and used in combination with gRNAs targeting the enhancer, the promoter, or the enhancer/promoter resulted in significantly increased Target Gene 2 expression compared to untreated control cells.
  • expression activators having epigenetic activating domains encoded by a fragment of gene VPR, VP64, VP16, NCOA2, MYBL1, LEUTX, CREB3L1, E2F2, KLF15, UBN1, INO80D, FOXO6, or TP53 and used in combination with gRNAs targeting the enhancer, promoter, or enhancer/promoter resulted in significantly increased Target Gene 3 expression compared to untreated control cells.
  • expression activators having epigenetic activating domains encoded by a fragment of gene MYBL1, E2F2, NCOA2, LEUTX, or CREB3L1 and used in combination with gRNAs targeting Site 1, Site 2, Site 3, Site 4, or Site 5 of Target Gene 4 in Hepa1-6 cells resulted in significantly increased Target Gene 4 expression compared to untreated control cells.
  • Expression activators with epigenetic activating domains encoded by a fragment of NCOA2 or LEUTX showed the highest activity at day 6.
  • activators having epigenetic activating domains encoded by a fragment of gene MYBL1, or E2F2 and used in combination with gRNAs targeting Site 1, Site 2, Site 3, Site 4, Site 5, and a combination of Site 1/ Site 3/ Site 5 of Target Gene 4 in Hepa1-6 cells resulted in significantly increased Target Gene 4 protein expression compared to control cells treated with a dCas9-SunTag or PIP2 domain.
  • expression activators having epigenetic activating domains encoded by a fragment of gene NCOA2 or LEUTX and used in combination with gRNAs targeting Target Gene 5 resulted in significantly increased Target Gene 5 expression compared to untreated control cells.
  • Cells were either sorted (FIG.6B or FIG.6D) or not sorted (FIG.6A or FIG.6C) using a cell sorter to isolate cells that have GFP signal which indicated expression of the epigenetic regulators. Cells were treated with a second dose of the effectors (re-transfected) on Day 15 (FIG.6C and FIG.6D) and upregulation of mRNA expression was induced.
  • expression activators having epigenetic activating domains encoded by a fragment of gene VP64, VP16, NCOA2, MYBL1, LEUTX, CREB3L1, E2F2, KLF15, FOXO3, UBN1, INO80D, or FIXO6 and used in combination FH12877464.1 Attorney Docket: OGY-02925 with gRNAs targeting Target Gene 6 resulted in significantly increased Target Gene 6 expression compared to untreated control.
  • the epigenetic activating domains encoded by the gene fragments of NCOA2, MYBL1, LEUTX, NCOA2, CREB3L1, and E2F2 and used in combination with gRNA pools targeting Site 1 or Site 2 in Hepa 1-6 cells resulted in significantly increased Target Gene 7 gene expression compared to untreated control cells out to 4-days post transfection. After day 4, activation returns to baseline over the course of 3 weeks.
  • the epigenetic activating domains encoded by the gene fragment of NCOA2 or PA.v1b and used in combination with gRNA pools targeting Site 1, Site 2, Site 3, or Site 4 in Hepa 1-6 cells resulted in significantly increased Target Gene 8 gene expression compared to untreated control cells out to 7-days post transfection.
  • the gRNA targeting Site 4 induced the highest up-regulation of Target Gene 8 expression 3-days post transfection.
  • Example 3 Targeted Modification Results in Modulation of Target Gene 4 in vivo This example describes the effect of transcriptional activation domains on Target Gene 4 expression from targeting the Target Gene 4 promoter with a gRNA (Site 1) and dCas9-SunTag and scFv-sfGFP-NCOA2 or scFv-sfGFP-LEUTX effector fusions in C57BL/J6 mice.
  • NNPs epigenetic activating domain polypeptides
  • LNPs were treated at a concentration of 3 mg/kg over the course of 7 days with a single administration on day 0.
  • Target Gene 4 serum expression was detected (FIG.11A) or normalized to day 0 baseline and presented as percentage of day 0 (FIG.11B).
  • NCOA2 and LEUTX significantly induced 111 FH12877464.1 Attorney Docket: OGY-02925 Target Gene 4 expression 3-days post administration and by 8-days post administration Target Gene 4 expression was statistically indistinguishable from PBS treated mice.
  • Target Gene 4 mRNA levels were assessed.
  • MC3-encapsulated NCOA2 and LEUTX LNPs were administered to C57BL/J6 mice as described above.
  • Example 4 Targeted Modification Results in Modulation of Target Gene 4 protein in vivo This example describes the effect of epigenetic activation domains, DNA demethylation combined with epigenetic activation domains, and PRDM9-mediated trimethylation of H3K4me and H3K36me on Target Gene 4 expression from targeting the Target Gene 4 promoter in C57BL/J6 mice.
  • NCOA2, PA.v1b, E2F2, MYBL1, and KLF15 epigenetic activating domain polypeptides
  • PBS negative control
  • LNPs consisting of dCas9-SunTag, gRNA (Site 1), and a scFv fused to an epigenetic activating domain of a polypeptide (NCOA2, PA.v1b, E2F2, MYBL1, and KLF15).
