WO2022132195A2 - Compositions and methods for modulation myc expression - Google Patents

Compositions and methods for modulation myc expression Download PDF

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Publication number
WO2022132195A2
WO2022132195A2 PCT/US2021/010059 US2021010059W WO2022132195A2 WO 2022132195 A2 WO2022132195 A2 WO 2022132195A2 US 2021010059 W US2021010059 W US 2021010059W WO 2022132195 A2 WO2022132195 A2 WO 2022132195A2
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Prior art keywords
expression
sequence
repressor
seq
nucleic acid
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English (en)
French (fr)
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WO2022132195A3 (en
WO2022132195A8 (en
Inventor
Abigail Elizabeth WITT
Jeremiah Dale FARELLI
Adam Walter SCHEIDEGGER
Jr. William Thomas SENAPEDIS
Jodi Michelle KENNEDY
Houda BELAGHZAL
Defne YARAR
Eugine Lee
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Flagship Pioneering Innovations V Inc
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Flagship Pioneering Innovations V Inc
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Priority to CN202180093730.9A priority Critical patent/CN116829175A/zh
Priority to JP2023536049A priority patent/JP2023553490A/ja
Priority to KR1020237024060A priority patent/KR20230120138A/ko
Priority to EP21907332.7A priority patent/EP4262849A4/en
Priority to US18/257,483 priority patent/US20240132559A1/en
Priority to CA3205133A priority patent/CA3205133A1/en
Priority to AU2021401231A priority patent/AU2021401231A1/en
Priority to MX2023007116A priority patent/MX2023007116A/es
Application filed by Flagship Pioneering Innovations V Inc filed Critical Flagship Pioneering Innovations V Inc
Priority to TW111112954A priority patent/TW202317601A/zh
Publication of WO2022132195A2 publication Critical patent/WO2022132195A2/en
Publication of WO2022132195A3 publication Critical patent/WO2022132195A3/en
Anticipated expiration legal-status Critical
Publication of WO2022132195A8 publication Critical patent/WO2022132195A8/en
Ceased legal-status Critical Current

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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
    • C07K14/4703—Inhibitors; Suppressors
    • 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
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/635—Externally inducible repressor mediated regulation of gene expression, e.g. tetR inducible by tetracyline
    • 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]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K45/00—Medicinal preparations containing active ingredients not provided for in groups A61K31/00 - A61K41/00
    • A61K45/06—Mixtures of active ingredients without chemical characterisation, e.g. antiphlogistics and cardiaca
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/12—Antivirals
    • A61P31/14—Antivirals for RNA viruses
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • 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
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/10—Transferases (2.)
    • C12N9/1003—Transferases (2.) transferring one-carbon groups (2.1)
    • C12N9/1007—Methyltransferases (general) (2.1.1.)
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/80—Fusion polypeptide containing a DNA binding domain, e.g. Lacl or Tet-repressor
    • C07K2319/81—Fusion polypeptide containing a DNA binding domain, e.g. Lacl or Tet-repressor containing a Zn-finger domain for DNA binding

Definitions

  • Mis-regulation of gene expression is the underlying cause of many diseases (e.g., in mammals, e.g., humans) e.g., neoplasia, neurological disorders, metabolic disorders and obesity.
  • the mis-regulation of the transcription factor MYC plays a central role in a variety of human tumors and chronic liver diseases.
  • MYC protein is considered “undruggable” due to various factors, e.g., lack of a defined ligand binding site, physiological function essential to the maintenance of normal tissues.
  • Techniques geared towards modulating the MYC gene expression provides a viable alternative approach in treating these diseases. There is a need for novel tools, systems, and methods to stably alter, e.g., decrease, expression of disease associated genes such as MYC.
  • the disclosure provides, among other things, expression repressors and expression repressor systems that may be used to modulate, e.g., decrease, expression of a target gene, e.g., MYC.
  • a target gene e.g., MYC.
  • an expression repressor comprises a targeting moiety that binds to a target gene promoter, e.g., MYC promoter, and optionally, an effector moiety, wherein the expression repressor is capable of decreasing expression of the target gene, e.g., MYC.
  • the disclosure provides an expression repressor comprising: a targeting moiety that binds a target gene locus, e.g., MYC, and an effector moiety comprising MQ1 or a fragment or variant thereof, wherein the expression repressor is capable of decreasing expression of target gene, e.g., MYC.
  • a targeting moiety that binds a target gene locus, e.g., MYC
  • an effector moiety comprising MQ1 or a fragment or variant thereof, wherein the expression repressor is capable of decreasing expression of target gene, e.g., MYC.
  • the disclosure provides an expression repressor comprising: a targeting moiety that binds to a regulatory element located in a super enhancer region of MYC, and optionally an effector moiety wherein the expression repressor is capable of decreasing expression of MYC.
  • the disclosure provides an expression repressor comprising: a targeting moiety that binds to a regulatory element located in a super enhancer region of a target gene, e.g., MYC, and an effector moiety (e.g., KRAB, or MQ1, or a fragment or variant thereof) wherein the expression repressor is capable of decreasing expression of the target gene, e.g., MYC.
  • a targeting moiety that binds to a regulatory element located in a super enhancer region of a target gene, e.g., MYC
  • an effector moiety e.g., KRAB, or MQ1, or a fragment or variant thereof
  • the disclosure provides an expression repressor comprising: a targeting moiety that binds a regulatory element located in a super enhancer region of a target gene, e.g., MYC, wherein the targeting moiety comprises a zinc finger domain, wherein the expression repressor is capable of decreasing expression of target gene, e.g., MYC.
  • the disclosure provides an expression repressor comprising: a targeting moiety that binds a regulatory element located in a super enhancer region of MY C, wherein the targeting moiety comprises a zinc finger domain or a TAL effector domain, and an effector moiety, wherein the effector moiety comprises a transcription repressor (e.g., KRAB or a fragment or variant thereof) or a DNA methyltransferase (e.g., MQ1 or a fragment or variant thereof); wherein the expression repressor is capable of decreasing expression of MYC.
  • a transcription repressor e.g., KRAB or a fragment or variant thereof
  • a DNA methyltransferase e.g., MQ1 or a fragment or variant thereof
  • the disclosure provides an expression repressor comprising: a targeting moiety that binds a target gene locus, e.g., MYC, wherein the targeting moiety comprises a zinc finger domain, wherein the expression repressor is capable of decreasing expression of target gene, e.g., MYC.
  • a targeting moiety that binds a target gene locus, e.g., MYC
  • the targeting moiety comprises a zinc finger domain
  • the expression repressor is capable of decreasing expression of target gene, e.g., MYC.
  • the disclosure provides expression repressor comprising: a targeting moiety that binds a genomic locus comprising at least 14, 15, 16, 17, 18, 19, or 20 nucleotides of the sequence of SEQ ID NO: 1, 3, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 109, 110, or 75, 76, 78, 79, 80, 81, 84, 85, 86, wherein the expression repressor is capable of decreasing expression of MYC.
  • the disclosure provides an expression repressor comprising: a targeting moiety that bind a genomic locus comprising at least 16, 17, 18, 19, or 20 nucleotides of the sequence of SEQ ID NO: 2 or 77, 82, 83 and wherein the expression repressor is capable of decreasing expression of target gene, e.g., MYC.
  • the expression expressor comprises an effector moiety.
  • the disclosure provides an expression repressor comprising a targeting moiety wherein the targeting moiety binds a genomic locus that is within 1400 nt upstream or downstream of SEQ ID NO: 4. In some aspects, the disclosure provides an expression repressor comprising a targeting moiety wherein, the targeting moiety binds a genomic locus comprising at least 14, 15, 16, 17, 18, 19, or 20 nucleotides of the sequence of SEQ ID NO: 4, 77, 82, or 83.
  • the disclosure provides an expression repressor comprising a targeting moiety wherein, the targeting moiety binds a genomic locus comprising at least 14, 15, 16, 17, 18, 19, or 20 nucleotides of the sequence of SEQ ID NO: 83, 96, or 108.
  • the disclosure provides a system comprising a first expression repressor comprising a first targeting moiety and optionally a first effector moiety, wherein the first expression repressor binds to a transcription regulatory element (e.g., a promoter or transcription start site (TSS)) operably linked to a target gene, e.g., MYC or to a sequence proximal to the transcription regulatory element, and a second expression repressor comprising a second targeting moiety and optionally a second effector moiety, wherein the second expression repressor binds to an anchor sequence of an anchor sequence mediated conjunction (ASMC) comprising a target gene, e.g., MYC or to a sequence proximal to the anchor sequence.
  • a transcription regulatory element e.g., a promoter or transcription start site (TSS)
  • TSS transcription start site
  • ASMC anchor sequence mediated conjunction
  • the disclosure provides a system comprising a first expression repressor comprising a first targeting moiety and optionally a first effector moiety, wherein the first expression repressor binds to a transcription regulatory element (e.g., a promoter or transcription start site (TSS)) operably linked to a target gene, e.g., MYC, or to a sequence proximal to the transcription regulatory element, and a second expression repressor comprising a second targeting moiety and optionally a second effector moiety, wherein the second expression repressor binds to a genomic locus located in a super enhancer region of a target gene, e.g., MYC.
  • the first targeting moiety specifically binds a first DNA sequence
  • the second targeting moiety specifically binds a second DNA sequence different from the first DNA sequence.
  • the first effector moiety is different from the second effector moiety.
  • the disclosure provides an expression repressor comprising: a targeting moiety comprising a CRISPR/Cas molecule, e.g., comprising a catalytically inactive CRISPR/Cas protein, that binds to a transcription regulatory element (e.g., a promoter or transcription start site (TSS)) operably linked to a target gene, e.g., MYC or a sequence proximal to said transcription regulatory element; and an effector moiety comprising MQ1 or a functional variant or fragment thereof.
  • a transcription regulatory element e.g., a promoter or transcription start site (TSS)
  • TSS transcription start site
  • the disclosure provides an expression repressor comprising: a targeting moiety comprising a CRISPR/Cas molecule, e.g., comprising a catalytically inactive CRISPR/Cas protein that binds to a genomic locus located in a super enhancer region of a target gene, e.g., MYC, and an effector moiety comprising KRAB, MQ1, or a functional variant or fragment thereof, wherein the expression repressor is capable of decreasing expression of target gene, e.g., MYC.
  • a targeting moiety comprising a CRISPR/Cas molecule, e.g., comprising a catalytically inactive CRISPR/Cas protein that binds to a genomic locus located in a super enhancer region of a target gene, e.g., MYC
  • an effector moiety comprising KRAB, MQ1, or a functional variant or fragment thereof
  • the disclosure provides an expression repressor comprising: a targeting moiety comprising a CRISPR/Cas molecule, e.g., comprising a catalytically inactive CRISPR/Cas protein, that binds to an anchor sequence of an anchor sequence mediated conjunction (ASMC) comprising a target gene, e.g., MYC or to a sequence proximal to the anchor sequence; and an effector moiety comprising KRAB or a functional variant or fragment thereof.
  • a targeting moiety comprising a CRISPR/Cas molecule, e.g., comprising a catalytically inactive CRISPR/Cas protein, that binds to an anchor sequence of an anchor sequence mediated conjunction (ASMC) comprising a target gene, e.g., MYC or to a sequence proximal to the anchor sequence
  • ASMC anchor sequence mediated conjunction
  • the disclosure provides an expression repressor comprising: a targeting moiety comprising a zinc finger molecule that binds to a transcription regulatory element (e.g., a promoter or transcription start site (TSS)) operably linked to a target gene, e.g., MYC or a sequence proximal to said transcription regulatory element; and an effector moiety comprising MQ1 or a functional variant or fragment thereof.
  • a transcription regulatory element e.g., a promoter or transcription start site (TSS)
  • TSS transcription start site
  • the disclosure provides an expression repressor comprising: a targeting moiety comprising a zinc finger molecule that binds to an anchor sequence of an anchor sequence mediated conjunction (ASMC) comprising a target gene, e.g., MYC or to a sequence proximal to the anchor sequence; and an effector moiety comprising KRAB or a functional variant or fragment thereof.
  • ASMC anchor sequence mediated conjunction
  • the disclosure provides an expression repressor comprising: a targeting moiety comprising a zinc finger molecule, that binds to a genomic locus located in a super enhancer region of a target gene, e.g., MYC, and an effector moiety comprising KRAB or a functional variant or fragment thereof.
  • the disclosure is directed to a nucleic acid encoding the first expression repressor, second expression repressor, both, or a component thereof (e.g., a gRNA, a mRNA).
  • the nucleic acid encoding the expression repressor system is a multi-cistronic sequence.
  • the multi-cistronic sequence is a bi-cistronic sequence.
  • the disclosure is directed to a vector comprising a nucleic acid, a system, or an expression repressor described herein.
  • the disclosure is directed to a lipid nanoparticle comprising a vector, a nucleic acid, a system, or an expression repressor described herein.
  • the disclosure is directed to a reaction mixture comprising an expression repressor, a system, a nucleic acid, a vector, or a lipid nanoparticle described herein.
  • the disclosure is directed to a pharmaceutical composition comprising an expression repressor, a system, a nucleic acid, a vector, a lipid nanoparticle, or a reaction mixture described herein.
  • the disclosure is directed to a method of decreasing expression of a target gene comprising providing an expression repressor or an expression repression system described herein and contacting the target gene and/or one or more operably linked transcription control elements with the expression repressor or expression repression system, thereby decreasing expression of the target gene.
  • the disclosure is directed to a method of treating a condition associated with over-expression of a target gene e.g., MYC in a subject, comprising administering an expression repressor, or a system, nucleic acid, or vector described herein to the subject, thereby treating the condition.
  • a target gene e.g., MYC
  • the disclosure is directed to a method of treating a condition associated with misregulation of a target gene, e.g., MYC, in a subject, comprising administering an expression repressor, system, nucleic acid, or vector described herein to the subject, thereby treating the condition.
  • a target gene e.g., MYC
  • the disclosure provides, a method of decreasing expression of a target gene, e.g., MYC in a cell, the method comprising: contacting the cell with a system comprising: a first expression repressor comprising a first targeting moiety and optionally a first effector moiety, wherein the first expression repressor binds to a transcription regulatory element (e.g., a promoter or transcription start site (TSS)) operably linked to a target gene, e.g., MYC, and a second expression repressor comprising a second targeting moiety and optionally a second effector moiety, wherein the second expression repressor binds to an anchor sequence of an anchor sequence mediated conjunction (ASMC) comprising a target gene, e.g., MYC or to a sequence proximal to the anchor sequence thereby decreasing expression of the target gene, e.g., MYC in the cell.
  • a transcription regulatory element e.g., a promoter or transcription
  • the disclosure provides a method of decreasing expression of a target gene, e.g., MYC, in a cell, the method comprising: contacting the cell with a system comprising: a first expression repressor comprising a first targeting moiety and optionally a first effector moiety, wherein the first expression repressor binds to a transcription regulatory element (e.g., a promoter or transcription start site (TSS)) operably linked to a target gene, e.g., MYC, and a second expression repressor comprising a second targeting moiety and optionally a second effector moiety, wheiein the second expression repressor binds to a genomic locus located in a super enhancer region of a target gene, e.g., MYC, thereby decreasing expression of the target gene, e.g., MYC, in the cell.
  • a transcription regulatory element e.g., a promoter or transcription start site (TSS)
  • TSS transcription
  • kits comprising: a) a container comprising a composition comprising an expression repressor comprising a targeting moiety that binds to a target gene, promoter, e.g., MYC, and an effector moiety capable of modulating, e.g., decreasing the expression of the target gene, e.g., MYC, and b) a set of instructions comprising at least one method for modulating the expression of a target gene, e.g., MYC within a cell with said composition.
  • a container comprising a composition comprising an expression repressor comprising a targeting moiety that binds to a target gene, promoter, e.g., MYC, and an effector moiety capable of modulating, e.g., decreasing the expression of the target gene, e.g., MYC
  • a set of instructions comprising at least one method for modulating the expression of a target gene, e.g., MYC within a cell with said composition.
  • kits comprising: a) a container comprising a composition comprising an expression repressor comprising a targeting moiety that binds to a locus located in a super enhancer region of a target gene, e.g., MYC, and an effector moiety capable of modulating, e.g., decreasing the expression of the target gene, e.g., MYC, and b) a set of instructions comprising at least one method for modulating the expression of a target gene, e.g., MYC within a cell with said composition.
  • a target gene e.g., MYC
  • a set of instructions comprising at least one method for modulating the expression of a target gene, e.g., MYC within a cell with said composition.
  • the kit comprises a) a container comprising a composition comprising a system comprising two expression repressors, comprising a first expression repressor comprising a first targeting moiety and optionally a first effector moiety, wherein the first expression repressor binds to a transcription regulatory element (e.g., a promoter or transcription start site (TSS)) operably linked to target gene, e.g., MYC or to a sequence proximal to the transcription regulatory element and an expression repressor comprising a second targeting moiety and optionally a second effector moiety, wherein the second expression repressor binds to an anchor sequence of an anchor sequence mediated conjunction (ASMC) comprising target gene, e.g., MYC or to a sequence proximal to the anchor sequence.
  • a transcription regulatory element e.g., a promoter or transcription start site (TSS)
  • TSS transcription start site
  • target gene e.g., MYC or to
  • the kit comprises a) a container comprising a composition comprising a system comprising two expression repressors, comprising a first expression repressor comprising a first targeting moiety and optionally a first effector moiety, wherein the first expression repressor binds to a transcription regulatory element (e.g., a promoter or transcription start site (TSS)) operably linked to target gene, e.g., MYC, or to a sequence proximal to the transcription regulatory element and an expression repressor comprising a second targeting moiety and optionally a second effector moiety, wherein the second expression repressor binds to a genomic locus located in a super enhancer region of a target gene, e.g., MYC.
  • a transcription regulatory element e.g., a promoter or transcription start site (TSS)
  • TSS transcription start site
  • the kit further comprises b) a set of instructions comprising at least one method for treating a disease or modulating, e.g., decreasing the expression of target gene, e.g., MYC within a cell with said composition.
  • the kits can optionally include a delivery vehicle for said composition (e.g., a lipid nanoparticle).
  • the reagents may be provided suspended in the excipient and/or delivery vehicle or may be provided as a separate component which can be later combined with the excipient and/or delivery vehicle.
  • the kits may optionally contain additional therapeutics to be co-administered with the compositions to affect the desired target gene expression, e.g., MYC gene expression modulation.
  • 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 by this invention. 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.
  • electronic storage media e.g., magnetic discs, tapes, cartridges, chips
  • optical media e.g., CD ROM
  • Such media may include addresses to internet sites that provide such instructional materials.
  • sequence database reference numbers All publications, patent applications, patents, and other references (e.g., sequence database reference numbers) mentioned herein are incorporated by reference in their entirety. For example, all GenBank, Unigene, and Entrez sequences referred to herein, e.g., in any Table herein, are incorporated by reference. Unless otherwise specified, the sequence accession numbers specified herein, including in any Table herein, refer to the database entries current as of December 15, 2020. When one gene or protein references a plurality of sequence accession numbers, all of the sequence variants are encompassed.
  • An expression repressor comprising: a targeting moiety that binds to a MYC promoter, and optionally, an effector moiety, wherein the expression repressor is capable of decreasing expression of MYC.
  • An expression repressor comprising: a targeting moiety that binds a genomic locus comprising at least 14, 15, 16, 17, 18, 19, or 20 nucleotides of the sequence of SEQ ID NO: 3, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 109, 110, 75, 76, 78, 79, 80, 81, 84, 85, 86, 190, 191 , 192, 200, or 202 and optionally, an effector moiety, wherein the expression repressor is capable of decreasing expression of MYC.
  • An expression repressor comprising: a targeting moiety that binds a genomic locus comprising at least 16, 17, 18, 19, or 20 nucleotides of the sequence of SEQ ID NO: 2, 77, 82, 83, 199, or 201 and optionally, an effector moiety, wherein the expression repressor is capable of decreasing expression of MYC.
  • An expression repressor comprising: a targeting moiety that binds a MYC locus, and an effector moiety comprising MQ1 or a fragment or variant thereof, wherein the expression repressor is capable of decreasing expression of MYC.
  • An expression repressor comprising: a targeting moiety that binds a locus in MYC super enhancer region, optionally an effector moiety, e.g., an effector moiety comprising a DNA methyltransferase, wherein optionally the effector moiety comprises MQ1 or a fragment or variant thereof, wherein the expression repressor is capable of decreasing expression of MYC.
  • An expression repressor comprising: a targeting moiety that binds a locus in MYC super enhancer region, an effector moiety comprising a transcription repressor, wherein optionally the effector moiety comprises KRAB or a fragment or variant thereof, wherein the expression repressor is capable of decreasing expression of MYC.
  • An expression repressor comprising: a targeting moiety that binds a locus, e.g., a MYC locus, a first effector moiety comprising EZH2 or a fragment or variant thereof, and a second effector moiety comprising KRAB or a fragment or variant thereof, wherein the expression repressor is capable of decreasing expression at the locus, e.g., decreasing expression of MYC.
  • the expression repressor of embodiment 13 or 14, wherein the targeting moiety comprises a TAL effector domain, a CRISPR/Cas domain, or a zinc finger domain.
  • An expression repressor comprising: a targeting moiety that binds a MYC locus, wherein the targeting moiety comprises a zinc finger domain, and optionally, an effector moiety, wherein the expression repressor is capable of decreasing expression of MYC.
  • An expression repressor comprising: a targeting moiety comprising a CRISPR/Cas domain, e.g., comprising a catalytically inactive CRISPR/Cas protein, that binds to a transcription regulatory element (e.g., a promoter, an enhancer, a super enhancer, or transcription start site (TSS)) operably linked to a MYC gene or a sequence proximal to said transcription regulatory element; and an effector moiety comprising MQ1 or a functional variant or fragment thereof.
  • a transcription regulatory element e.g., a promoter, an enhancer, a super enhancer, or transcription start site (TSS)
  • An expression repressor comprising: a targeting moiety comprising a CRISPR/Cas domain, e.g., comprising a catalytically inactive CRISPR/Cas protein, that binds to a transcription regulatory element (e.g., a promoter, an enhancer, or transcription start site (TSS)) operably linked to a MYC gene or a sequence proximal to said transcription regulatory element; and an effector moiety comprising MQ 1 or a functional variant or fragment thereof.
  • a transcription regulatory element e.g., a promoter, an enhancer, or transcription start site (TSS)
  • An expression repressor comprising: a targeting moiety comprising a CRISPR/Cas domain, e.g., comprising a catalytically inactive CRISPR/Cas protein, that binds to a transcription regulatory element (e.g., a promoter, an enhancer, or transcription start site (TSS)) operably linked to a MYC gene or a sequence proximal to said transcription regulatory element; and an effector moiety comprising KRAB or a functional variant or fragment thereof.
  • a transcription regulatory element e.g., a promoter, an enhancer, or transcription start site (TSS)
  • An expression repressor comprising: a targeting moiety comprising a CRISPR/Cas domain, e.g., comprising a catalytically inactive CRISPR/Cas protein, that binds to an anchor sequence of an anchor sequence mediated conjunction (ASMC) comprising a MYC gene or to a sequence proximal to the anchor sequence; and an effector moiety comprising KRAB or a functional variant or fragment thereof.
  • a targeting moiety comprising a CRISPR/Cas domain, e.g., comprising a catalytically inactive CRISPR/Cas protein, that binds to an anchor sequence of an anchor sequence mediated conjunction (ASMC) comprising a MYC gene or to a sequence proximal to the anchor sequence
  • ASMC anchor sequence mediated conjunction
  • An expression repressor comprising: a targeting moiety comprising a zinc finger domain that binds to a transcription regulatory element (e.g., a promoter, an enhancer, or transcription start site (TSS)) operably linked to a MYC gene or a sequence proximal to said transcription regulatory element; and an effector moiety comprising MQ1 or a functional variant or fragment thereof.
  • a transcription regulatory element e.g., a promoter, an enhancer, or transcription start site (TSS)
  • TSS transcription start site
  • An expression repressor comprising: a targeting moiety comprising a zinc finger domain that binds to a transcription regulatory element (e.g., a promoter, an enhancer, or transcription start site (TSS)) operably linked to a MYC gene or a sequence proximal to said transcription regulatory element; and an effector moiety comprising KRAB or a functional variant or fragment thereof.
  • a transcription regulatory element e.g., a promoter, an enhancer, or transcription start site (TSS)
  • TSS transcription start site
  • An expression repressor comprising: a targeting moiety that binds a mouse genomic locus comprising at least 14, 15, 16, 17, 18, 19, or
  • the expression repressor of embodiments 24 or 25, wherein the targeting moiety comprises a TAL effector domain, a CRISPR/Cas domain, a zinc finger domain, a tetR domain, a meganuclease domain, or an oligonucleotide.
  • the targeting moiety comprises an amino acid sequence according to any of SEQ ID NOs: 154-156, or a sequence with at least 80, 85, 90, 95, 99, or 100% identity thereto, or having no more than 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 positions of difference thereto.
  • the targeting moiety comprises a nucleic acid sequence according to any of SEQ ID NOs: 157-159, or a sequence with at least 80, 85, 90, 95, 99, or 100% identity thereto, or having no more than 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 positions of difference thereto.
  • the effector moiety is a durable effector moiety.
  • the targeting moiety comprises a zinc finger domain
  • the effector moiety comprises an epigenetic modifying moiety, e.g., a DNA methyltransferase, e.g., MQ1 or a fragment or variant thereof.
  • SNP disease-associated single nucleotide polymorphism
  • An expression repressor comprising: a targeting moiety comprising a zinc finger domain that binds to an anchor sequence of an anchor sequence mediated conjunction (ASMC) comprising a MYC gene or to a sequence proximal to the anchor sequence; and an effector moiety comprising KRAB or a functional variant or fragment thereof.
  • ASMC anchor sequence mediated conjunction
  • the expression repressor of any of embodiments 1-23 or 36-43 wherein the expression repressor comprises an amino acid sequence chosen from any of SEQ ID NOs: 22-37, 129, 133, 134, 139-149, or 177-186, or a sequence with at least 80, 85, 90, 95, 99, or 100% identity thereto, or having no more than 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 positions of difference thereto.
  • any of embodiments 1-23 or 36-45 wherein the expression repressor is encoded by a nucleotide sequence chosen from any of SEQ ID NOs: 55-70, 130, 189, or 193-197, or a sequence with at least 80, 85, 90, 95, 99, or 100% identity thereto, or having no more than 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 positions of difference thereto.
  • the targeting moiety comprises an amino acid sequence according to any of SEQ ID NOs: 5-16, or 169-172, or a sequence with at least 80, 85, 90, 95, 99, or 100% identity thereto, or having no more than 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 positions of difference thereto.
  • effector moiety comprises an amino acid sequence according to SEQ ID NO: 18 19, or 87, or a sequence with at least 80, 85, 90, 95, 99, or 100% identity thereto, or having no more than 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6,
  • the targeting moiety comprises a TAL effector domain, a CRISPR/Cas domain, a zinc finger domain, a tetR domain, a meganuclease domain, or an oligonucleotide.
  • gRNA e.g., a gRNA that binds a genomic locus comprising at least 14, 15, 16, 17, 18, 19, or 20 nucleotides of the sequence of any of SEQ ID NOs: 1-4, e.g., wherein the gRNA comprises a sequence that comprises at least 14, 15, 16, 17, 18, 19, or 20 nucleotides of the sequence of any of SEQ ID NOs: 1-4.
  • gRNA e.g., a gRNA that binds a genomic locus comprising at least 14, 15, 16, 17, 18, 19, or 20 nucleotides of the sequence of any of SEQ ID NOs: 96-110, e.g., wherein the gRNA comprises a sequence that comprises at least 14, 15, 16, 17, 18, 19, or 20 nucleotides of the sequence of any of SEQ ID NOs: 96-110.
  • the zinc finger domain comprises 1-10, 1-9, 1-8, 1-7, 1-6, 1-5, 1-4, 1-3, 1-2, 2-10, 2-9, 2-8, 2-7, 2-6, 2-5, 2-4, 2-3, 3-10, 3-9, 3-8, 3-7, 3-6, 3-5, 3-4, 4-10, 4-9, 4-8, 4-7, 4-6, 4-5, 5-10, 5-9, 5-8, 5-7, 5-6, 6-10, 6-9, 6-8, 6-7, 7-10, 7-9, 7-8, 8-10, 8-9, or 9-10 zinc fingers.
  • the targeting moiety comprises a catalytically inactive CRISPR/Cas domain (e.g., dCas9) and the effector moiety comprises a transcription repressor, e.g., KRAB or a fragment or variant thereof.
  • a catalytically inactive CRISPR/Cas domain e.g., dCas9
  • the effector moiety comprises a transcription repressor, e.g., KRAB or a fragment or variant thereof.
  • 66 The expression repressor of any of embodiments 17, 36-43, 45-47, 53, or 58-63, wherein the targeting moiety comprises a zinc finger domain, and the expression repressor does not comprise an effector moiety.
  • 67 The expression repressor of any of embodiments 1-12, 18-19, 22, 36-43, 45-49, 51, or 53-57 wherein the targeting moiety comprises a catalytically inactive CRISPR/Cas domain (e.g., dCas9) and the effector moiety comprises an epigenetic modifying moiety, e.g., a DNA methyltransferase, e.g., MQ1 or a fragment or variant thereof.
  • an epigenetic modifying moiety e.g., a DNA methyltransferase, e.g., MQ1 or a fragment or variant thereof.
  • the targeting moiety comprises a zinc finger domain
  • the effector moiety comprises an epigenetic modifying moiety, e.g., a DNA methyltransferase, e.g., MQ1 or a fragment or variant thereof.
  • the expression repressor of any of the preceding embodiments which: (i) comprises one or more nuclear localization signal sequences (NLS), or (ii) does not comprise an NLS.
  • anchor sequence comprises a sequence according to SEQ ID NO: 73 or 74, or a sequence with no more than 8, 7, 6, 5, 4, 3, 2, or 1 alterations relative thereto.
  • anchor sequence is upstream of the MYC gene (e.g., upstream of the TSS or upstream of the promoter).
  • the anchor sequence is at least 1, 5, 10, 50, 100, or 1000 kilobases away from the MYC gene (e.g., from the TSS or promoter of the MYC gene).
  • the anchor sequence is 0.1 -0.5, 0.1-1, 0.1-5, 0.1-10, 0.1-50, 0.1-100, 0.1-500, 0.1-1000, 0.5-1, 0.5-5, 0.5-10, 0.5-50, 0.5-100, 0.5-500, 0.5-1000, 1-5, 1-10, 1-50, 1-100, 1-500, 1-1000, 5-10, 5-50, 5-100, 5-500, 5-1000, 10-50, 10-100, 10-500, 10-1000, 50-100, 50-500, 50-1000, 100-500, 100-1000, or 500-1000 kilobases away from the MYC gene (e.g., from the TSS or promoter of the MYC gene).
  • the MYC gene e.g., from the TSS or promoter of the MYC gene.
  • 91 The expression repressor of any of embodiments 24-43 49, 51,53, 56-57, 59-62, 66-68, 70-89, wherein the targeting moiety binds to a mouse genomic locus.
  • 92 The expression repressor of any of the preceding embodiments, wherein binding of the expression repressor to the MYC locus decreases the viability of a cell comprising the MYC locus (e.g., cancer cells).
  • the plurality of cells comprises a plurality of cancer cells and a plurality of non-cancer cells and/or a plurality of infected cells and a plurality of uninfected cells.
  • HCC hepatocellular carcinoma
  • FHCC fibrolamellar hepatocellular carcinoma
  • FHCC cholangiocarcinoma
  • angiosarcoma secondary liver cancer
  • NSCLC non-small cell lung cancer
  • SCLC small cell lung cancer
  • large cell (undifferentiated) carcinoma triple negative breast cancer
  • gastric adenocarcinoma endometrial carcinoma
  • pancreatic carcinoma hepatocellular carcinoma
  • FHCC fibrolamellar hepatocellular carcinoma
  • SCLC small cell lung cancer
  • large cell (undifferentiated) carcinoma triple negative breast cancer
  • gastric adenocarcinoma endometrial carcinoma
  • pancreatic carcinoma pancreatic carcinoma
  • the expression repressor of any of the preceding embodiments which has an EC50 of 0.04 - 0.4, 0.04 - 0.1, 0.1 - 0.2, 0.2 - 0.3, or 0.3 - 0.4 ⁇ g/mL when tested in an assay for viability of cancer cells (e.g., HCC cells) using LNP delivery of mRNA encoding the expression repressor, e.g., in an assay according to Example 12.
  • the expression repressor of any of embodiments 1-104 which has an EC50 of 0.1- 2.5, 0.5-2.2, 1.0- 1.5, 1.2-2 ⁇ g/mL when tested in an assay for viability of cancer cells (e.g., lung cancer cells) using LNP delivery of mRNA encoding the expression repressor, e.g., in an assay according to Example 18.
  • the expression repressor of any of the preceding embodiments which has an EC50 of 0.004 - 0.08, 0.004 - 0.01, 0.01 - 0.02, 0.02 - 0.04, or 0.04 - 0.08 ⁇ g/mL when tested in an assay for reducing MYC mRNA levels in cancer cells (e.g., HCC cells) using LNP delivery of mRNA encoding the expression repressor, e.g., in an assay according to Example 12.