  • LNPs were treated at a concentration of 3 mg/kg over the course of 6 days with a single administration on day 0.
  • 14–15-week-old male C57BL/J6 were treated with PBS (negative control) and MC3 coformulated LNPS of TET2-XTEN80-dCas9, dCas9-SunTag: scFv NCOA2 fusion, and gRNA.
  • LNPs were treated at a concentration of 3 mg/kg over the course of 6 days with a FH12877464.1 Attorney Docket: OGY-02925 single administration on day 0.
  • LNPs were treated at a concentration of 3 mg/kg over the course of 6 days with a single administration on day 0. Serum was isolated from the respective groups at day -1-, 2-, and 6-days post- administration.
  • Target Gene 4 serum expression was detected and quantified using the Mouse/Rat FGF-21 Quantikine ELISA (R&D Systems #MF2100) according to manufacturer’s instructions.
  • Target Gene 4 protein levels were calculated as pg/ml (FIG.12) or normalized to day 0 baseline and presented as percentage of day 0 (FIGs.13A-13C).
  • activators having epigenetic activating domains encoded by a fragment of gene NCOA2 and the viral IRF2 (PA.v1b) protein and used in combination with gRNA targeting Site 1 of Target Gene 4 in C57BL/J6 mice resulted in significantly increased serum Target Gene 4 protein expression at 2-days post administration and decreased to levels comparable to PBS treated mice at 6-days post administration.
  • the combination of NCOA2 and TET2-mediated DNA demethylation similarly significantly increased serum Target Gene 4 protein expression at 2-days post administration and decreased to levels comparable to PBS treated mice 6-days post administration (FIGs.12 and 13B).
  • PRDM9 treatment significantly reduced Target Gene 4 protein expression 2-days post administration and expression then increased to comparable levels to PBS treated mice 6-days post administration (FIGs.12 and 13C). These results indicate the transcriptional activating effector domains, NCOA2 and PA.v1b, induce short-term (2-days post administration) Target Gene 4 expression in the liver that is secreted into circulation. Additionally, combining NCOA2 with TET2-mediated DNA demethylation induces short-term (2-days post administration) Target Gene 4 expression in the liver that is secreted into circulation. Finally, PRDM9 treatment promoted short-term (2- days post administration) reductions in circulating Target Gene 4 protein expression.
  • Example 5 Targeted Modification Results in Modulation of Target Gene 8 mRNA in vivo This example describes the effect of transcriptional activation domains on Target Gene 8 expression from targeting the Target Gene 8 promoter with a gRNA (Site 4) and dCas9-SunTag and scFv-sfGFP-NCOA2 effector fusions in C57BL/J6 mice.
  • FH12877464.1 Attorney Docket: OGY-02925
  • NNPs epigenetic activating domain polypeptide
  • LNPs were treated with a single administration at a concentration of 3 mg/kg for mice sacrificed at 3 days post administration or two administrations (once at day 0 and another at day 8) for mice sacrificed at 22- or 36- days post administration.
  • mRNA was isolated from mouse liver punches at day 3-, 22-, and 36-days post- administration using Macherey-Nagel NuceleoSpin 96 RNA kit in accordance with the manufacturer’s instructions. RNA was reverse transcribed to cDNA and analyzed by multiplexed qPCR using TaqMan® primer probes specific to B2M (housekeeper control) and Target Gene 8. Relative Target Gene 8 mRNA expression is determined through the comparative ⁇ Ct method.
  • Target Gene 8 mRNA expressed relative to untreated cells indicates the LNP-formulated mRNA functioned for upregulation of Target Gene 8.
  • the NCOA2 activating domain fragment used in combination with gRNA targeting Site 4 of Target Gene 8 in C57BL/6 mice resulted in significantly increased Target Gene 8 mRNA expression at 3-, and 22-days post administrations with expression returning to baseline 36-days post administration.
  • the NCOA2 activating domain fragment used in combination with gRNA targeting Site 4 of Target Gene 8 in C57BL/6 mice did not result in significant changes in body weight compared to PBS-treated control mice out to 36-days post administration.
  • Example 6 Targeted Modification Results in Modulation of Target Gene 6 mRNA
  • ZF zinc finger DNA binding domain
  • ZF-NCOA2 activator domain ZF-NCOA2
  • ZF-LEUTX LEUTX activator domain
  • a region encompassing a portion of the zinc finger DNA binding sites at the Target Gene 6 promoter was scanned for identification of target sequences for ZFs and a bioinformatics approach was taken to select suitable target sequences based upon FH12877464.1 Attorney Docket: OGY-02925 criteria that included likelihood of off-target binding.
  • ZFs were designed to target sequences within the promoter of Target Gene 6 and were tested for Target Gene 6 upregulation. Each ZF was modified by tethering it to NCOA2 (a nuclear receptor coactivator) or LEUTX (a homeobox transcriptional activator) to generate a ZF-NCOA2 or ZF-LEUTX fusion.