  • the expression repressor of any of the preceding embodiments which has an EC50 of 0.04 - 0.1, 0.04 - 0.09, 0.05 - 0.09, or 0.06 - 0.8 ⁇ g/mL when tested in an assay for reducing MYC mRNA levels in cancer cells (e.g., lung cancer cells) using LNP delivery of mRNA encoding the expression repressor, e.g., in an assay according to Example 18.
  • a target gene e.g., MYC in a cell by at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, or at least 90% compared to the protein level in an untreated cell.
  • the expression repressor of any of the preceding embodiments which is capable of reducing tumor volume, e.g., in a human subject or in a mammalian model.
  • the expression repressor of any of preceding embodiments, wherein the expression repressor is capable of reducing tumor volume to a similar or greater degree compared to a chemotherapeutic agent, e.g., in a mammalian model, e.g., when measured at day 20 after initiation of treatment, e.g., wherein the expression repressor is administered every 5 days at a dose of 3mg/kg.
  • the expression repressor of any of preceding embodiments wherein the expression repressor is capable of reducing tumor volume compared to a PBS control, e.g., in a mammalian model, e.g., when measured at day 20 after initiation of treatment e.g., wherein the expression repressor is administered every 5 days for 4 doses followed by every 3 days for 3 doses at Img/kg, 1.5 mg/kg, or 3mg/kg.
  • a system comprising: a first expression repressor of any of the preceding embodiments, and a second expression repressor, e.g., a second expression repressor described herein, e.g., a second expression repressor of any of the preceding embodiments.
  • a system comprising: a first expression repressor comprising a first targeting moiety and optionally a first effector moiety, wherein the first expression repressor binds to a transcription regulatory element (e.g., a promoter, enhancer, or transcription start site (TSS)) operably linked to a MYC gene or to a sequence proximal to the transcription regulatory element, and a second expression repressor comprising a second targeting moiety and optionally a second effector moiety, wherein the second expression repressor binds to an anchor sequence of an anchor sequence mediated conjunction (ASMC) comprising a MYC gene or to a sequence proximal to the anchor sequence.
  • a transcription regulatory element e.g., a promoter, enhancer, or transcription start site (TSS)
  • TSS transcription start site
  • first expression repressor comprises a targeting moiety that binds a genomic locus comprising at least 16, 17, 18, 19, or 20 nucleotides of the sequence of SEQ ID NO: 2, 3, 4, 71-86, or 200-206; and the second expression repressor comprises a targeting moiety that binds a genomic locus comprising at least 16, 17, 18, 19, or 20 nucleotides of the sequence of SEQ ID NO: 2, 3, 4, 71-86, or 200-206.
  • the first expression repressor comprises a targeting moiety that binds a genomic locus comprising at least 16, 17, 18, 19, or 20 nucleotides of the sequence of any of SEQ ID NO: 96-110.
  • the first expression repressor comprises a targeting moiety that binds a genomic locus comprising at least 16, 17, 18, 19, or 20 nucleotides of the sequence of SEQ ID NO: 71, SEQ ID NO: 72, or SEQ ID NO: 83; and the second expression repressor comprises a targeting moiety that binds a genomic locus comprising at least 16, 17, 18, 19, or 20 nucleotides of the sequence of SEQ ID NO: 77.
  • a system comprising: a first expression repressor comprising a first targeting moiety and optionally a first effector moiety, wherein the first expression repressor binds to a promoter operably linked to a MYC gene or to a sequence proximal to the promoter, and a second expression repressor comprising a second targeting moiety and optionally a second effector moiety, wherein the second expression repressor binds to an enhancer (e.g., a super-enhancer) of the MYC gene.
  • an enhancer e.g., a super-enhancer
  • the first expression repressor comprises a targeting moiety that binds a genomic locus comprising at least 16, 17, 18, 19, or 20 nucleotides of the sequence of SEQ ID NO: 204
  • the second expression repressor comprises a targeting moiety that binds a genomic locus comprising at least 16, 17, 18, 19, or 20 nucleotides of the sequence of any of SEQ ID NOs: 199 or 201. 128.
  • a system for reducing MYC expression comprising: a) a first expression repressor comprising: i) a first targeting moiety having an amino acid sequence according to SEQ ID NO: 13 or a sequence with at least 80, 85, 90, 95, 99, or 100% identity thereto, or having no more than 20,
  • a first effector moiety having an amino acid sequence according to SEQ ID NO: 19 or 87 or a sequence with at least 80, 85, 90, 95, 99, or 100% identity thereto, or having no more than
  • a second expression repressor comprising: i) a second targeting moiety having an amino acid sequence according to SEQ ID NO: 7 169, or 171 or a sequence with at least 80, 85, 90, 95, 99, or 100% identity thereto, or having no more than 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 positions of difference thereto, and ii) a second effector moiety having an amino acid sequence according to SEQ ID NO: 18, or a sequence with at least 80, 85, 90, 95, 99, or 100% identity thereto, or having no more than 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 positions of difference thereto.
  • first expression repressor further comprises a first nuclear localization signal, e.g., an SV40 NLS, e.g., a sequence according to SEQ ID NO: 135 or a sequence with at least 80, 85, 90, 95, 99, or 100% identity thereto, e.g., situated N-terminal of the first targeting moiety.
  • a first nuclear localization signal e.g., an SV40 NLS, e.g., a sequence according to SEQ ID NO: 135 or a sequence with at least 80, 85, 90, 95, 99, or 100% identity thereto, e.g., situated N-terminal of the first targeting moiety.
  • first expression repressor further comprises a second nuclear localization signal, e.g., a nucleoplasmin NLS, e.g., a sequence according to SEQ ID NO: 136 or a sequence with at least 80, 85, 90, 95, 99, or 100% identity thereto, e.g., situated C-terminal of the first effector moiety.
  • a second nuclear localization signal e.g., a nucleoplasmin NLS, e.g., a sequence according to SEQ ID NO: 136 or a sequence with at least 80, 85, 90, 95, 99, or 100% identity thereto, e.g., situated C-terminal of the first effector moiety.
  • the second expression repressor further comprises a first nuclear localization signal, e.g., an SV40 NLS, e.g., a sequence according to SEQ ID NO: 135 or a sequence with at least 80, 85, 90, 95, 99, or 100% identity thereto, e.g., situated N-terminal of the second targeting moiety.
  • a first nuclear localization signal e.g., an SV40 NLS, e.g., a sequence according to SEQ ID NO: 135 or a sequence with at least 80, 85, 90, 95, 99, or 100% identity thereto, e.g., situated N-terminal of the second targeting moiety.
  • the second expression repressor further comprises a second nuclear localization signal, e.g., a nucleoplasmin NLS, e.g., a sequence according to SEQ ID NO: 136 or a sequence with at least 80, 85, 90, 95, 99, or 100% identity thereto, e.g., situated C- terminal of the second effector moiety.
  • a second nuclear localization signal e.g., a nucleoplasmin NLS, e.g., a sequence according to SEQ ID NO: 136 or a sequence with at least 80, 85, 90, 95, 99, or 100% identity thereto, e.g., situated C- terminal of the second effector moiety.
  • first expression repressor further comprises a first linker situated between the first targeting moiety and the first effector moiety, wherein optionally the first linker has an amino acid sequence according to SEQ ID NO: 137 or a sequence with at least 80, 85, 90, 95, 99, or 100% identity thereto.
  • the second expression repressor further comprises a second linker situated between the second targeting moiety and the second effector moiety, wherein optionally the second linker has an amino acid sequence according to SEQ ID NO: 138 or a sequence with at least 80, 85, 90, 95, 99, or 100% identity thereto.
  • first expression repressor further comprises an amino acid sequence C-terminal of the first effector moiety, e.g., a sequence of up to 30, 25, 20, or 18 amino acids, e.g., a sequence according to SEQ ID NO: 126 or a sequence with at least 80, 85, 90, 95, 99, or 100% identity thereto.
  • amino acid sequence C-terminal of the first effector moiety e.g., a sequence of up to 30, 25, 20, or 18 amino acids, e.g., a sequence according to SEQ ID NO: 126 or a sequence with at least 80, 85, 90, 95, 99, or 100% identity thereto.
  • the second expression repressor further comprises an amino acid sequence N-terminal of the second targeting moiety, e.g., a sequence of up to 30, 25, 20, or 18 amino acids, e.g., a sequence according to SEQ ID NO: 128 or a sequence with at least 80, 85, 90, 95, 99, or 100% identity thereto.
  • the second targeting moiety comprises an amino acid sequence according to SEQ ID NO: 171, or a sequence with at least 80, 85, 90, 95, 99, or 100% identity thereto.
  • a nucleic acid encoding a system for reducing MYC expression comprising: a) a first region encoding a first expression repressor, the first expression repressor comprising: i) a first targeting moiety having an amino acid sequence according to SEQ ID NO: 13 or a sequence with at least 80, 85, 90, 95, 99, or 100% identity thereto, or having no more than 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 positions of difference thereto, and ii) a first effector moiety having an amino acid sequence according to SEQ ID NO: 19 or 87 or a sequence with at least 80, 85, 90, 95, 99, or 100% identity thereto, or having no more than 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 positions of difference thereto, and b) a second region encoding a second expression repressor, the second expression repressor comprising: i) a second region encoding a second expression
  • nucleic acid of embodiment 145 or 146, wherein the first region further comprises a nucleotide sequence encoding a first nuclear localization signal, e.g., an SV40 NLS, e.g., a sequence according to SEQ ID NO: 135 or a sequence with at least 80, 85, 90, 95, 99, or 100% identity thereto, e.g., situated N- terminal of the first targeting moiety.
  • a first nuclear localization signal e.g., an SV40 NLS, e.g., a sequence according to SEQ ID NO: 135 or a sequence with at least 80, 85, 90, 95, 99, or 100% identity thereto, e.g., situated N- terminal of the first targeting moiety.
  • a second nuclear localization signal e.g., a nucleoplasmin NLS, e.g., a sequence according to SEQ ID NO: 136 or a sequence with at least 80, 85, 90, 95, 99, or 100% identity thereto, e.g., situated C-terminal of the first effector moiety.
  • a first nuclear localization signal e.g., an SV40 NLS, e.g., a sequence according to SEQ ID NO: 135 or a sequence with at least 80, 85, 90, 95, 99, or 100% identity thereto, e.g., situated N-terminal of the second targeting moiety.
  • a second nuclear localization signal e.g., a nucleoplasmin NLS, e.g., a sequence according to SEQ ID NO: 136 or a sequence with at least 80, 85, 90, 95, 99, or 100% identity thereto, e.g., situated C-terminal of the second effector moiety.
  • nucleic acid of any of embodiments 145-154, wherein the first expression repressor has an amino acid sequence according SEQ ID NO: 30 or 129, or a sequence with at least 80, 85, 90, 95, 99, or 100% identity thereto.
  • nucleic acid of any of embodiments 145-155, wherein the second expression repressor has an amino acid sequence according to SEQ ID NO: 24, or a sequence with at least 80, 85, 90, 95, 99, or 100% identity thereto.
  • nucleic acid of any of embodiments 145-156 wherein the first region comprises a nucleotide sequence encoding the first targeting moiety, wherein the nucleotide sequence encoding the first targeting moiety comprises a sequence according to SEQ ID NO: 46 or 131 or a sequence with at least 80, 85, 90, 95, 99, or 100% identity thereto, or having no more than 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 positions of difference thereto.
  • nucleic acid of any of embodiments 145-157 wherein the first region comprises a nucleotide sequence encoding the first effector moiety, wherein the nucleotide sequence encoding the first effector moiety comprises a sequence according to SEQ ID NO: 52 or 132, or a sequence with at least 80, 85, 90, 95, 99, or 100% identity thereto, or having no more than 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 positions of difference thereto.
  • nucleic acid of any of embodiments 145-159 wherein the first region comprises a nucleotide sequence encoding the first effector moiety, wherein the nucleotide sequence encoding the first effector moiety comprises a sequence according to SEQ ID NO: 51, or a sequence with at least 80, 85, 90, 95, 99, or 100% identity thereto, or having no more than 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 positions of difference thereto.
  • nucleic acid of any of embodiments 145-160 wherein the first region comprises a nucleotide sequence according to SEQ ID NO: 63 or 130, or a sequence with at least 80, 85, 90, 95, 99, or 100% identity thereto, or having no more than 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 positions of difference thereto, wherein a poly-A sequence is optional.
  • nucleic acid of any of embodiments 145-161, wherein the second region comprises a nucleotide sequence according to SEQ ID NO: 57, or a sequence with at least 80, 85, 90, 95, 99, or 100% identity thereto, or having no more than 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 positions of difference thereto, wherein a poly-A sequence is optional.
  • a nucleic acid encoding a system for reducing MYC expression comprising: a) a first region encoding a first expression repressor, the first expression repressor comprising: i) a first targeting moiety having an amino acid sequence according to SEQ ID NO: 13 or a sequence with at least 80, 85, 90, 95, 99, or 100% identity thereto, or having no more than 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 positions of difference thereto, and ii) a first effector moiety having an amino acid sequence according to SEQ ID NO: 19 or 87 or a sequence with at least 80, 85, 90, 95, 99, or 100% identity thereto, or having no more than 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 positions of difference thereto, and b) a second region encoding a second expression repressor, the second expression repressor comprising: i) a second targeting moiety having an amino acid sequence
  • a nucleic acid encoding a system for reducing MYC expression comprising: a) a first region encoding a first expression repressor, the first expression repressor comprising: i) a first targeting moiety having an amino acid sequence according to SEQ ID NO: 13 or a sequence with at least 80, 85, 90, 95, 99, or 100% identity thereto, or having no more than 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 positions of difference thereto, and ii) a first effector moiety having an amino acid sequence according to SEQ ID NO: 19 or 87 or a sequence with at least 80, 85, 90, 95, 99, or 100% identity thereto, or having no more than 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 positions of difference thereto, and b) a second region encoding a second expression repressor, the second expression repressor comprising: i) a second region encoding a second expression
  • a first nuclear localization signal e.g., an SV40 NLS, e.g., a sequence according to SEQ ID NO: 135 or a sequence with at least 80, 85, 90, 95, 99, or 100% identity thereto, e.g., situated N-terminal of the first targeting moiety.
  • a second nuclear localization signal e.g., a nucleoplasmin NLS, e.g., a sequence according to SEQ ID NO: 136 or a sequence with at least 80, 85, 90, 95, 99, or 100% identity thereto, e.g., situated C-terminal of the first effector moiety.
  • a first nuclear localization signal e.g., an SV40 NLS, e.g., a sequence according to SEQ ID NO: 135 or a sequence with at least 80, 85, 90, 95, 99, or 100% identity thereto, e.g., situated N-terminal of the second targeting moiety.
  • a second nuclear localization signal e.g., a nucleoplasmin NLS, e.g., a sequence according to SEQ ID NO: 136 or a sequence with at least 80, 85, 90, 95, 99, or 100% identity thereto, e.g., situated C-terminal of the second effector moiety.
  • a nucleotide sequence encoding an amino acid sequence C-terminal of the first effector moiety, e.g., a sequence of up to 30, 25, 20, or 18 amino acids, e.g., a sequence according to SEQ ID NO: 126 or a sequence with at least 80, 85, 90, 95, 99, or 100% identity thereto.
  • nucleic acid of any of embodiments 163-174, wherein the first expression repressor has an amino acid sequence according SEQ ID NO: 30 or 129, or a sequence with at least 80, 85, 90, 95, 99, or 100% identity thereto.
  • nucleic acid of any of embodiments 144-175, wherein the second expression repressor has an amino acid sequence according to SEQ ID NO: 177, or 183 or a sequence with at least 80, 85, 90, 95, 99, or 100% identity thereto.
  • nucleic acid of any of embodiments 144-176, wherein the second expression repressor has an amino acid sequence according to SEQ ID NO: 179, or 185, or a sequence with at least 80, 85, 90, 95, 99, or 100% identity thereto.
  • first expression repressor comprises an amino acid sequence according to SEQ ID NO: 30, or 129, or a sequence with at least 80, 85, 90, 95, 99, or 100% identity thereto and the second expression repressor has an amino acid sequence according to SEQ ID NO: 24, 141, 177, 179, 183, or 185, or a sequence with at least 80, 85, 90, 95, 99, or 100% identity thereto.
  • nucleic acid of any of embodiments 144-179 wherein the first region comprises a nucleotide sequence encoding the first effector moiety, wherein the nucleotide sequence encoding the first effector moiety comprises a sequence according to SEQ ID NO: 52 or 132, or a sequence with at least 80, 85, 90, 95, 99, or 100% identity thereto, or having no more than 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 positions of difference thereto.
  • nucleic acid of any of embodiments 144-180, wherein the second region comprises a nucleotide sequence according to SEQ ID NO: 173 or a sequence with at least 80, 85, 90, 95, 99, or 100% identity thereto, or having no more than 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 positions of difference thereto, wherein a poly-A sequence is optional.
  • nucleic acid of any of embodiments 144-181, wherein the second region comprises a nucleotide sequence according to SEQ ID NO: 175 or a sequence with at least 80, 85, 90, 95, 99, or 100% identity thereto, or having no more than 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 positions of difference thereto, wherein a poly-A sequence is optional.
  • nucleic acid of any of embodiments 144-183, wherein the first region comprises a nucleotide sequence according to SEQ ID NO: 63 or 130, or a sequence with at least 80, 85, 90, 95, 99, or 100% identity thereto, or having no more than 20, 19, 18, 17, 16, 15, 14, 13, 12, 1 1, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 positions of difference thereto, wherein a poly-A sequence is optional.
  • nucleic acid of any of embodiments 144-184, wherein the second region comprises a nucleotide sequence according to SEQ ID NO: 189, or a sequence with at least 80, 85, 90, 95, 99, or 100% identity thereto, or having no more than 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 positions of difference thereto, wherein a poly-A sequence is optional.
  • SEQ ID NO: 189 or a sequence with at least 80, 85, 90, 95, 99, or 100% identity thereto, or having no more than 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 positions of difference thereto, wherein a poly-A sequence is optional.
  • nucleic acid of any of embodiments 144-185 wherein the second region comprises a nucleotide sequence according to SEQ ID NO: 194, or a sequence with at least 80, 85, 90, 95, 99, or 100% identity thereto, or having no more than 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 positions of difference thereto, wherein a poly-A sequence is optional
  • nucleic acid of any of embodiments 144-189, wherein the second region comprises a nucleotide sequence according to SEQ ID NO: 189, or a sequence with at least 80, 85, 90, 95, 99, or 100% identity thereto, or having no more than 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 positions of difference thereto.
  • nucleic acid of any of embodiments 144-190, wherein the second region comprises a nucleotide sequence according to SEQ ID NO: 194, or a sequence with at least 80, 85, 90, 95, 99, or 100% identity thereto, or having no more than 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 positions of difference thereto.
  • nucleic acid of any of embodiments 144-192 which has a nucleotide sequence according to SEQ ID NO: 196 or 197, or a sequence with at least 80, 85, 90, 95, 99, or 100% identity thereto, or having no more than 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 positions of difference thereto.
  • the system or nucleic acid of embodiment 200, wherein the first effector moiety comprises a protein chosen from SETDB1, SETDB2, EHMT2 (i.e., G9A), EHMT1 (i.e., GLP), SUV39H1, EZH2, EZH1, SUV39H2, SETD8, SUV420H1, SUV420H2, or a functional variant or fragment of any thereof, e.g., a SET domain of any thereof.
  • EHMT2 i.e., G9A
  • EHMT1 i.e., GLP
  • the system or nucleic acid of embodiment 202, wherein the first effector moiety comprises a protein chosen from KDM1A (i.euze LSD1), KDM1B (i.e., LSD2), KDM2A, KDM2B, KDM5A, KDM5B, KDM5C, KDM5D, KDM4B, NO66 (or a functional variant or fragment of any thereof).
  • the system or nucleic acid of embodiment 204, wherein the first effector moiety comprises a protein chosen from HDAC1, HDAC2, HDAC3, HDAC4, HDAC5, HDAC6, HDAC7, HDAC8, HDAC9, HDAC10, HDAC11, SIRT1, SIRT2, SIRT3, SIRT4, SIRT5, SIRT6, SIRT7, SIRT8, SIRT9, or a functional variant or fragment of any thereof.
  • the system or nucleic acid of embodiment 206, wherein the first effector moiety comprises a protein chosen from MQ1, DNMT1, DNMT3A1, DNMT3A2, DNMT3B1, DNMT3B2, DNMT3B3, DNMT3B4, DNMT3B5, DNMT3B6, DNMT3L, or a functional variant or fragment of any thereof.
  • the system or nucleic acid of embodiment 216, wherein the second effector moiety comprises a protein chosen from SETDB1, SETDB2, EHMT2 (i.e., G9A), EHMT1 (i.e., GLP), SUV39H1, EZH2, EZH1, SUV39H2, SETD8, SUV420H1, SUV420H2, or a functional variant or fragment of any thereof, e.g., a SET domain of any thereof.
  • the system or nucleic acid of embodiment 218, wherein the second effector moiety comprises a protein chosen from KDM1A (i.e., LSD1), KDM1B (i.e., LSD2), KDM2A, KDM2B, KDM5A, KDM5B, KDM5C, KDM5D, KDM4B, NO66 (or a functional variant or fragment of any thereof.
  • KDM1A i.e., LSD1
  • KDM1B i.e., LSD2
  • KDM5A KDM5B
  • KDM5C KDM5C
  • KDM5D KDM4B
  • NO66 or a functional variant or fragment of any thereof.
  • the system or nucleic acid of embodiment 222, wherein the second effector moiety comprises a protein chosen from MQ1, DNMT1, DNMT3A1, DNMT3A2, DNMT3B1, DNMT3B2, DNMT3B3, DNMT3B4, DNMT3B5, DNMT3B6, DNMT3L, or a functional variant or fragment of any thereof.
  • the first effector moiety comprises a histone methyltransferase, histone demethylase, histone deacetylase, DNA methyltransferase, a functional variant or fragment of any thereof, or a combination of any thereof
  • the second effector moiety comprises a transcription repressor or a functional variant or fragment of any thereof.
  • the first effector moiety comprises a SETDB1, SETDB2, EHMT2 (i.e., G9A), EHMT1 (i.e., GLP), SUV39H1, EZH2, EZH1, SUV39H2, SETD8, SUV420H1, SUV420H2, KDM1A (i.e., LSD1), KDM1B (i.e., LSD2), KDM2A, KDM2B, KDM5A, KDM5B, KDM5C, KDM5D, KDM4B, NO66, HDAC1, HDAC2, HDAC3, HDAC4, HDAC5, HDAC6, HDAC7, HDAC8, HDAC9, HDAC10, HDAC11, SIRT1, SIRT2, SIRT3, SIRT4, SIRT5, SIRT6, SIRT7, SIRT8, SIRT9, MQ1, DNMT1, DN
  • the system or nucleic acid of any of embodiments 118-200 wherein the first expression repressor comprises an amino acid sequence chosen from any of SEQ ID NOs: 22-37, 129, 133, 134, 139-149, 177- 180, or 183-186, or a sequence with at least 80, 85, 90, 95, or 99% identity thereto, or a sequence with no more than 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 positions of difference thereto.
  • the second expression repressor comprises an amino acid sequence chosen from any of SEQ ID NOs: 22-37, 129, 133, 134, 139-149, 177-180, or 183-186, or a sequence with at least 80, 85, 90, 95, or 99% identity thereto, or a sequence with no more than 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 positions of difference thereto.
  • the system or nucleic acid of embodiment 244 comprising an amino acid sequence of SEQ ID NO: 91, 92, 121, 122, 181, 182, 187, or 188, or a sequence with at least 80, 85, 90, 95, or 99% identity thereto, or a sequence with no more than 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 positions of difference thereto.
  • a first nuclear localization signal e.g., a SV40 NLS; e.g., a sequence according to SEQ ID NO: 135;
  • a first targeting moiety e.g., a zinc finger binding domain, e.g., ZF9; e.g., a sequence according to SEQ ID NO: 13;
  • a first effector moiety e.g., a DNA methyltransferase, e.g., MQ1; e.g., a sequence according to SEQ ID NO: 19 or 87;
  • a second nuclear localization signal e.g., a nucleoplasmin NLS; e.g., a sequence according to SEQ ID NO: 136; and the second expression repressor comprises, from N-terminus to C-terminus:
  • a third nuclear localization signal e.g., a SV40NLS; e.g., a sequence according to SEQ ID NO: 135;
  • a second targeting moiety e.g., a zinc finger binding domain, e.g., ZF3; e.g., a sequence according to SEQ ID NO: 7;
  • a second effector moiety e.g., KRAB, e.g., a sequence according to SEQ ID NO: 18;
  • a fourth nuclear localization signal e.g., a nucleoplasmin NLS, e.g., a sequence according to SEQ ID NO: 136.
  • the first expression repressor comprises from N-terminus to C-terminus:
  • a first nuclear localization signal e.g., a SV40 NLS; e.g., a sequence according to SEQ ID NO: 135;
  • a first targeting moiety e.g., a zinc finger binding domain, e.g., ZF9; e.g., a sequence according to SEQ ID NO: 13;
  • a first effector moiety e.g., a DNA methyltransferase, e.g., MQ1; e.g., a sequence according to SEQ ID NO: 19 or 87;
  • a second nuclear localization signal e.g., a nucleoplasmin NLS; e.g., a sequence according to SEQ ID NO: 136; and the second expression repressor comprises, from N-terminus to C-terminus:
  • a third nuclear localization signal e.g., a SV40NLS; e.g., a sequence according to SEQ ID NO: 135;
  • a second targeting moiety e.g., a zinc finger binding domain, e.g., ZF54; e.g., a sequence according to SEQ ID NO: 169;
  • a second effector moiety e.g., KRAB, e.g., a sequence according to SEQ ID NO: 18;
  • a fourth nuclear localization signal e.g., a nucleoplasmin NLS, e.g., a sequence according to SEQ ID NO: 136. 248.
  • the first expression repressor comprises from N-terminus to C-terminus:
  • a first nuclear localization signal e.g., a SV40 NLS; e.g., a sequence according to SEQ ID NO: 135;
  • a first targeting moiety e.g., a zinc finger binding domain, e.g., ZF9; e.g., a sequence according to SEQ ID NO: 13;
  • a first effector moiety e.g., a DNA methyltransferase, e.g., MQ1 ; e.g., a sequence according to SEQ ID NO: 19 or 87;
  • a second nuclear localization signal e.g., a nucleoplasmin NLS; e.g., a sequence according to SEQ ID NO: 136; and the second expression repressor comprises, from N-terminus to C-terminus:
  • a third nuclear localization signal e.g., a SV40NLS; e.g., a sequence according to SEQ ID NO: 135;
  • a second targeting moiety e.g., a zinc finger binding domain, e.g., ZF67; e.g., a sequence according to SEQ ID NO: 171;
  • a second effector moiety e.g., KRAB, e.g., a sequence according to SEQ ID NO: 18;
  • a fourth nuclear localization signal e.g., a nucleoplasmin NLS, e.g., a sequence according to SEQ ID NO: 136. 249.
  • a fourth nuclear localization signal e.g., a nucleoplasmin NLS, e.g., a sequence according to SEQ ID NO: 136. 249.
  • the system of any of embodiments 118-250 which is capable of reducing tumor volume, e.g., in a human subject or in a mammalian model. 252.
  • chemotherapeutic agent is sorafenib or cisplatin.
  • the second targeting moiety is selected from a TAL effector domain, a CRISPR/Cas domain, a zinc finger domain, a tetR domain, a meganuclease, or an oligonucleotide.
  • the first targeting moiety comprises a CRISPR/Cas domain (e.g., a first CRISPR/Cas domain).
  • first CRISPR/Cas domain comprises an amino acid sequence of a Cas protein or Cpfl protein chosen from Table 1 or a variant (e.g., mutant) of any thereof
  • second CRISPR/Cas domain comprises an amino acid sequence of a different Cas protein or Cpfl protein chosen from Table 1 or a variant (e.g., mutant) of any thereof.
  • the system or nucleic acid of any of embodiments 268-275, wherein the second zinc finger domain comprises at least 1, 2, 3, 4, 5, 7, 8, 9, or 10 zinc fingers (and optionally no more than 11, 10, 9, 8, 7, 6, or 5 zinc fingers).
  • a TAL effector domain e.g., a first TAL effector domain
  • the system or nucleic acid of any of embodiments 279 or 280, wherein the first TAL effector domain comprises at least 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, or 40 central repeats (and optionally, no more than 45, 40, 35, 30, 25, 20, 15, or 10 central repeats).
  • the system or nucleic acid of any of embodiments 279-2 1, wherein the first TAL effector domain comprises 2-40, 5-40, 10-40, 15-40, 20-40, 25-40, 30-40, 35-40, 2-35, 5-35, 10-35, 15-35, 20-35, 25-35, 30-35, 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 central repeats.
  • the system or nucleic acid of any of embodiments 279-282, wherein the second TAL effector domain comprises at least 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, or 40 central repeats (and optionally, no more than 45, 40, 35, 30, 25, 20, 15, or 10 central repeats).
  • the system or nucleic acid of any of embodiments 279-283, wherein the second TAL effector domain comprises 2-40, 5-40, 10-40, 15-40, 20-40, 25-40, 30-40, 35-40, 2-35, 5-35, 10-35, 15-35, 20-35, 25-35, 30-35, 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 central repeats.
  • nucleic acid comprises a sequence that is complementary to the transcriptional regulatory element or a sequence proximal thereto, or comprises no more than 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 mismatches relative to the transcriptional regulatory element or a sequence proximal thereto.
  • nucleic acid comprises a sequence that is complementary to the anchor sequence or a sequence proximal thereto, or comprises no more than 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 mismatches relative to the anchor sequence or a sequence proximal thereto.
  • nucleic acid comprises DNA, a peptide nucleic acid (PNA), a peptide-oligonucleotide conjugate, a locked nucleic acid (LNA), a bridged nucleic acid (BNA), a polyamide, a triplex-forming oligonucleotide, an antisense oligonucleotide, tRNA, mRNA, rRNA, miRNA, gRNA, siRNA, or other RNAi molecule.
  • PNA peptide nucleic acid
  • LNA locked nucleic acid
  • BNA bridged nucleic acid
  • nucleic acid comprises a gRNA. 295.
  • nucleic acid comprises a sequence with at least 80, 85, 90, 95, 99, or 100% identity to any of SEQ ID NOs: 1-4, or has no more than 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 positions of difference thereto. 296.
  • the first nucleic acid comprises a sequence with at least 80, 85, 90, 95, 99, or 100% identity to any of SEQ ID NOs: 1-4 or has no more than 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 positions of difference thereto
  • the second nucleic acid comprises a sequence with at least 80, 85, 90, 95, 99, or 100% identity to any of SEQ ID NOs: 1-4 or has no more than 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 positions of difference thereto.
  • the first nucleic acid comprises a sequence with at least 80, 85, 90, 95, 99, or 100% identity to any of SEQ ID NOs: 96-110 or has no more than 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 positions of difference thereto
  • the second nucleic acid comprises a sequence with at least 80, 85, 90, 95, 99, or 100% identity to any of SEQ ID NOs: 96-110 or has no more than 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 positions of difference thereto.
  • transcriptional regulatory element comprises an enhancer; e.g., a super enhancer.
  • anchor sequence comprises the sequence of SEQ ID NO: 71 or 72, or a sequence with no more than 8, 7, 6, 5, 4, 3, 2, or 1 alterations relative thereto.
  • anchor sequence comprises a sequence according to SEQ ID NO: 73 or 74, or a sequence with no more than 8, 7, 6, 5, 4, 3, 2, or 1 alterations relative thereto.
  • anchor sequence is at least 1, 5, 10, 50, 100, or 1000 kilobases away from the MYC gene (e.g., from the TSS or promoter of the MYC gene).
  • the first expression repressor comprises a targeting moiety comprising a first CRISPR/Cas molecule, e.g., comprising a first catalytically inactive CRISPR/Cas protein, and an effector moiety comprising an epigenetic modifying moiety; and the second expression repressor comprises a targeting moiety comprising a second CRISPR/Cas molecule, e.g., comprising a second catalytically inactive CRISPR/Cas protein, and an optionally an effector moiety comprising a transcription repressor.
  • the first expression repressor comprises a targeting moiety comprising a first zinc finger domain, and an effector moiety comprising an epigenetic modifying moiety; and the second expression repressor comprises a targeting moiety comprising a second zinc finger domain, and optionally an effector moiety comprising a transcription repressor.
  • the first expression repressor comprises a targeting moiety comprising a CRISPR/Cas molecule, e.g., comprising a catalytically inactive CRISPR/Cas protein, and an effector moiety comprising an epigenetic modifying moiety; and the second expression repressor comprises a targeting moiety comprising a zinc finger domain, and optionally an effector moiety comprising a transcription repressor.
  • the first expression repressor comprises a targeting moiety comprising a zinc finger domain, and an effector moiety comprising an epigenetic modifying moiety; and the second expression repressor comprises a targeting moiety comprising a CRISPR/Cas domain, e.g., comprising a catalytically inactive CRISPR/Cas protein, and optionally an effector moiety comprising a transcription repressor.