  • Each ZF also comprised a nuclear localization sequence (NLS), coupled to the ZF via a short spacer.
  • mRNA encoding the fusions comprised an ORF encoding, from 5 ⁇ linker; (iii) NCOA2; and (iv) LEUTX.
  • the mRNA further included a 5 ⁇ UTR, a 3 ⁇ UTR, and a 3 ⁇ poly-A sequence.
  • the mRNA sequences were prepared by in vitro-transcription and fully modified with N1-methyl-pseudouridine (m1 ⁇ ). Furthermore, the mRNAs were synthesized to have a polyA-tail and a Cap1 structure.
  • epigenetic activating domain polypeptides (NCOA2 or LEUTX) on Target Gene 8 expression
  • K- 562 cells (ATCC CCL-243) were treated with 0.625 ⁇ g/ml of the indicated mRNA in lipid. Untreated cells were utilized as a reference control.

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Abstract

La présente invention concerne des compositions et des procédés pour augmenter l'expression de gènes cibles dans une cellule, par exemple , à l'aide d'un activateur d'expression qui comprend une fraction de ciblage qui se lie à un gène cible et un domaine effecteur comprenant un domaine d'activation épigénétique. L'invention concerne également des systèmes comprenant au moins deux activateurs d'expression.
PCT/US2025/028069 2024-05-07 2025-05-07 Modulation épigénétique pour la régulation positive de gènes Pending WO2025235563A1 (fr)

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Citations (58)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US789538A (en) 1904-11-11 1905-05-09 Colin E Ham Dumb-bell.
WO1995019431A1 (fr) 1994-01-18 1995-07-20 The Scripps Research Institute Derives de proteine a doigts zinciques et procedes associes
WO1996006166A1 (fr) 1994-08-20 1996-02-29 Medical Research Council Ameliorations concernant des proteines de liaison permettant de reconnaitre l'adn
US5789538A (en) 1995-02-03 1998-08-04 Massachusetts Institute Of Technology Zinc finger proteins with high affinity new DNA binding specificities
WO1998037186A1 (fr) 1997-02-18 1998-08-27 Actinova Limited Bibliotheque d'expression in vitro de proteine ou de peptide
WO1998053059A1 (fr) 1997-05-23 1998-11-26 Medical Research Council Proteines de liaison d'acide nucleique
WO1998053060A1 (fr) 1997-05-23 1998-11-26 Gendaq Limited Proteines de liaison d'acide nucleique
WO1998054311A1 (fr) 1997-05-27 1998-12-03 The Scripps Research Institute Derives de proteines a doigts de zinc et procedes associes
US5925523A (en) 1996-08-23 1999-07-20 President & Fellows Of Harvard College Intraction trap assay, reagents and uses thereof
GB2338237A (en) 1997-02-18 1999-12-15 Actinova Ltd In vitro peptide or protein expression library
WO2000027878A1 (fr) 1998-11-09 2000-05-18 Gendaq Limited Systeme de criblage de polypeptides a motifs en doigt de zinc, destine a mettre en evidence une certaine capacite de liaison
US6140815A (en) 1998-06-17 2000-10-31 Dover Instrument Corporation High stability spin stand platform
US6242568B1 (en) 1994-01-18 2001-06-05 The Scripps Research Institute Zinc finger protein derivatives and methods therefor
WO2001060970A2 (fr) 2000-02-18 2001-08-23 Toolgen, Inc. Domaines a doigts de zinc et leurs procedes d'identification
WO2001088197A2 (fr) 2000-05-16 2001-11-22 Massachusetts Institute Of Technology Methodes et compositions de dosage de piegeage par interaction
WO2002016536A1 (fr) 2000-08-23 2002-02-28 Kao Corporation Detergent bactericide antisalissures, destine aux surfaces dures
US6410248B1 (en) 1998-01-30 2002-06-25 Massachusetts Institute Of Technology General strategy for selecting high-affinity zinc finger proteins for diverse DNA target sites
US6453242B1 (en) 1999-01-12 2002-09-17 Sangamo Biosciences, Inc. Selection of sites for targeting by zinc finger proteins and methods of designing zinc finger proteins to bind to preselected sites
WO2002077227A2 (fr) 2000-11-20 2002-10-03 Sangamo Biosciences, Inc. Optimisation iterative de la conception de proteines de liaison
US6479626B1 (en) 1998-03-02 2002-11-12 Massachusetts Institute Of Technology Poly zinc finger proteins with improved linkers
WO2002099084A2 (fr) 2001-04-04 2002-12-12 Gendaq Limited Polypeptides de liaison composites
US6503717B2 (en) 1999-12-06 2003-01-07 Sangamo Biosciences, Inc. Methods of using randomized libraries of zinc finger proteins for the identification of gene function
WO2003016496A2 (fr) 2001-08-20 2003-02-27 The Scripps Research Institute Domaines de fixation en doigt de zinc pour cnn
US6534261B1 (en) 1999-01-12 2003-03-18 Sangamo Biosciences, Inc. Regulation of endogenous gene expression in cells using zinc finger proteins