  • the zinc finger domain (e.g., the first or second zinc finger domain) comprises 1-10, 1-9, 1-8, 1-7, 1-6, 1-5, 1-4, 1-3, 1-2, 2-10, 2-9, 2- 8, 2-7, 2-6, 2-5, 2-4, 2-3, 3-10, 3-9, 3-8, 3-7, 3-6, 3-5, 3-4, 4-10, 4-9, 4-8, 4-7, 4-6, 4-5, 5-10, 5-9, 5-8, 5- 7, 5-6, 6-10, 6-9, 6-8, 6-7, 7-10, 7-9, 7-8, 8-10, 8-9, or 9-10 zinc fingers, e.g., 3 or 9 zinc fingers.
  • the zinc finger domain comprises 1-10, 1-9, 1-8, 1-7, 1-6, 1-5, 1-4, 1-3, 1-2, 2-10, 2-9, 2- 8, 2-7, 2-6, 2-5, 2-4, 2-3, 3-10, 3-9, 3-8, 3-7, 3-6, 3-5, 3-4, 4-10, 4-9, 4-8, 4-7
  • the first expression repressor comprises an amino acid sequence of any of SEQ ID NOS: 28-33 or 35-37, 145-149, 151, 152, or a sequence with at least 80, 85, 90, 95, 99, or 100% identity thereto, or having no more than 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 positions of difference thereto.
  • binding of the first expression repressor to the transcription regulatory element or a sequence proximal thereto decreases expression of MYC in a cell.
  • binding of the first expression repressor to the transcription regulatory element or a sequence proximal thereto and binding of the second expression repressor to the anchor sequence or a sequence proximal thereto appreciably decreases expression of MYC at 1, 2, 3, 4, 5, 6, 7, 8, 10, 12, 16, 20, 24, 28, 32, 36, 40, 44, 48, 52, 56, 60, 64, 68, 72, 76, 80, or 96 hours post-transfection.
  • binding of the first expression repressor to the transcription regulatory element or a sequence proximal thereto and the second expression repressor to the anchor sequence or a sequence proximal thereto appreciably decreases expression of MYC for a time period of at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 hours, or at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 days, or at least 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 cell divisions, e.g., as measured by QPCR or ELISA.
  • binding of the first expression repressor to the promoter or a sequence proximal thereto and the second expression repressor to the super-enhancer or a sequence proximal thereto appreciably decreases expression of MYC for a time period of at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 hours, or at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 days, or at least 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 cell divisions, e.g., as measured by QPCR or ELISA.
  • the expression repressor or system of any preceding embodiments which does not reduce viability of non-cancer cells (e.g., primary hepatocytes) by more than 5, 10, 15, or 20%, e.g., when assayed according to Example 29.
  • HCC hepatocellular carcinoma
  • FHCC Fibrolamellar Hepatocellular Carcinoma
  • Cholangiocarcinoma Angiosarcoma
  • secondary liver cancer Non-small cell lung cancer (NSCLC)
  • NSCLC Non-small cell lung cancer
  • SCLC Small cell lung cancer
  • Large cell (undifferentiated) carcinoma triple negative breast cancer, gastric adenocarcinoma, endometrial carcinoma, or pancreatic carcinoma.
  • cancer cells are lung cancer cells, gastric cancer cells, gastrointestinal cancer cells, colorectal cancer cells, pancreatic cancer cells, or hepatic cancer cells.
  • a fusion protein comprising: a first amino acid region comprising a sequence encoding the first expression repressor of a system of any of embodiments 118-383; and a second amino acid region comprising a sequence encoding the second expression repressor of a system of any of embodiments 118-383.
  • the fusion protein of embodiment 384 which comprises a third amino acid region, wherein the third amino acid region is situated between the first amino acid region and the second amino acid region.
  • the fusion protein of embodiment 385, wherein the third amino acid region comprises a protease cleavage peptide sequence, e.g., a self-cleaving peptide sequence, e.g., a T2A self-cleaving peptide sequence, e.g., a sequence according to SEQ ID NO: 120.
  • a protease cleavage peptide sequence e.g., a self-cleaving peptide sequence, e.g., a T2A self-cleaving peptide sequence, e.g., a sequence according to SEQ ID NO: 120.
  • the fusion protein of embodiment 386, wherein the third amino acid region comprises a protease cleavage peptide sequence, e.g., a self-cleaving peptide sequence, e.g., a tandem 2A peptide sequence, e.g., a tPT2A sequence, e.g., a sequence according to SEQ ID NO: 124.
  • a protease cleavage peptide sequence e.g., a self-cleaving peptide sequence, e.g., a tandem 2A peptide sequence, e.g., a tPT2A sequence, e.g., a sequence according to SEQ ID NO: 124.
  • the second expression repressor comprises an amino acid sequence according to SEQ ID NO: 24 or 142, or a sequence with at least 80, 85, 90, 95, or 99% identity thereto.
  • the second expression repressor comprises an amino acid sequence according to SEQ ID NO: 177 or 183, or a sequence with at least 80, 85, 90, 95, or 99% identity thereto.
  • the fusion protein of any of embodiments 384-391 which comprises an amino acid sequence of SEQ ID NO: 91, 92, 121, or 122, or a sequence with at least 80, 85, 90, 95, or 99% identity thereto, or a sequence with no more than 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 positions of difference thereto.
  • the fusion protein of any of embodiments 384-392 which comprises an amino acid sequence of SEQ ID NO: 181, 182, 187, or 188, or a sequence with at least 80, 85, 90, 95, or 99% identity thereto, or a sequence with no more than 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 positions of difference thereto.
  • a nucleic acid comprising a sequence encoding the system of any of embodiments 118-393.
  • a nucleic acid comprising a sequence encoding the system of embodiment 394.
  • nucleic acid of embodiment 394 or 395 which comprises: a first region comprising a sequence encoding the first expression repressor of a system of any of embodiments 118-393; and a second region comprising a sequence encoding the second expression repressor of a system of any of embodiments 118-393.
  • nucleic acid of any of embodiments 394-396 which comprises a third region, wherein the third region is situated between the first region and the second region.
  • nucleic acid of embodiment 397 or 398, wherein the third region encodes a tPT2A peptide sequence e.g., a sequence according to SEQ ID NO: 124.
  • a protease cleavage peptide sequence e.g., a self-cleaving peptide sequence, e.g., a T2A self-cleaving peptide sequence, e.g., a sequence according to SEQ ID NO: 95. 401.
  • a protease cleavage peptide sequence e.g., a self-cleaving peptide sequence, e.g., a tandem 2A peptide sequence, e.g., a tPT2A peptide sequence, e.g., a sequence according to SEQ ID NO: 124.
  • nucleic acid of any of embodiments 394-401 wherein the first expression repressor comprises an amino acid sequence according to SEQ ID NO: 30, 129 or a sequence with at least 80, 85, 90, 95, or 99% identity thereto; and the second expression repressor comprises an amino acid sequence according to SEQ ID NO: 177, 179, 183, or 185 or a sequence with at least 80, 85, 90, 95, or 99% identity thereto.
  • nucleic acid of any of embodiments 394-404 which encodes an amino acid sequence of SEQ ID NO: 181, 182, 187, 188, or a sequence with at least 80, 85, 90, 95, or 99% identity thereto, or a sequence with no more than 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 positions of difference thereto.
  • a nucleic acid comprising a sequence encoding the expression repressor or the expression repressor system of any of embodiments 1-407.
  • nucleic acid of any of embodiments 394-409 which comprises an N7-methylated guanosine, e.g., linked to the 5’ end of the RNA, e.g., via a reverse 5' to 5' triphosphate linkage.
  • nucleic acid of any of embodiments 394-410 which comprises a 5’ UTR.
  • nucleic acid of any of embodiments 394-411 which comprises a Kozak sequence, e.g., between the 5’ UTR and the sequence encoding the expression repressor.
  • a system comprising: a first nucleic acid comprising a sequence encoding the first expression repressor of a system of any of embodiments 118-393; and a second nucleic acid comprising a sequence encoding a second expression repressor, e.g., the second expression repressor of a system of any of embodiments 118-393.
  • the system of embodiment 414 wherein the first nucleic acid has a nucleotide sequence of SEQ ID NO: 63, 130, or a sequence with at least 80, 85, 90, 95, or 99% identity thereto, or a sequence with no more than 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 positions of difference thereto, and the second nucleic acid having a nucleotide sequence of SEQ ID NO: 189, or 194, or a sequence with at least 80, 85, 90, 95, or 99% identity thereto, or a sequence with no more than 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 positions of difference thereto.
  • the system of embodiment 415 wherein the first nucleic acid has a nucleotide sequence of SEQ ID NO: 189, 194, or a sequence with at least 80, 85, 90, 95, or 99% identity thereto, or a sequence with no more than 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 positions of difference thereto, and the second nucleic acid having a nucleotide sequence of SEQ ID NO: 63, 130, or a sequence with at least 80, 85, 90, 95, or 99% identity thereto, or a sequence with no more than 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 positions of difference thereto.
  • a vector comprising the nucleic acid encoding the system, or expression repressor of any of the preceding embodiments.
  • a lipid nanoparticle comprising the system, nucleic acid, mRNA, or vector of any of the preceding embodiments.
  • the lipid nanoparticle of embodiment 419 comprising an ionizable lipid, e.g., a cationic lipid, e.g., MC3, SSOP.
  • the lipid nanoparticle of embodiment 419 or 420 further comprising one or more of neutral lipids, ionizable amine-containing lipids, biodegradable alkyn lipids, steroids, phospholipids, polyunsaturated lipids, structural lipids (e.g., sterols), PEG, cholesterol, or polymer conjugated lipids.
  • reaction mixture comprising the expression repressor, system, nucleic acid, vector, or lipid nanoparticle of any of the preceding embodiments.
  • reaction mixture of embodiment 422, further comprising a cell further comprising a cell.
  • a pharmaceutical composition comprising the expression repressor, system, nucleic acid, vector, lipid nanoparticle or the reaction mixture of any preceding embodiments. 425.
  • a method of decreasing expression of a MYC gene in a cell comprising: contacting the cell (e.g., a cancer cell) with an expression repressor, a system, one or more nucleic acids encoding said system or expression repressor, a vector, a lipid nanoparticle, or a pharmaceutical composition of any of embodiments 1-424, thereby decreasing expression of the MYC gene in the cell.
  • a method of treating cancer in a subject in need thereof comprising: administering the expression repressor, system, nucleic acid, vector, lipid nanoparticle, or a pharmaceutical composition of any of embodiments 1-424 to the subject, thereby treating the cancer in the subject.
  • a method of reducing tumor growth in a subject in need thereof comprising: administering the expression repressor, system, nucleic acid, vector, lipid nanoparticle, or a pharmaceutical composition of any of embodiments 1-424 to the subject, thereby reducing the tumor size in the subject.
  • a method of increasing or restoring sensitivity of a cancer to a kinase inhibitor comprising administering an expression repressor or system described herein to a subject having the cancer.
  • a method of increasing or restoring sensitivity of a cancer to a bromodomain inhibitor e.g., a BET inhibitor, e.g., JQ1
  • the method comprising administering an expression repressor, system, or nucleic acid described herein (e.g., of any of embodiments 1-423) to a subject having the cancer, wherein optionally administration of the expression repressor or system lowers the IC 50 of the bromodomain inhibitor by 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 95%, e.g., in a cancer cell viability assay, e.g., an assay according to Example 39.
  • bromodomain inhibitor is or comprises JQ1, BET672, or birabresib.
  • a method of increasing or restoring sensitivity of a cancer to a MEK inhibitor comprising administering an expression repressor, system, or nucleic acid described herein (e.g., of any of embodiments 1-423) to a subject having the cancer, wherein optionally administration of the expression repressor or system lowers the IC 50 of the MEK inhibitor by 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 95%, e.g., in a cancer cell viability assay, e.g., an assay according to Example 51.
  • a cancer cell viability assay e.g., an assay according to Example 51.
  • the method of reducing tumor size in a subject in need thereof comprising: administering the expression repressor, system, nucleic acid, vector, lipid nanoparticle, or a pharmaceutical composition of 1-424 to the subject, wherein the reduction in tumor size is greater than or similar to a tumor size reduction when the subject is treated with a chemotherapeutic agent.
  • chemotherapeutic agent is sorafenib or cisplatin. 441. The method of any of the preceding embodiments wherein the subject does not experience any significant side effects compared to when treated with a chemotherapeutic agent or a small molecule MYC inhibitor.
  • a method of treating a liver disease in a subject in need thereof comprising: administering an expression repressor to the subject, wherein the expression repressor comprises targeting moiety that binds a MYC locus (e.g., a transcribed region of MYC, a MYC promoter, or an anchor sequence of an anchor sequence mediated conjunction (ASMC) comprising a MYC gene or to a sequence proximal to the anchor sequence), and optionally, an effector moiety, e.g., an effector moiety described herein; thereby treating the liver disease in the subject.
  • a MYC locus e.g., a transcribed region of MYC, a MYC promoter, or an anchor sequence of an anchor sequence mediated conjunction (ASMC) comprising a MYC gene or to a sequence proximal to the anchor sequence
  • ASMC anchor sequence mediated conjunction
  • invention 449 The method of embodiment 447, which further comprises administering to the subject a second expression repressor, the second expression repressor comprising a targeting moiety that binds to an anchor sequence of an anchor sequence mediated conjunction (ASMC) comprising a target gene, e.g., MYC, and optionally, a second effector moiety, e.g., an effector moiety described herein; e.g., KRAB; thereby treating the liver disease in the subject.
  • ASMC anchor sequence mediated conjunction
  • a method of treating a liver disease in a subject in need thereof comprising: administering the expression repressor, system, nucleic acid, vector, lipid nanoparticle, or a pharmaceutical composition, of any of embodiments 1-424 to the subject, thereby treating the liver disease in the subject.
  • liver disease is a chronic liver disease.
  • liver disease is viral or alcohol related.
  • liver disease is hepatitis of hepatocellular carcinoma.
  • hepatocellular carcinoma is selected from HCC subtype SI, HCC subtype S2, or HCC subtype S3.
  • liver disease is caused by a hepatitis B virus or hepatitis C virus.
  • a method of treating a pulmonary disease in a subject in need thereof comprising: administering an expression repressor to the subject, wherein the expression repressor comprises targeting moiety that binds a MYC locus (e.g., a transcribed region of MYC, a MYC promoter, or an anchor sequence of an anchor sequence mediated conjunction (ASMC) comprising a MYC gene or to a sequence proximal to the anchor sequence), and optionally, an effector moiety, e.g., an effector moiety described herein; thereby treating the pulmonary disease in the subject.
  • a MYC locus e.g., a transcribed region of MYC, a MYC promoter, or an anchor sequence of an anchor sequence mediated conjunction (ASMC) comprising a MYC gene or to a sequence proximal to the anchor sequence
  • ASMC anchor sequence mediated conjunction
  • the method of embodiment 458, which further comprises administering to the subject a second expression repressor, the second expression repressor comprising a targeting moiety that binds a genomic locus located in a super enhancer region of a target gene, e.g., MYC, and optionally, a second effector moiety, e.g., an effector moiety described herein; e.g., KRAB; thereby treating the pulmonary disease in the subject
  • a targeting moiety that binds a genomic locus located in a super enhancer region of a target gene, e.g., MYC
  • a second effector moiety e.g., an effector moiety described herein; e.g., KRAB
  • a method of treating a pulmonary disease in a subject in need thereof comprising: administering the expression repressor, system, nucleic acid, vector, lipid nanoparticle, or a pharmaceutical composition, of any of embodiments 1-424 to the subject, thereby treating the pulmonary disease in the subject.
  • pulmonary disease is a cancer, e.g., a lung cancer, e.g., a lung carcinoma, e.g., non-small cell lung carcinoma or small cell lung carcinoma.
  • contacting or administering comprises intra- tumoral delivery (e.g., injection).
  • cancer selected from colorectal cancer, breast cancer, AML, prostate cancer, neuroblastoma, lung cancer, endometrial cancer, liver cancer, a lymphoma (e.g., Burkitt lymphoma), carcinoma of the cervix, or stomach cancer.
  • a lymphoma e.g., Burkitt lymphoma
  • carcinoma of the cervix or stomach cancer.
  • AFP alpha-fetoprotein
  • cells of the cancer are characterized by the presence of a super enhancer, e.g., comprising the MYC gene or comprising the anchor-sequence mediated conjunction comprising the MYC gene, wherein optionally the cancer is selected from liver cancer, colorectal cancer, breast cancer, AML, prostate cancer, neuroblastoma, lung cancer, or endometrial cancer.
  • a super enhancer e.g., comprising the MYC gene or comprising the anchor-sequence mediated conjunction comprising the MYC gene
  • the cancer is selected from liver cancer, colorectal cancer, breast cancer, AML, prostate cancer, neuroblastoma, lung cancer, or endometrial cancer.
  • the cancer comprises cells characterized by increased MYC expression relative to a reference level (e.g., relative to a reference cell’s MYC expression, e.g., an otherwise similar non-cancerous cell of the subject), and cells not characterized by increased MYC expression relative to a reference level (e.g., relative to a reference cell’s MYC expression, e.g., an otherwise similar non-cancerous cell of the subject), e.g., having normal MYC expression.
  • a reference level e.g., relative to a reference cell’s MYC expression, e.g., an otherwise similar non-cancerous cell of the subject
  • any of embodiments 426-477 which comprises administering a plurality of doses of the expression repressor, system, nucleic acid, vector, lipid nanoparticle, or a pharmaceutical composition to the subject, e.g., at least 2, 3, 4, 5, or 6 doses.
  • any of embodiments 426-479 comprising: a) first, administering to the subject a first plurality of doses of an expression repressor or system described herein (e.g., of any of embodiments 1-424), wherein optionally each subsequent dose in the first plurality is administered 5 days after the previous dose in the first plurality; b) second, withdrawing the expression repressor or system for a period of time (a “drug holiday”), e.g., for about 2 weeks), and c) third, administering to the subject a second plurality of doses of the expression repressor or system, wherein optionally a subsequent dose of the second plurality is administered 5 days after the previous dose in the second plurality.
  • a drug holiday e.g., for about 2 weeks
  • the second therapeutic agent is an immunotherapy, one or both of immune checkpoint and anti-vascular-endothelial-growth-factor therapy, systemic chemotherapy, a tyrosine kinase inhibitor, e.g., sorafenib, a mitogen-activated protein kinase kinase inhibitor (MEK inhibitor), e.g., trametinib, or a bromodomain inhibitor, e.g., a BET inhibitor, e.g., JQ1 or birabresib.
  • a tyrosine kinase inhibitor e.g., sorafenib
  • MEK inhibitor mitogen-activated protein kinase kinase inhibitor
  • BET inhibitor e.g., JQ1 or birabresib.
  • the second therapeutic agent is a bromodomain inhibitor, e.g., a BET inhibitor, e.g., JQ1, birabresib, or BET 672.
  • a bromodomain inhibitor e.g., a BET inhibitor, e.g., JQ1, birabresib, or BET 672.
  • the second therapeutic agent is a mitogen- activated protein kinase kinase inhibitor (MEK inhibitor), e.g., trametinib.
  • MEK inhibitor mitogen- activated protein kinase kinase inhibitor
  • the method further comprises administering an additional therapy to the subject.
  • the additional therapy comprises surgical resection orthotopic liver transplantation, radiofrequency ablation, photodynamic therapy (PDT), laser therapy, brachytherapy, radiation therapy, trans-catheter arterial chemo- or radio-embolization, or stereotactic radiation therapy.
  • the second therapeutic agent is a chemotherapeutic agent, e.g., a kinase inhibitor or a bromodomain inhibitor, e.g., a BET inhibitor.
  • a kinase inhibitor e.g., a kinase inhibitor that inhibits one or more of VEGFR, PDGFR, or RAF kinase, e.g., sorafenib.
  • a kit comprising a container comprising a composition comprising an expression repressor, a system, one or more nucleic acids encoding said system or expression repressor, a vector, a lipid nanoparticle, reaction mixture, or a pharmaceutical composition of any of embodiments 1-424 and a set of instructions comprising at least one method for modulating, e.g., decreasing the expression of a MYC gene within a cell with said composition.
  • 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 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.
  • an anchor sequence comprises one or more CTCF binding motifs.
  • an anchor sequence is not located within a gene coding region.
  • an anchor sequence is located within an intergenic region.
  • an anchor sequence is not located within either of an enhancer or a promoter.
  • 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 Ikb away from any transcription start site.
  • 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 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 sequences (see, e.g. Figure 1).
  • 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.
  • 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.
  • 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.). In some embodiments, a domain is or comprises a section of a polypeptide. In some such embodiments, 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.).
  • 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.
  • a particular functional feature e.g., binding activity, enzymatic activity, folding activity, signaling activity, etc.
  • effector moiety refers to a domain that is capable of altering the expression of a target gene when localized to an appropriate site in the nucleus of a cell.
  • an effector moiety recruits components of the transcription machinery.
  • an effector moiety inhibits recruitment of components of transcription factors or expression repressing factors.
  • an effector moiety comprises an epigenetic modifying moiety (e.g., epigenetically modifies a target DNA sequence).
  • Epigenetic modifying moiety refers to a domain that alters: i) the structure, e.g., two dimensional structure, of chromatin; and/or ii) an epigenetic marker (e.g., one or more of DNA methylation, histone methylation, histone acetylation, histone sumoylation, histone phosphorylation, and RNA-associated silencing), when the epigenetic modifying moiety is appropriately localized to a nucleic acid (e.g., by a targeting moiety).
  • an epigenetic marker e.g., one or more of DNA methylation, histone methylation, histone acetylation, histone sumoylation, histone phosphorylation, and RNA-associated silencing
  • an epigenetic modifying moiety comprises an enzyme, or a functional fragment or variant thereof, that affects (e.g., increases or decreases the level of) one or more epigenetic markers.
  • an epigenetic modifying moiety comprises a DNA methyltransferase, a histone methyltransferase, CREB-binding protein (CBP), or a functional fragment of any thereof.
  • Expression control sequence refers to a nucleic acid sequence that increases or decreases transcription of a gene and includes (but is not limited to) a promoter and an enhancer.
  • An “enhancing sequence” refers to a subtype of expression control sequence and increases the likelihood of gene transcription.
  • a “silencing or repressor sequence” refers to a subtype of expression control sequence and decreases the likelihood of gene transcription.
  • Expression repressor refers to an agent or entity with one or more functionalities that decreases 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 repressor comprises at least one targeting moiety and optionally one effector moiety.
  • an expression repression system refers to a plurality of expression repressors which decrease expression of a target gene in a cell.
  • an expression repression system comprises a first expression repressor and a second expression repressor, wherein the first expression repressor and second expression repressor (or nucleic acids encoding the first expression repressor and second expression repressor) are present together in a single composition, mixture, or pharmaceutical composition.
  • an expression repression system comprises a first expression repressor and a second expression repressor, wherein the first expression repressor and second expression repressor (or nucleic acids encoding the first expression repressor and second expression repressor) are present in separate compositions or pharmaceutical compositions.
  • the first expression repressor and the second expression repressor are present in the same cell at the same time.
  • the first expression repressor and the second expression repressor are not present in the same cell at the same time, e.g., they are present sequentially.
  • the first expression repressor may be present in a cell for a first time period, and then the second expression repressor may be present in the cell for a second time period, wherein the first and second time periods may be overlapping or non-overlapping.
  • fusion molecule refers to a compound comprising two or more moieties, e.g., a targeting moiety and an effector moiety, that are covalently linked.
  • a fusion molecule and its moieties may comprise any combination of polypeptide, nucleic acid, glycan, small molecule, or other components described herein (e.g., a targeting moiety may comprise a nucleic acid and an effector moiety may comprise a polypeptide).
  • a fusion molecule is a fusion protein, e.g., comprising one or more polypeptide domains covalently linked via peptide bonds.
  • a fusion molecule is a conjugate molecule that comprises a targeting moiety and effector moiety that are linked by a covalent bond other than a peptide bond or phosphodiester bond (e.g., a targeting moiety that comprises a nucleic acid and an effector moiety comprising a polypeptide linked by a covalent bond other than a peptide bond or phosphodiester bond).
  • an expression repressor is or comprises a fusion molecule.
  • 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).
  • the genomic sequence elements are anchor sequences to which one or more protein components of the complex binds.
  • a genomic complex may comprise an anchor sequence-mediated conjunction.
  • 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).
  • colocalization 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, noncoding 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 (e.g., Mediator, P300, enhancer-binding proteins, repressor-binding proteins, histone modifiers, etc.), etc.
  • CTCF noncoding RNA
  • eRNA noncoding RNA
  • 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 e.g., Mediator, P300, enhancer-binding proteins, repressor-binding
  • 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
  • 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, e.g., MYC.
  • MYC locus refers to the portion of the human genome that encodes a MYC polypeptide (e.g., the polypeptide disclosed in NCBI Accession Number NP002458.2, or a mutant thereof), the promoter operably linked to MYC (“MYC promoter”), and the anchor sequences that form an ASMC comprising the MYC gene.
  • MYC locus encodes a nucleic acid having NCBI Accession Number NM — 002467.
  • the MYC gene is a protooncogene, and in some embodiments the MYC gene is an oncogene.
  • a MYC gene is found on chromosome 8, at 8q24.21. In certain instances, a MYC gene begins at 128,816,862 bp from pter and ends at 128,822,856 bp from pter. In certain instances, a MYC gene is about 6 kb. In certain instances, a MYC gene encodes at least eight separate mRNA sequences — 5 alternatively spliced variants and 3 un-spliced variants.
  • 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-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)-methylguanine, 2-thiocytidine, methylated bases, intercal
  • 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, 1 10, 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 As used herein, the term “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.
  • an anchor sequence-mediated conjunction e.g., in some cases, a DNA loop
  • an assembles collection of two or more conjunction nucleating polypeptides (which may, in some embodiments, include multiple copies of the same agent and/or in some embodiments one or more of each of a plurality of different agents) may be referred to as a “complex”, a “dimer” a “multimer”, etc.
  • 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 1000, 500, 100, 50, or 10 base pairs of each other or directly adjacent to each other.
  • Peptide, Polypeptide, Protein refers 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.
  • composition refers to an active agent (e.g., a modulating agent, e.g., a disrupting agent), formulated together with one or more pharmaceutically acceptable carriers.
  • active agent e.g., a modulating agent, e.g., a disrupting agent
  • 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 repressor at the first site and/or modification of the first site by an expression repressor 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 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 5000, 4000, 3000, 2000, 1000, 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).
  • Specific binding refers to an ability to discriminate between possible binding partners in the environment in which binding is to occur.
  • 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 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.
  • the term “substantially” refers to the qualitative condition of exhibiting total or near-total extent or degree of a characteristic or property of interest.
  • One of ordinary skill in the art will understand that biological and chemical phenomena rarely, if ever, go to completion and/or proceed to completeness or achieve or avoid an absolute result.
  • the term “substantially” may therefore be used in some embodiments herein to capture potential lack of completeness inherent in many biological and chemical phenomena.
  • Symptoms are reduced may be used when one or more symptoms of a particular disease, disorder or condition is reduced in magnitude (e.g., intensity, severity, etc.) and/or frequency. In some embodiments, a delay in the onset of a particular symptom is considered one form of reducing the frequency of that symptom.
  • Target 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 repression system, e.g., expression repressor(s), described herein.
  • a target gene is aberrantly expressed (e.g., overexpressed) 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).
  • 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 (e.g., MYC).
  • a 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 condition.
  • a therapeutic agent comprises an expression repression system, e.g., an expression repressor, described herein.
  • a therapeutic agent comprises a nucleic acid encoding an expression repression system, e.g., an expression repressor, described herein.
  • a therapeutic agent comprises a pharmaceutical composition described herein.
  • 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.
  • a therapeutically effective amount is administered in a single dose; in some embodiments, multiple unit doses are required to deliver a therapeutically effective amount.
  • Fig. 1 A depicts a schematic representation of a dual target approach based on a durable block of the MYC promoter using a DBD fused to a DNA methyltransferase, and a transient (48/72 Hours) block of CTCF/TF sites using a DBD or a DBD fused to a short-term effector.
  • Fig. IB depicts guide RNA localization and chromatin context of target sites (CTCF and promoter) for the MYC gene. From top to bottom, the graphs represent, for the MYC locus in HepG2 cells, H3K4me3 (histone H3 Ky trimethylation) levels; H3K9me3 (histone H3 K9 trimethylation) levels (replicate 1); H3K9me3 (histone H3 K9 trim ethylation) levels (replicate 2); H3K27me3 (histone H3 K27 trimethylation) levels; H3K27ac (histone H3 K27 acetylation) levels; GROseq_fwdStrand levels (binding of transcriptionally active RNA pol II on the forward strand); GROseq_revStrand levels (binding of transcriptionally active RNA pol II on the reverse strand); RNAseq_rep2 levels (MYC transcript levels measured using RNAseq, replicate 2); DNA
  • gRNAs GD-28859, GD-28616, GD-28862 target at or near the anchor site upstream of MYC, and gRNA GD-28617 targets the MYC promoter.
  • GD-28859 is also referred to as GD-59;
  • GD- 28616 is also referred to as GD-16;
  • GD-28862 is also referred to as GD-62; and
  • GD-28617 is also referred to as GD-17.
  • Fig. 1C shows a schematic diagram of an exemplary bi-cistronic construct.
  • the 5’ end of the construct possesses a cap structure defined by an N7-methylated guanosine linked to the first nucleotide of the mRNA via a reverse 5' to 5' triphosphate linkage.
  • the cap structure promotes protein translation and stability. Downstream of the cap structure is an un-translated region (5’ UTR) designed to promote high levels of protein translation, followed by the canonical “Kozak” sequence that is recognized by the ribosome to start translation of the protein.
  • UTR un-translated region
  • the CDS is a single continuous sequence comprising the first expression repressor comprising a first targeting moiety and a first effector moiety and the second expression repressor comprising the second targeting moiety and the second effector moiety separated by a tPT2A “ribosome skipping” sequence (the linker).
  • a ribosome reaches the tPT2A linker, it begins translating the linker into amino acids.
  • the first 18 amino acids produced from the P2A linker remain at the C-terminal end of the first expression repressor (e.g., comprising a ZF DBD and MQ1), which the ribosome then releases.
  • the ribosome then continues on until it reaches the T2A linker, and the first 17 residues of the T2A linker are translated and released.
  • the second polypeptide is translated, comprising a single amino acid and then the beginning of the second expression repressor (e.g., comprising a second ZF DBD and KRAB).
  • the CDS is a 3’ UTR which is designed to aid in high levels of translation and to also stabilize the mRNA.
  • the polyA tail promotes protein translation and mRNA stability.
  • Fig. 2 A shows that Cas9-Nuclease editing of the CTCF motif results in down-regulation of MYC expression.
  • Disruption of the CTCF motif with Cas9 resulted in a 32- 39% down-regulation in MYC expression in all three HCC cell lines (HepG2, Hep3B and SKHEP1).
  • Disruption of the region adjacent to the CTCF motif GD-28859 regulated MYC expression 35-45% in two (HepG2 and Hep3B) of the three cell lines.
  • Fig. 2B shows that editing efficiency as assessed by AmpSeq confirmed 77-100% editing of all the cell lines.
  • Fig. 3 shows that dCas9-KRAB down-regulates MYC expression when directed to the promotor or associated CTCF motif.
  • LNP-mediated transfection of dCas9-KRAB/GD-28616 down-regulated MYC expression by 11-34% at 48/72-hour timepoints in Hep3B and SKHEP1.
  • LNP-mediated transfection of dCas9-KRAB/GD-28859 down-regulated MYC expression by 18-44% at 48/72-hour timepoints in all 3 HCC models.
  • Directing dCas9-KRAB to the MYC promoter via dCas9-KRAB/GD-28617 down- regulated MYC expression by 24-58% at 48/72-hour timepoints in all 3 HCC models.
  • Fig. 4 A depicts sgRNA localization and zinc finger design at the promoter associated CTCF motif.
  • Fig. discloses SEQ ID NO: 208.
  • Fig. 4B shows that ZF-KRAB constructs directed to the promoter associated CTCF effected MYC down-regulation in Hep3B.
  • ZF2-KRAB, ZF3-KRAB and ZF4-KRAB down-regulated MYC to an equivalent or greater degree than dCas9-KRAB/GD-28859 in Hep3B cells, with ZF3-KRAB having the strongest down-regulatory effect.
  • Fig. 4C shows thatZF3-No Effector and ZF3-KRAB down-regulated MYC expression in multiple human HCC Models (HepG2, Hep3B, and SKHEP1).
  • ZF3-KRAB was also shown to down-regulate MYC to an equivalent or greater degree than ZF3-No Effector and ZF5-No Effector in the other two HCC models, HepG2 and SKHEP1.
  • Fig. 4D shows that ZF3-No Effector and ZF3-KRAB demonstrated equivalent effects on MYC expression and viability in Hep3B cells at different time points (24 hours, 72 hours, and 120 hours).
  • Fig. 5 shows that dCas9-MQl down-regulated MYC expression when directed at the MYC promoter in multiple HCC models (HepG2, Hep3B, and SKHEP1).
  • Fig. 6A depicts sgRNA localization and zinc finger design at the MYC promoter.
  • Fig. discloses SEQ ID NO: 209.
  • Fig. 6B depicts 6 ZF-MQ1 constructs directed to the MYC promoter that were screened for effects on MYC down-regulation in Hep3B.