US6599692B1 (en) 1999-09-14 2003-07-29 Sangamo Bioscience, Inc. Functional genomics using zinc finger proteins
US6689558B2 (en) 2000-02-08 2004-02-10 Sangamo Biosciences, Inc. Cells for drug discovery
US6693086B1 (en) 1998-06-25 2004-02-17 National Jewish Medical And Research Center Systemic immune activation method using nucleic acid-lipid complexes
US20050064474A1 (en) 2003-08-08 2005-03-24 Sangamo Biosciences, Inc. Methods and compositions for targeted cleavage and recombination
US20050267061A1 (en) 2004-04-08 2005-12-01 Sangamo Biosciences, Inc. Methods and compositions for treating neuropathic and neurodegenerative conditions
US7013219B2 (en) 1999-01-12 2006-03-14 Sangamo Biosciences, Inc. Regulation of endogenous gene expression in cells using zinc finger proteins
US7030215B2 (en) 1999-03-24 2006-04-18 Sangamo Biosciences, Inc. Position dependent recognition of GNN nucleotide triplets by zinc fingers
US7067317B2 (en) 2000-12-07 2006-06-27 Sangamo Biosciences, Inc. Regulation of angiogenesis with zinc finger proteins
US7070934B2 (en) 1999-01-12 2006-07-04 Sangamo Biosciences, Inc. Ligand-controlled regulation of endogenous gene expression
US7253273B2 (en) 2004-04-08 2007-08-07 Sangamo Biosciences, Inc. Treatment of neuropathic pain with zinc finger proteins
US7262054B2 (en) 2002-01-22 2007-08-28 Sangamo Biosciences, Inc. Zinc finger proteins for DNA binding and gene regulation in plants
US20070218528A1 (en) 2004-02-05 2007-09-20 Sangamo Biosciences, Inc. Methods and compositions for targeted cleavage and recombination
US7361635B2 (en) 2002-08-29 2008-04-22 Sangamo Biosciences, Inc. Simultaneous modulation of multiple genes
US20090226470A1 (en) 2007-12-11 2009-09-10 Mauro Vincent P Compositions and methods related to mRNA translational enhancer elements
US20100293625A1 (en) 2007-09-26 2010-11-18 Interexon Corporation Synthetic 5'UTRs, Expression Vectors, and Methods for Increasing Transgene Expression
WO2012019168A2 (fr) 2010-08-06 2012-02-09 Moderna Therapeutics, Inc. Acides nucléiques modifiés et leurs procédés d'utilisation
WO2012045075A1 (fr) 2010-10-01 2012-04-05 Jason Schrum Nucléosides, nucléotides et acides nucléiques modifiés et leurs utilisations
WO2012135805A2 (fr) 2011-03-31 2012-10-04 modeRNA Therapeutics Administration et formulation d'acides nucléiques génétiquement modifiés
WO2013052523A1 (fr) 2011-10-03 2013-04-11 modeRNA Therapeutics Nucléosides, nucléotides et acides nucléiques modifiés, et leurs utilisations
WO2013151665A2 (fr) 2012-04-02 2013-10-10 modeRNA Therapeutics Polynucléotides modifiés destinés à la production de protéines associées à une maladie humaine
WO2013151666A2 (fr) 2012-04-02 2013-10-10 modeRNA Therapeutics Polynucléotides modifiés destinés à la production de produits biologiques et de protéines associées à une maladie humaine
WO2014093924A1 (fr) 2012-12-13 2014-06-19 Moderna Therapeutics, Inc. Molécules d'acide nucléique modifiées et leurs utilisations
WO2014144767A1 (fr) 2013-03-15 2014-09-18 Moderna Therapeutics, Inc. Purification d'arnm par échange d'ions
WO2014152030A1 (fr) 2013-03-15 2014-09-25 Moderna Therapeutics, Inc. Elimination de fragments d'adn dans des procédés de production d'arnm
WO2014152031A1 (fr) 2013-03-15 2014-09-25 Moderna Therapeutics, Inc. Purification d'acide ribonucléique
WO2014164253A1 (fr) 2013-03-09 2014-10-09 Moderna Therapeutics, Inc. Régions non traduites hétérologues pour arnm
WO2015038892A1 (fr) 2013-09-13 2015-03-19 Moderna Therapeutics, Inc. Compositions polynucléotididiques contenant des acides aminés
WO2015089511A2 (fr) 2013-12-13 2015-06-18 Moderna Therapeutics, Inc. Molécules d'acides nucléiques modifiés et leurs utilisations
WO2015196118A1 (fr) 2014-06-19 2015-12-23 Moderna Therapeutics, Inc. Molécules d'acide nucléique différentes et utilisations
WO2015196128A2 (fr) 2014-06-19 2015-12-23 Moderna Therapeutics, Inc. Molécules d'acide nucléique alternatives et leurs utilisations
WO2015196130A2 (fr) 2014-06-19 2015-12-23 Moderna Therapeutics, Inc. Molécules d'acide nucléique alternatives et leurs utilisations
WO2016011306A2 (fr) 2014-07-17 2016-01-21 Moderna Therapeutics, Inc. Modifications de terminal de polynucléotides
CN115627272A (zh) * 2021-07-14 2023-01-20 中国科学院遗传与发育生物学研究所 一种靶向去除dna甲基化的植物表观遗传修饰编辑器
WO2023247789A1 (fr) * 2022-06-24 2023-12-28 European Molecular Biology Laboratory Outil modulaire basé sur une crispr pour l'introduction spécifique de modifications épigénétiques sur des loci cibles

Patent Citations (82)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US789538A (en) 1904-11-11 1905-05-09 Colin E Ham Dumb-bell.