  • ZF8-MQ1, ZF9-MQ1 and ZF11-MQ1 down-regulated MYC to the greatest degree in Hep3B cells, with ZF9-KRAB MQ1 having the strongest down-regulatory effect.
  • Fig. 7 A shows that ZF9-MQ1 significantly down-regulated MYC expression and reduces viability in Hep3B compared to ZF12-MQ1.
  • Fig. 7B shows that ZF9-MQ1 significantly down-regulated MYC expression and reduces viability in HepG2 compared to ZF12-MQ1.
  • Fig. 7C shows that ZF9-MQ1 significantly down-regulated MYC expression and reduces viability in SKHEP1 compared to ZF12-MQ1.
  • Fig.7D shows that ZF9-MQ1 is more efficient in down-regulating MYC expression and reducing viability in Hep3B compared to ZF8-MQ1.
  • Fig.7E shows that ZF9-MQ1 is more efficient in down-regulating MYC expression and reducing viability in HepG2 compared to ZF8-MQ1.
  • Fig.7F shows that ZF9-MQ1 is more efficient in down-regulating MYC expression and reducing viability in SKHEP1 compared to ZF8-MQ1.
  • Fig. 7G shows that ZF9-MQ1 significantly down-regulated MYC expression and reduces viability in Hep3B compared to dCas9-MQl/GD17.
  • Fig. 7H shows that ZF9-MQ1 significantly down-regulated MYC expression and reduces viability in HepG2 compared to dCas9-MQl/GD17.
  • Fig. 71 shows that ZF9-MQ1 significantly down-regulated MYC expression and reduces viability in SKHEP1 compared to dCas9-MQl/GD17.
  • Fig. 8 A shows that dCas9-MQl effected a 50-90% decrease in mRNA at 72 hours across the three cell lines (Hep3B, HepG2, and SKHEP1).
  • Fig. 8B shows that MYC down-regulation dramatically decreased viability in HepG2 and Hep3B at 72 and 168 hours, although SK-HEP-1 viability was minimally affected by MYC down-regulation.
  • Fig.8C shows that at day 7 and day 11, MYC mRNA was decreased ⁇ 70% and ⁇ 55% respectively. As far out as day 15, an ⁇ 40% down-regulation in transcript was maintained.
  • Fig.8D shows that that treatment with dCas9-MQl/GD-28617 directs de novo methylation to the targeted region and that these transcriptional changes tightly correlate with the percentage of CpG methylation in the target region and confirmed methylation persists out to day 15.
  • Fig. 9 shows that treatment with dCas9-MQl/GD-17 inhibited tumor growth in vivo.
  • Fig. 10 shows that dCas9-MQl/GD-17 down-regulated MYC in the context of Hepatitis B infection in human hepatocytes.
  • Fig. 11 shows that targeting KRAB effector (or no-effector or NE) fused to zinc-finger domain to the upstream region directly adjacent to the CTCF motif (ZF3-NE or ZF3-KRAB) and targeting MQ1 effector fused to Zinc-Finger domains to the MYC promoter (ZF9-MQ1) downregulated MYC1 mRNA expression.
  • KRAB effector or no-effector or NE
  • ZF9-MQ1 Zinc-Finger domains to the MYC promoter
  • Fig. 12 shows that ZF3-KRAB plus ZF9-MQ1 down-regulated MYC to a greater degree than ZF9-MQ1 alone or ZF3-NE plus ZF9-MQ1 combination.
  • Fig. 13A shows that ZF9-MQ1 designed to bind and target the MYC promoter was dosed at multiple concentration in five HCC cells line, Hep3B, HepG2, SKHEP1, SNU-182 and SNU-449.
  • Fig. 13B-F shows that ZF9-MQ1 downregulated MYC expression and reduced viability in all five HCC cell lines tested and ZF9-MQ1 downregulated MYC expression with a median EC50 of 0.028 ug/ml LNP/mRNA with a ⁇ 10-fold higher median EC50 effect on viability (0.13 ug/ml) in vitro at 72 hours in a HepG2 cell line.
  • Fig.14 shows that ZF9-MQ1 was able to significantly reduced tumor growth (from day 6 forward) when compared to PBS control treated mice and ZF9-MQ1 reduced tumor growth more than the small molecule comparator (MYCi975) (A). ZF9-MQ1 had minimal effect on overall animal weight compared to PBS or MYCi975 (B).
  • Fig.l5A shows that combination of ZF9-MQ1 and ZF3-KRAB 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 reduced tumor growth at a comparable level to sorafenib.
  • Fig. 15B shows that treatment with a combination of ZF9-MQ1 and ZF3-KRAB had minimal effect on overall animal weight compared to the effect on overall animal weight when treated with sorafenib.
  • Fig.l6A shows that that ZF9-MQ1 (from day 13 forward) and co-formulation of ZF9-MQ1 and ZF3-KRAB (from day 6 forward) at 1 mg/kg was able to significantly reduce tumor growth when compared to negative control treated mice.
  • Fig.l6B shows that ZF9-MQ1 individually and the co-formulation of ZF9-MQ1 and ZF3-KRAB at 3 mg/kg was able to reduce tumor growth when compared to negative control treated mice.
  • Fig.l6C shows that the co-formulation of ZF9-MQ1 and ZF3-KRAB was able to reduce tumor growth at a similar or a greater level than cisplatin or the small molecule comparator (MYCi975) at both 1 mg/kg and 3 mg/kg dosage.
  • Fig.l6D shows that treatment with a co-formulation of ZF9-MQ1 and ZF3-KRAB at both 1 mg/kg and 3 mg/kg dosage had minimal effect on overall animal weight compared to the effect on overall animal weight when treated with either cisplatin or MYCi975.
  • Fig. 17A shows that ZF9-MQ1 reduced MYC mRNA level by over 80% in A549 cell line 120 hours post-treatment.
  • Fig. 17B shows that ZF9-MQ1 reduced MYC mRNA level by over 80% in NCI-H2009 cell line 120 hours post-treatment.
  • Fig. 17C shows that ZF9-MQ1 reduced MYC mRNA level by over 80% in NCI-H358 cell line 120 hours post-treatment.
  • Fig. 17D shows that ZF9-MQ1 reduced MYC mRNA level by over 80% in HCC95 cell line 72 hours post-treatment.
  • Fig. 17E shows that ZF9-MQ1 caused loss of cell viability in A549 cell line 120 hours posttreatment.
  • Fig. 17F shows that ZF9-MQ1 caused loss of cell viability in NCI-H2009 cell line 120 hours post-treatment.
  • Fig. 17G shows that ZF9-MQ1 caused loss of cell viability in NCI-H358 cell line 120 hours posttreatment.
  • Fig. 17H shows that ZF9-MQ1 caused loss of cell viability in HCC95 cell line 72 hours posttreatment.
  • Fig. 18A shows that 96 hours post-treatment, about ⁇ 17.5% cells were apoptotic in the untreated cell population.
  • Fig. 18B shows that 96 hours post-treatment, about ⁇ 18% cells were apoptotic in the cell population treated with ZF9-NE.
  • Fig. 18C shows that 96 hours post-treatment, about ⁇ 38.9% cells were apoptotic in the cell population treated with ZF9-MQ1.
  • Fig. 18D shows that 96 hours post-treatment, about ⁇ 38.9% cells were apoptotic in the cell population treated with ZF9-MQ1 in contrast to ⁇ 18% apoptotic cells in both untreated cells and ZF9-NE treated cell population indicating that ZF9-MQ1 is capable of inducing cellular apoptosis of lung cancer cells.
  • Figs. 19 A and B show ZF9-MQ1 down-regulated MYC with an EC50 of 0.08 ug/ml LNP/mRNA with a ⁇ 25-fold higher EC50 effect on viability (2 ug/ml) in vitro at 72 hours in these A549 (Fig. 19A) and HCC95 (Fig. 19B) cell line.
  • Figs. 20A and B show that ZF9-MQ1 treatment reduces MYC protein levels over 80% at 96 hours post-treatment in lung cancer cell lines.
  • Fig. 21 shows that ZF9-MQ1 was able to significantly reduce tumor growth (from day 8 forward ⁇ when compared to PBS control treated mice. It was also observed that ZF9-MQ1 had minimal effect on overall animal weight.
  • Fig. 22 shows that guide RNA GD-29833 and 29914 could effectively downregulate MYC mRNA levels when delivered with a dCAS9-KRAB effector mRNA using LNP delivery with SSOP, highlighting the ability to decrease oncogenic MYC using this distal regulatory element.
  • Fig. 23 shows that guide RNA GD-29833 and 29914 could effectively downregulate MYC mRNA levels when delivered with a dCAS9-KRAB effector mRNA using LNP delivery with MC3, highlighting the ability to decrease oncogenic MYC using this distal regulatory element.
  • Fig. 24A shows that guide RNA GD-29833 and 29914 could effectively downregulate MYC mRNA levels when delivered with all 3 effector proteins (EZH2, EZH2-KRAB, and MQ1) in A549 cell line.
  • Fig. 24B shows that guide RNA GD-29833 and 29914 could effectively downregulate MYC mRNA levels when delivered with all 3 effector proteins (EZH2, EZH2-KRAB, and MQ1) in NCI-H2009 cell line.
  • Fig. 25A shows that guide RNA GD-29833 and 29914 delivered with KRAB or MQ1 could significantly downregulate MYC mRNA levels in A549 cell line 120 hours post treatment.
  • Fig. 25B shows that guide RNA GD-29833 and 29914 delivered with KRAB or MQ1 could significantly downregulate MYC mRNA levels in NCI-H2009 cell line 120 hours post treatment and the downregulation is comparable to the downregulation observed after ZF9-MQ1 treatment.
  • Fig. 26A shows that dCas9-MQl increased target site methylation inNSCLC to about 60%.
  • Fig. 26B shows that dCas9-MQl directed methylation to the distal promoter region (increased to about 50%).
  • Figs. 27A-B show that directing guides to the MYC lung super-enhancer with transcriptional repressors reduces MYC protein levels at 96 hours in NCI-H2009 lung cancer cell lines.
  • Fig. 28A shows the ZF9-MQ1 protein presence in whole cell lysate decreases gradually after treating the Hep3B cell with ZF9-MQ1.
  • Fig. 28B shows the MYC protein expression in whole cell lysate downregulates gradually after treating the Hep3B cell with ZF9-MQ1.
  • Fig. 28C shows the ZF9-MQ1 protein presence in whole cell lysate correlates with down regulation of MYC protein after treating the Hep3B cell with ZF9-MQ1.
  • Fig. 29A shows that down regulation of mRNA expression with a 45% down-regulation in MYC transcript at several timepoints through Day 15 in SK-HEP cell line after treatment with ZF9-MQ1.
  • Fig. 29B shows that MYC transcriptional changes correlated with the percentage of methylation out to day 15.
  • Fig. 30A shows that primary hepatocytes treated with ZF9-MQ1, ZF9-MQ1 + ZF3-KRAB, or bi- cistronic ZF9-MQ1 ZF3-KRAB at concentrations 0.6 ⁇ g/ml, 1.25 ⁇ g/ml, and 2.5 ⁇ g/ml showed a decrease of MYC mRNA expression when compared to GFP, ZF-NE, or ZF3-KRAB alone.
  • Fig.30B shows that treatment with ZF9-MQ1, ZF9-MQ1 + ZF3-KRAB, or bi-cistronic ZF9- MQ1 ZF3-KRAB at concentrations 0.6 ⁇ g/ml, 1.25 ⁇ g/ml, and 2.5 ⁇ g/ml had minimal effect on viability of primary hepatocytes, demonstrating that the decrease in MYC expression is less consequential to normal cells when compared to HCC cell lines.
  • Fig. 30C shows that primary hepatocytes treated with ZF9-MQ1, ZF9-MQ1 + ZF3-KRAB, or bi- cistronic ZF9-MQ1_ZF3-KRAB at concentrations 0.5 ⁇ g/ml, 1.0 ⁇ g/ml, and 2.0 ⁇ g/ml showed a decrease of MYC mRNA expression when compared to GFP, ZF-NE, or ZF3-KRAB alone.
  • Fig.30D shows that treatment with ZF9-MQ1, ZF9-MQ1 + ZF3-KRAB, or bi-cistronic ZF9- MQ1_ZF3-KRAB at concentrations 0.5 ⁇ g/ml, 1.0 ⁇ g/ml, and 2.0 ⁇ g/ml had minimal effect on viability of primary hepatocytes, demonstrating that the decrease in MYC expression is less consequential to normal cells when compared to HCC cell lines.
  • Fig. 31A shows that treatment with ZF9-MQ1+ZF3-KRAB showed a statistically significant reduction in tumor size following three administrations, resulting in a 63% lower tumor volume at Day 25 compared to control and that ZF9-MQ1+ZF3-KRAB treatment was associated with an equivalent effect on tumor volume to treatment with cisplatin.
  • Fig. 3 IB showed that mice treated with ZF9-MQ1+ZF3-KRAB did not experience a significant decrease in body weight.
  • Fig. 32A shows that treatment with ZF9-MQ1+ZF3-KRAB at 1.5 mg/kg was associated with a statistically significant reduction in tumor size following two administrations, resulting in 63% inhibition of tumor growth by Day 23 compared to negative control.
  • ZF9-MQ1 + ZF3-KRAB at 3 mg/kg was associated with a statistically significant reduction in tumor size following two administrations, resulting in 54% inhibition of tumor growth by Day 23 compared to negative control
  • treatment with a 6 mg/kg dose of ZF9-MQ1 + ZF3-KRAB is associated with a statistically significant reduction in tumor size following two administrations, resulting in 63% lower tumor volume at Day 23 compared to negative control.
  • Fig. 32B shows that mice treated with ZF9-MQ1 + ZF3-KRAB did not experience a significant decrease in body weight. Mice treated with sorafenib experienced an initial drop in body weight with a later gain in overall body weight potentially due to an increase in tumor mass.
  • Fig. 33A shows that the bi-cistronic construct ZF9-MQ1_ ZF3-KRAB downregulated MYC mRNA expression at concentrations 0.6 ⁇ g/ml and 2.0 ⁇ g/ml in Hep 3B cells to a greater extent than the single constructs (ZF3-KRAB or ZF9-MQ1) alone.
  • Bi-cistronic ZF9-MQ1_ ZF3-KRAB reduced total MYC mRNA levels by 99% at 48 hours at both 0.6 ⁇ g/ml and 2 ⁇ g/ml concentrations.
  • Fig. 33B shows that the bi-cistronic construct ZF9-MQ1_ ZF3-KRAB downregulated cell viability in Hep 3B cells to a greater extent than the single constructs (ZF3-KRAB or ZF9-MQ1) alone.
  • Bi-cistronic ZF9-MQ1_ ZF3-KRAB reduced the viability of Hep3B cells by about 80% and 27% respectively at both 0.6 ⁇ g/ml and 2 ⁇ g/ml concentrations.
  • Fig. 34A shows that the bi-cistronic construct ZF9-MQ1_ ZF3-KRAB downregulated MYC mRNA and cell viability in Hep3B cells in a dose-dependent manner.
  • Fig. 34B shows that the bi-cistronic construct ZF9-MQ1_ ZF3-KRAB downregulated MYC mRNA and cell viability in HepG2 cells in a dose-dependent manner.
  • Fig. 34C shows that the bi-cistronic construct ZF9-MQ1_ ZF3-KRAB downregulated MYC mRNA and cell viability in SKHEP1 cells in a dose-dependent manner.
  • Fig. 34D shows bi-cistronic ZF9-MQ1 ZF3-KRAB bi-cistronic construct ZF9-MQ1_ ZF3- KRAB was effective against both HCC S 1 and S2 subtype.
  • Fig. 35 shows at 48 hours of treatment with bi-cistronic ZF9-MQ1 ZF3-KRAB > 75% apoptotic cells were detected in the Hep 3B and Hep G2 cell lines and about 15% apoptotic cells were detected in the SK-HEP-1 cell line. Cells were unaffected by non-coding mRNA control compared to untreated cells (5-20% background apoptosis).
  • Fig. 36 shows, in SKHEP1 cells, after 1 treatment with the bi-cistronic ZF9-MQ1 ZF3-KRAB construct the MYC mRNA levels were reduced at day one and remained repressed up to fifteen days following the treatment.
  • Fig. 37 shows, bi-cistronic ZF9-MQ1 ZF3-KRAB treatment decreased MYC mRNA and protein expression at 6 hours which remained down 96 hours later when compared to short non-coding mRNA or untreated cells in both Hep3B and SKHEP1 cell line.
  • Fig. 38 shows at both 6 and 24 hour timepoints following transfection, both OEC ZF3-KRAB and ZF9-MQ1 proteins encoded by bi-cistronic ZF9-MQ1_ZF3-KRAB mRNA were visualized by HA tag on a western blot.