WO1995019431A1 (fr) 1994-01-18 1995-07-20 The Scripps Research Institute Derives de proteine a doigts zinciques et procedes associes
US6140466A (en) 1994-01-18 2000-10-31 The Scripps Research Institute Zinc finger protein derivatives and methods therefor
US6242568B1 (en) 1994-01-18 2001-06-05 The Scripps Research Institute Zinc finger protein derivatives and methods therefor
WO1996006166A1 (fr) 1994-08-20 1996-02-29 Medical Research Council Ameliorations concernant des proteines de liaison permettant de reconnaitre l'adn
US6013453A (en) 1994-08-20 2000-01-11 Medical Research Council Binding proteins for recognition of DNA
US6007988A (en) 1994-08-20 1999-12-28 Medical Research Council Binding proteins for recognition of DNA
US5789538A (en) 1995-02-03 1998-08-04 Massachusetts Institute Of Technology Zinc finger proteins with high affinity new DNA binding specificities
US5925523A (en) 1996-08-23 1999-07-20 President & Fellows Of Harvard College Intraction trap assay, reagents and uses thereof
US6200759B1 (en) 1996-08-23 2001-03-13 President And Fellows Of Harvard College Interaction trap assay, reagents and uses thereof
WO1998037186A1 (fr) 1997-02-18 1998-08-27 Actinova Limited Bibliotheque d'expression in vitro de proteine ou de peptide
GB2338237A (en) 1997-02-18 1999-12-15 Actinova Ltd In vitro peptide or protein expression library
WO1998053057A1 (fr) 1997-05-23 1998-11-26 Gendaq Limited Bibliotheque de polypeptides de fixation d'acide nucleique
WO1998053058A1 (fr) 1997-05-23 1998-11-26 Gendaq Limited Proteines de liaison d'acide nucleique
WO1998053060A1 (fr) 1997-05-23 1998-11-26 Gendaq Limited Proteines de liaison d'acide nucleique
WO1998053059A1 (fr) 1997-05-23 1998-11-26 Medical Research Council Proteines de liaison d'acide nucleique
WO1998054311A1 (fr) 1997-05-27 1998-12-03 The Scripps Research Institute Derives de proteines a doigts de zinc et procedes associes
US6410248B1 (en) 1998-01-30 2002-06-25 Massachusetts Institute Of Technology General strategy for selecting high-affinity zinc finger proteins for diverse DNA target sites
US7153949B2 (en) 1998-03-02 2006-12-26 Massachusetts Institute Of Technology Nucleic acid encoding poly-zinc finger proteins with improved linkers
US6903185B2 (en) 1998-03-02 2005-06-07 Massachusetts Institute Of Technology Poly zinc finger proteins with improved linkers
US6479626B1 (en) 1998-03-02 2002-11-12 Massachusetts Institute Of Technology Poly zinc finger proteins with improved linkers
US6140815A (en) 1998-06-17 2000-10-31 Dover Instrument Corporation High stability spin stand platform
US6693086B1 (en) 1998-06-25 2004-02-17 National Jewish Medical And Research Center Systemic immune activation method using nucleic acid-lipid complexes
WO2000027878A1 (fr) 1998-11-09 2000-05-18 Gendaq Limited Systeme de criblage de polypeptides a motifs en doigt de zinc, destine a mettre en evidence une certaine capacite de liaison
US7163824B2 (en) 1999-01-12 2007-01-16 Sangamo Biosciences, Inc. Regulation of endogenous gene expression in cells using zinc finger proteins
US6453242B1 (en) 1999-01-12 2002-09-17 Sangamo Biosciences, Inc. Selection of sites for targeting by zinc finger proteins and methods of designing zinc finger proteins to bind to preselected sites
US7070934B2 (en) 1999-01-12 2006-07-04 Sangamo Biosciences, Inc. Ligand-controlled regulation of endogenous gene expression
US6824978B1 (en) 1999-01-12 2004-11-30 Sangamo Biosciences, Inc. Regulation of endogenous gene expression in cells using zinc finger proteins
US6534261B1 (en) 1999-01-12 2003-03-18 Sangamo Biosciences, Inc. Regulation of endogenous gene expression in cells using zinc finger proteins
US6607882B1 (en) 1999-01-12 2003-08-19 Sangamo Biosciences, Inc. Regulation of endogenous gene expression in cells using zinc finger proteins
US6979539B2 (en) 1999-01-12 2005-12-27 Sangamo Biosciences, Inc. Regulation of endogenous gene expression in cells using zinc finger proteins
US6933113B2 (en) 1999-01-12 2005-08-23 Sangamo Biosciences, Inc. Modulation of endogenous gene expression in cells
US7013219B2 (en) 1999-01-12 2006-03-14 Sangamo Biosciences, Inc. Regulation of endogenous gene expression in cells using zinc finger proteins
US7030215B2 (en) 1999-03-24 2006-04-18 Sangamo Biosciences, Inc. Position dependent recognition of GNN nucleotide triplets by zinc fingers
US6599692B1 (en) 1999-09-14 2003-07-29 Sangamo Bioscience, Inc. Functional genomics using zinc finger proteins
US6503717B2 (en) 1999-12-06 2003-01-07 Sangamo Biosciences, Inc. Methods of using randomized libraries of zinc finger proteins for the identification of gene function
US6689558B2 (en) 2000-02-08 2004-02-10 Sangamo Biosciences, Inc. Cells for drug discovery
WO2001060970A2 (fr) 2000-02-18 2001-08-23 Toolgen, Inc. Domaines a doigts de zinc et leurs procedes d'identification
WO2001088197A2 (fr) 2000-05-16 2001-11-22 Massachusetts Institute Of Technology Methodes et compositions de dosage de piegeage par interaction
WO2002016536A1 (fr) 2000-08-23 2002-02-28 Kao Corporation Detergent bactericide antisalissures, destine aux surfaces dures
US6794136B1 (en) 2000-11-20 2004-09-21 Sangamo Biosciences, Inc. Iterative optimization in the design of binding proteins
WO2002077227A2 (fr) 2000-11-20 2002-10-03 Sangamo Biosciences, Inc. Optimisation iterative de la conception de proteines de liaison
US7067317B2 (en) 2000-12-07 2006-06-27 Sangamo Biosciences, Inc. Regulation of angiogenesis with zinc finger proteins
WO2002099084A2 (fr) 2001-04-04 2002-12-12 Gendaq Limited Polypeptides de liaison composites
WO2003016496A2 (fr) 2001-08-20 2003-02-27 The Scripps Research Institute Domaines de fixation en doigt de zinc pour cnn
US7262054B2 (en) 2002-01-22 2007-08-28 Sangamo Biosciences, Inc. Zinc finger proteins for DNA binding and gene regulation in plants
US7361635B2 (en) 2002-08-29 2008-04-22 Sangamo Biosciences, Inc. Simultaneous modulation of multiple genes
US20050064474A1 (en) 2003-08-08 2005-03-24 Sangamo Biosciences, Inc. Methods and compositions for targeted cleavage and recombination
US20070218528A1 (en) 2004-02-05 2007-09-20 Sangamo Biosciences, Inc. Methods and compositions for targeted cleavage and recombination
US7253273B2 (en) 2004-04-08 2007-08-07 Sangamo Biosciences, Inc. Treatment of neuropathic pain with zinc finger proteins
US20050267061A1 (en) 2004-04-08 2005-12-01 Sangamo Biosciences, Inc. Methods and compositions for treating neuropathic and neurodegenerative conditions
US20100293625A1 (en) 2007-09-26 2010-11-18 Interexon Corporation Synthetic 5'UTRs, Expression Vectors, and Methods for Increasing Transgene Expression
US20090226470A1 (en) 2007-12-11 2009-09-10 Mauro Vincent P Compositions and methods related to mRNA translational enhancer elements
WO2012019168A2 (fr) 2010-08-06 2012-02-09 Moderna Therapeutics, Inc. Acides nucléiques modifiés et leurs procédés d'utilisation
WO2012045075A1 (fr) 2010-10-01 2012-04-05 Jason Schrum Nucléosides, nucléotides et acides nucléiques modifiés et leurs utilisations
WO2012135805A2 (fr) 2011-03-31 2012-10-04 modeRNA Therapeutics Administration et formulation d'acides nucléiques génétiquement modifiés
WO2013052523A1 (fr) 2011-10-03 2013-04-11 modeRNA Therapeutics Nucléosides, nucléotides et acides nucléiques modifiés, et leurs utilisations
WO2013151669A1 (fr) 2012-04-02 2013-10-10 modeRNA Therapeutics Polynucléotides modifiés pour la production de protéines cytoplasmiques et cytosquelettiques
WO2013151672A2 (fr) 2012-04-02 2013-10-10 modeRNA Therapeutics Polynucléotides modifiés destinés à la production de protéines et de peptides associés à l'oncologie
WO2013151665A2 (fr) 2012-04-02 2013-10-10 modeRNA Therapeutics Polynucléotides modifiés destinés à la production de protéines associées à une maladie humaine
WO2013151668A2 (fr) 2012-04-02 2013-10-10 modeRNA Therapeutics Polynucléotides modifiés destinés à la production de protéines sécrétées
WO2013151666A2 (fr) 2012-04-02 2013-10-10 modeRNA Therapeutics Polynucléotides modifiés destinés à la production de produits biologiques et de protéines associées à une maladie humaine
WO2013151664A1 (fr) 2012-04-02 2013-10-10 modeRNA Therapeutics Polynucléotides modifiés pour la production de protéines
WO2013151663A1 (fr) 2012-04-02 2013-10-10 modeRNA Therapeutics Polynucléotides modifiés pour la production de protéines membranaires
WO2013151671A1 (fr) 2012-04-02 2013-10-10 modeRNA Therapeutics Polynucléotides modifiés pour la production de protéines et de peptides cosmétiques
WO2013151670A2 (fr) 2012-04-02 2013-10-10 modeRNA Therapeutics Polynucléotides modifiés destinés à la production de protéines nucléaires
WO2013151667A1 (fr) 2012-04-02 2013-10-10 modeRNA Therapeutics Polynucléotides modifiés
WO2013151736A2 (fr) 2012-04-02 2013-10-10 modeRNA Therapeutics Production in vivo de protéines
WO2014093924A1 (fr) 2012-12-13 2014-06-19 Moderna Therapeutics, Inc. Molécules d'acide nucléique modifiées et leurs utilisations
US20160022840A1 (en) 2013-03-09 2016-01-28 Moderna Therapeutics, Inc. Heterologous untranslated regions for mrna
WO2014164253A1 (fr) 2013-03-09 2014-10-09 Moderna Therapeutics, Inc. Régions non traduites hétérologues pour arnm
WO2014152030A1 (fr) 2013-03-15 2014-09-25 Moderna Therapeutics, Inc. Elimination de fragments d'adn dans des procédés de production d'arnm
WO2014152031A1 (fr) 2013-03-15 2014-09-25 Moderna Therapeutics, Inc. Purification d'acide ribonucléique
WO2014144767A1 (fr) 2013-03-15 2014-09-18 Moderna Therapeutics, Inc. Purification d'arnm par échange d'ions
WO2015038892A1 (fr) 2013-09-13 2015-03-19 Moderna Therapeutics, Inc. Compositions polynucléotididiques contenant des acides aminés
WO2015089511A2 (fr) 2013-12-13 2015-06-18 Moderna Therapeutics, Inc. Molécules d'acides nucléiques modifiés et leurs utilisations
WO2015196118A1 (fr) 2014-06-19 2015-12-23 Moderna Therapeutics, Inc. Molécules d'acide nucléique différentes et utilisations
WO2015196128A2 (fr) 2014-06-19 2015-12-23 Moderna Therapeutics, Inc. Molécules d'acide nucléique alternatives et leurs utilisations
WO2015196130A2 (fr) 2014-06-19 2015-12-23 Moderna Therapeutics, Inc. Molécules d'acide nucléique alternatives et leurs utilisations
WO2016011306A2 (fr) 2014-07-17 2016-01-21 Moderna Therapeutics, Inc. Modifications de terminal de polynucléotides
CN115627272A (zh) * 2021-07-14 2023-01-20 中国科学院遗传与发育生物学研究所 一种靶向去除dna甲基化的植物表观遗传修饰编辑器
WO2023247789A1 (fr) * 2022-06-24 2023-12-28 European Molecular Biology Laboratory Outil modulaire basé sur une crispr pour l'introduction spécifique de modifications épigénétiques sur des loci cibles

Non-Patent Citations (45)

* Cited by examiner, † Cited by third party
Title
"Mammalian Cell Cultures for Biologies Manufacturing (Advances in Biochemical Engineering/Biotechnology", 2014, SPRINGER
ADUSUMALLI ET AL., BRIEF BIOINFORM, vol. 16, 2015, pages 369 - 79
ALVAREZ ET AL., HUM. GENE THER., vol. 5, 1997, pages 597 - 613
BEERLI ET AL., NATURE BIOTECHNOL., vol. 20, 2002, pages 135 - 141
BIRD ET AL., CELL, vol. 40, 1985, pages 91 - 99
BRIAN BRAY, NATURE REVIEWS DRUG DISCOVERY, vol. 2, 2003, pages 587 - 593
CHEN ET AL.: "Fusion Protein Linkers: Property, Design and Functionality", ADV DRUG DELIV REV., vol. 65, no. 10, 2013, pages 1357 - 1369, XP028737352, DOI: 10.1016/j.addr.2012.09.039
CHOO ET AL., CURR. OPIN. STRUCT. BIOL., vol. 10, 2000, pages 411 - 416
CICIR� YLENIA ET AL: "MYB oncoproteins: emerging players and potential therapeutic targets in human cancer", ONCOGENESIS, vol. 10, no. 2, 26 February 2021 (2021-02-26), US, XP093213173, ISSN: 2157-9024, DOI: 10.1038/s41389-021-00309-y *
COIN, I. ET AL., NATURE PROTOCOLS, vol. 2, 2007, pages 3247 - 3256
CUTLER: "Protein Purification Protocols (Methods in Molecular Biology", 2010, HUMANA PRESS
DATABASE UniProtKB reviewed (Swiss [online] 1 July 1989 (1989-07-01), N/A N/A: "Myb-related protein A (Homo sapiens)", XP093301180, retrieved from https://www.uniprot.org/uniprotkb/P10243/entry Database accession no. P10243 *
DEATHERAGE ET AL., METHODS MOL BIOL, vol. 507, 2009, pages 325 - 139
DOWEN ET AL., CELL, vol. 159, 2014, pages 374 - 87
GARDINER-GARDEN MFROMMER M: "CpG islands in vertebrate genomes", JOURNAL OF MOLECULAR BIOLOGY, vol. 196, no. 2, 1987, pages 261 - 282, XP024021238, DOI: 10.1016/0022-2836(87)90689-9
GREENSAMBROOK: "Therapeutic Protein Drug Products: Practical Approaches to formulation in the Laboratory, Manufacturing, and the Clinic", 2012, COLD SPRING HARBOR LABORATORY PRESS
ISALAN ET AL., NATURE BIOTECHNOL., vol. 19, 2001, pages 656 - 660
JOHN MORROW STEWARTJANIS DILLAHA YOUNG: "Solid Phase Peptide Syntheses", 2ND ECI., PIERCE CHEMICAL COMPANY, 1984
KEARNS ET AL., GENE THER., vol. 9, 1996, pages 748 - 55
KIMURA ET AL., J. HU. GENETICS, vol. 58, 2013, pages 445
LI ET AL., CURRENT BIOLOGY, vol. 15, 2005, pages 15061 - 1507
MEZGER A ET AL., NATURE COMM., 2018
PABO ET AL., ANN. REV. BIOCHEM., vol. 70, 2001, pages 313 - 340
PFLUEGER CHRISTIAN ET AL: "A modular dCas9-SunTag DNMT3A epigenome editing system overcomes pervasive off-target activity of direct fusion dCas9-DNMT3A constructs", GENOME RESEARCH, vol. 28, no. 8, 15 June 2018 (2018-06-15), US, pages 1193 - 1206, XP055975093, ISSN: 1088-9051, DOI: 10.1101/gr.233049.117 *
REMADE ET AL., EMBO JOURNAL, vol. 18, no. 18, 1999, pages 5073 - 5084
ROSENECKER ET AL., INFECTION, vol. 24, 1996, pages 15 - 10
ROSIN ET AL., MOLECULAR THERAPY, vol. 19, no. 12, December 2011 (2011-12-01), pages 1286 - 2200
S. M. BERGE ET AL., J. PHARMACEUTICAL SCIENCES, vol. 66, 1977, pages 1 - 19
SAXONOV ET AL., PNAS, vol. 103, 2006, pages 1412S - 17
SAXONOV SBERG PBRUTLAG DL: "A genome-wide analysis of CpG dinucleotides in the human genome distinguishes two distinct classes of promoters", PROC NATL ACAD SCI USA., vol. 103, no. 5, 2006, pages 1412 - 1417, XP055002395, DOI: 10.1073/pnas.0510310103
SCHUMACHER ET AL., NUCLEIC ACIDS RES, vol. 34, 2006, pages 528 - 42
SEGAL, CURR. OPIN. BIOTECHNOL., vol. 12, 2001, pages 632 - 637
SPUCHNAVARRO, JOURNAL OF DRUG DELIVERY, vol. 2011, 2011, pages 12
STERMAN ET AL., HUM. GENE THER., vol. 7, 1998, pages 71083 - 1089
SUMIYO MORITA ET AL: "Targeted DNA demethylation in vivo using dCas9-peptide repeat and scFv-TET1 catalytic domain fusions", NATURE BIOTECHNOLOGY, vol. 34, no. 10, 1 October 2016 (2016-10-01), New York, pages 1060 - 1065, XP055459761, ISSN: 1087-0156, DOI: 10.1038/nbt.3658 *
TEMPLETON ET AL., NATURE BIOTECH, vol. 15, 1997, pages 647 - 652
TOPF ET AL., GENE THER., vol. 5, 1998, pages 507 - 513
UI-TEI ET AL., FEBS LETTERS, vol. 479, 2000, pages 79 - 82
VAISVILA ET AL., GENOME RES, vol. 31, 2021, pages 1280
WAGNER ET AL., LANCET, vol. 351, 1998, pages 91171702 - 3
WEFERS ANNIKA K ET AL: "Isomorphic diffuse glioma is a morphologically and molecularly distinct tumour entity with recurrent gene fusions oforand a benign disease course", ACTA NEUROPATHOLOGICA, SPRINGER VERLAG, BERLIN, DE, vol. 139, no. 1, 28 September 2019 (2019-09-28), pages 193 - 209, XP036977492, ISSN: 0001-6322, [retrieved on 20190928], DOI: 10.1007/S00401-019-02078-W *
WEISSBACHWEISSBACH: "Methods for Plant Molecular Biology", 1988, ACADEMIC PRESS, pages: 421 - 463
WELSH ET AL., HUM. GENE THER., vol. 2, 1995, pages 205 - 18
WILLHELM-BENARTZI, BR J CANCER, vol. 109, 2013, pages 1394 - 1402
ZETSCHE ET AL., CELL, vol. 163, 2015, pages 759 - 771

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