  • Fig. 39A shows the IC50 of sorafenib in SKHEP1 reduced from 12.3 to 10.7 ⁇ M when sorafenib was administered in combination with 0.6 ⁇ g/ml bi-cistronic ZF9-MQ1 ZF3-KRAB.
  • the IC 50 of sorafenib did not change significantly in SKHEP1 cells when sorafenib was administered in combination with 0.1 ⁇ g/ml bi-cistronic ZF9-MQ1 ZF3-KRAB.
  • Fig. 39B shows the IC50 of sorafenib in Hep 3B reduced from 4.4 to 2.9 ⁇ M when sorafenib was administered in combination with 0.6 ⁇ g/ml bi-cistronic ZF9-MQ1 ZF3-KRAB.
  • the IC 50 of sorafenib did not change significantly in Hep 3B cells when sorafenib was administered in combination with O.l ⁇ g/ml bi-cistronic ZF9-MQ1 ZF3-KRAB.
  • Fig. 40A shows, the IC 50 of JQ1 in SKHEP1 cells reduced when treated with bi-cistronic ZF9- MQ1 ZF3-KRAB at concentrations 0.6 ⁇ g/ml and 0.1 ⁇ g/ml respectively.
  • Fig. 40B shows the IC 50 of JQ1 in Hep 3B cells reduced when treated with bi-cistronic ZF9- MQ1_ZF3-KRAB at concentrations 0.6 ⁇ g/ml and 0.1 ⁇ g/ml respectively.
  • Fig. 41A shows ZF17-MQ1 was able to downregulate mouse MYC mRNA expression in Hepal- 6 cells compared to untreated cells at both 0.6 and 1.2 ⁇ g/ml concentrations.
  • Fig. 41B shows ZF17-MQ1 was able to reduce cell viability in mouse Hepal-6 cells compared to untreated cells at both 0.6 and 1.2 ⁇ g/ml concentrations.
  • Fig. 42A shows ZF17-MQ1 treatment in mouse HCC cells Hepal-6 showed significant downregulation of MYC protein at 24 and 48 hours.
  • Fig. 42B shows ZF17-MQ1 treatment in mouse HCC cells Hepal-6 showed significant downregulation of MYC protein at 24 and 48 hours.
  • Fig. 42C shows ZF17-MQ1 treatment in mouse HCC cells Hepal-6 showed significant downregulation of MYC mRNA at 96 hours.
  • Fig. 42D shows ZF17-MQ1 treatment in mouse HCC cells Hepal-6 showed significant loss of cell viability at 96 hours.
  • Fig. 43 shows ZF17-MQ1 significantly reduced animal tumor burden after 4 doses and following a drug holiday of two weeks, re-treatment of the mice with ZF17-MQ1 resulted in full tumor depletion after ⁇ 4 weeks.
  • Fig. 44A shows ZF17-MQ1 treated cells showed reduced MYC protein levels in LL2 cells in comparison to untreated or GFP-treated cells.
  • Fig. 44B shows compared to levels observed in untreated cells, ZF17-MQ1 and ZF16-MQ1 reduced MYC mRNA levels by >99.9% or 74%, respectively in LL2 cells.
  • Fig. 44C shows all three constructs, ZF15-MQ1, ZF16-MQ1, and ZF17-MQ1 were able to reduce cell viability in LL2 cell to a greater extent than untreated and GFP-treated cells.
  • Fig. 45A shows ZF17-MQ1 reduced MYC mRNA level at both 1.25 ⁇ g/mL and 2.5 ⁇ g/mL concentrations. Compared to levels observed in untreated cells, at 2.5 ⁇ g/mL ZF17-MQ1 reduced MYC mRNA levels by 93% and 85% in LL2 and CT26 cells, respectively.
  • Fig. 45B shows ZF17-MQ1 reduced cell viability at both concentrations. Compared to untreated cells, under these conditions, ZF17-MQ1 reduced cell viability by 87% and 93% in LL2 and CT26 cells, respectively.
  • Fig. 46 shows ZF17-MQ1 downregulated MYC mRNA and reduces cell viability in CMT167 and LL2 cells to. a greater extent than untreated and GFP-treated cells (negative control). Compared to levels observed in untreated cells, ZF17-MQ1 reduced MYC mRNA levels by 62% and 73% in CMT167 and LL2 cells, respectively. Furthermore, compared to untreated cells, under these conditions, ZF17-MQ1 reduced cell viability by 54% and 57% in CMT167 and LL2 cells, respectively.
  • Fig. 47 shows ZF9-MQ1 downregulated MYC mRNA levels by 94%, 96%, 96% levels compared to untreated cells in primary small airway epithelial cells, primary lobar epithelial cells, and primary lung fibroblasts respectively. However, viability was only reduced by 16%, 9%, and 22% compared to control cells.
  • Fig. 48A shows ZF9-MQ1 and JQ1 each separately inhibited cell viability of A549 cells.
  • Fig. 48B shows ZF9-MQ1 (0.5 ⁇ g/ml) and JQ1 (concentrations up to 6.25uM) showed a greater than additive effect on the inhibition of A549 viability than what was predicted by their individual activities.
  • Fig. 48C shows ZF9-MQ1 (1.0 ⁇ g/ml) and JQ1 (concentrations up to 6.25uM) showed a greater than additive effect on the inhibition of A549 viability than what was predicted by their individual activities.
  • Fig. 49A shows ZF9-MQ1 and BET762 each separately inhibited cell viability of A549 cells.
  • Fig. 49B shows ZF9-MQ1 (0.5 ⁇ g/ml) and BET762 (concentrations up to 1.25uM) showed a greater than additive effect on the inhibition of A549 viability than what was predicted by their individual activities.
  • Fig. 49C shows ZF9-MQ1 (1.0 ⁇ g/ml) and BET762 (concentrations up to 0.625uM) showed a greater than additive effect on the inhibition of A549 viability than what was predicted by their individual activities.
  • Fig. 50A shows ZF9-MQ1 and Birabresib each separately inhibited cell viability of A549 cells.
  • Fig. 50B shows ZF9-MQ1 (0.5 ⁇ g/ml) and Birabresib (concentrations up to 0.625uM) showed a greater than additive effect on the inhibition of A549 viability than what was predicted by their individual activities.
  • Fig. 50C shows ZF9-MQ1 (1.0 ⁇ g/ml) and Birabresib (concentrations up to 0.313uM) showed a greater than additive effect on the inhibition of A549 viability than what was predicted by their individual activities.
  • Fig. 51A shows ZF9-MQ1 and Trametinib each separately inhibited cell viability of A549 cells.
  • Fig. 5 IB shows ZF9-MQ1 (0.5 ⁇ g/ml) and Trametinib (concentrations up to 0.05 uM) showed a greater than additive effect on the inhibition of A549 viability than what was predicted by their individual activities.
  • Fig. 51C shows ZF9-MQ1 (1.0 ⁇ g/ml) and Trametinib (concentrations up to 0.05 uM) showed a greater than additive effect on the inhibition of A549 viability than what was predicted by their individual activities.
  • Fig. 52A shows all the constructs ZF9-MQ1, ZF54-KRAB, ZF67-KRAB, and ZF68-KRAB were able to downregulate MYC mRNA levels in H2009 cells by at least 42% at 72 hours post-treatment compared to untreated cells.
  • Fig. 52B shows the constructs ZF9-MQ1, ZF67-KRAB, and ZF68-KRAB were able to downregulate MYC mRNA levels in H226 cells by at least 27% at 72 hours post-treatment compared to untreated cells.
  • Fig. 52C shows both the constructs ZF9-MQ1 and ZF54-KRAB were able to downregulate MYC mRNA levels in H226 cells by at least 27% at 72 hours post-treatment compared to untreated cells.
  • Fig. 52D shows the constructs ZF9-MQ1, ZF61-KRAB, ZF67-KRAB, and ZF68-KRAB were able to downregulate MYC mRNA levels in H460 cells by at least 26% at 72 hours post-treatment compared to untreated cells.
  • Fig. 53 shows at the highest concentration tested, ZF9-MQ1 and ZF54-KRAB each separately downregulated MYC mRNA in H2009 cells by 99% or 62% respectively, relative to untreated control cells.
  • ZF9-MQ1 and ZF54-KRAB each separately downregulated MYC mRNA in H2009 cells by 99% or 62% respectively, relative to untreated control cells.
  • MYC mRNA is downregulated to a greater extent than that observed for either treatment alone.
  • Fig. 54 shows ZF9-MQ1 downregulated MYC mRNA in H1299 cells by 95% relative to untreated control cells by 48 hours and maintained downregulation at 90% of control levels at 144 hours. Combination of ZF9-MQ1 plus ZF54-KRAB reduced MYC mRNA levels to 98% at 48 hours and maintained downregulation to 93% of control levels at 144 hours (Fig. 54). Further, the data showed ZF9- MQ1 and ZF9-MQ1 combined with ZF54-KRAB downregulated MYC mRNA levels in H1299 cells for at least 6 days.
  • Fig. 55 shows 24 hours after introduction to H2009 cells, ZF9-MQ1 and ZF54-KRAB downregulated MYC mRNA levels by up to 83% and 55%, respectively, in comparison to untreated cells. MYC mRNA levels were further reduced by another 13% in ZF9-MQ1 -treated cells to 96% of untreated controls 48 hours after treatment, whereas ZF54-KRAB does not further downregulate MYC levels. MYC mRNA levels in cells treated with ZF9-MQ1 ZF54-KRAB and ZF54-KRAB ZF9-MQ1 were reduced to 95% and 96% of control cells, respectively, at 24 hours post-treatment.
  • Fig. 56 shows ZF9-MQ1 treatment inhibited tumor growth in the H460 subcutaneous tumor model at a similar or higher level compared to sorafenib induced tumor growth inhibition.
  • the present disclosure provides technologies for modulating, e.g., decreasing, expression of a target gene e.g., MYC in cell, e.g., in a subject or patient, through the use of an expression repressor or a system described herein.
  • a target gene e.g., MYC in cell, e.g., in a subject or patient
  • MYC a transcription factor and master cell regulator
  • MYC a transcription factor and master cell regulator
  • MYC typically upregulates gene expression.
  • MYC is the most frequently amplified oncogene, and the elevated expression of its gene product is associated with tumor aggression and poor clinical outcome. Elevated levels of c-MYC can promote tumorigenesis in a wide range of tissues. Most tumor cells depend on the transcription factor c-MYC for their growth and proliferation.
  • MYC overexpression is also associated in chronic liver disease e.g., viral and alcohol related liver disease. MYC overexpression level varies in specific cancer subtypes.
  • modulating e.g., decreasing the levels of MYC in a subject (e.g., overall, or in a specific target tissue or tissues) suffering from MYC mis-regulation disorder may lessen or eliminate the symptoms of the MYC mis-regulation disorder.
  • an expression repressor comprising a targeting moiety that binds to a target gene promoter, e.g., MYC promoter or operably linked to the target gene, e.g., MYC gene and an effector moiety capable of modulating (e.g., decreasing) expression of the target gene, e.g., MYC when localized by the targeting moiety.
  • a target gene promoter e.g., MYC promoter or operably linked to the target gene, e.g., MYC gene
  • an effector moiety capable of modulating (e.g., decreasing) expression of the target gene, e.g., MYC when localized by the targeting moiety.
  • the expression repressors disclosed herein specifically bind to an expression control element (e.g., a promoter or enhancer, repressor or silencer) operably linked to the target gene, e.g., MYC via the targeting moiety and the effector moiety modulates expression of the target gene, e.g., MYC.
  • the expression repressors disclosed herein specifically bind to an anchor sequence of an anchor sequence mediated conjunction (ASMC) comprising a target gene, e.g., MYC or to a sequence proximal to the anchor sequence via the targeting moiety and the effector moiety modulates expression of the target gene, e.g., MYC.
  • ASMC anchor sequence mediated conjunction
  • the expression repressors disclosed herein specifically bind to a genomic locus located in a super enhancer region of a target gene, e.g., MYC and the effector moiety modulates expression of the target gene, e.g., MYC.
  • an expression repression system comprising two or more expression repressors, each comprising a targeting moiety and optionally an effector moiety.
  • the targeting moieties target two or more different sequences (e.g., each expression repressor may target a different sequence).
  • the first expression repressor binds to a transcription regulatory element (e.g., a promoter or transcription start site (TSS)) operably linked to a target gene, e.g., MYC and the second expression repressor binds to an anchor sequence of an anchor sequence mediated conjunction (ASMC) comprising a target gene, e.g., MYC.
  • a transcription regulatory element e.g., a promoter or transcription start site (TSS)
  • TSS transcription start site
  • ASMC anchor sequence mediated conjunction
  • the first expression repressor binds to a transcription regulatory element (e.g., a promoter or transcription start site (TSS)) operably linked to a target gene, e.g., MYC and the second expression repressor binds to an expression control element (e.g., an enhancer, a super-enhancer, a repressor, or a silencer) operably linked to a target gene, e.g., MYC.
  • a transcription regulatory element e.g., a promoter or transcription start site (TSS)
  • TSS transcription start site
  • the first expression repressor binds to an anchor sequence of an anchor sequence mediated conjunction (ASMC) comprising a target gene, e.g., MYC and the second expression repressor binds to an expression control element (e.g., an enhancer, a superenhancer, a repressor, or a silencer) operably linked to a target gene.
  • ASMC anchor sequence mediated conjunction
  • an expression control element e.g., an enhancer, a superenhancer, a repressor, or a silencer
  • modulation of expression of a target gene, e.g., MYC by an expression repression system involves the binding of the first expression repressor and second expression repressor to the first and second DNA sequences, respectively. Binding of the first and second DNA sequences localizes the functionalities of the first and second effector moieties to those sites.
  • the expression repressor system is encoded by a bi-cistronic nucleic acid sequence.
  • the disclosure further provides nucleic acids encoding said expression repressors and/or expression repressor systems, compositions comprising expression repressors and/or expression repressor systems, and methods for delivering said nucleic acids. Further provided are methods for increasing target gene expression, e.g., MYC gene expression in a cell using the expression repressors or expression repressor systems described herein.
  • an expression repressor may comprise a targeting moiety that binds to a target gene promoter, e.g., MYC promoter and optionally an effector moiety.
  • the targeting moiety specifically binds a target DNA sequence, e.g., MYC DNA sequence, thereby localizing the expression repressor’s functionality to the DNA sequence.
  • an expression repressor comprises a targeting moiety and one effector moiety.
  • an expression repressor comprises a targeting moiety and a plurality of effector moieties (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or more effector domains (and optionally, less than 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, or 2 effector domains)).
  • effector moieties e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or more effector domains (and optionally, less than 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, or 2 effector domains
  • An expression repressor may comprise a plurality of effector moieties, where each effector moiety comprises a different functionality than the other effector moieties.
  • an expression repressor may comprise two effector moieties, where the first effector moiety comprises DNA methylase functionality and the second effector moiety comprises a transcriptional repressor functionality.
  • an expression repressor comprises effector moieties whose functionalities are complementary to one another with regard to decreasing expression of a target gene, e.g., MYC, where the functionalities together enable inhibition of expression and, optionally, do not inhibit or negligibly inhibit expression when present individually.
  • an expression repressor comprises a plurality of effector moieties, wherein each effector moiety complements each other effector moiety, each effector moiety decreases expression of a target gene, e.g., MYC.
  • an expression repressor comprises a combination of effector moieties whose functionalities synergize with one another with regard to decreasing expression of a target gene, e.g., MYC.
  • epigenetic modifications to a genomic locus are cumulative, in that multiple transcription activating epigenetic markers (e.g., multiple different types of epigenetic markers and/or more extensive marking of a given type) individually together inhibit expression more effectively than individual modifications alone (e.g., producing a greater decrease in expression and/or a longer-lasting decrease in expression).
  • an expression repressor comprises a plurality of effector moieties, wherein each effector moiety synergizes with each other effector moiety, e.g., each effector moiety decreases expression of a target gene, e.g., MYC.
  • an expression repressor (comprising a plurality of effector moieties which synergize with one another) is more effective at inhibiting expression of a target gene, e.g., MYC than an expression repressor comprising an individual effector moiety.
  • an expression repressor comprising said plurality of effector moieties is at least 1.05x (i.e., 1.05 times), 1.1x, 1.15x, 1.2x, 1.25x, 1.3x, 1.35x, 1.4x, 1.45x, 1.5x, 1.55x, 1.6x, 1.65x, 1.7x, 1.75x, 1.8x, 1.85x, 1.9x, 1.95x, 2x, 3x, 4x, 5x, 6x, 7x, 8x, 9x, 10x, 20x, 30x, 40x, 50x, 60x, 70x, 80x, 90x, or 100x as effective at decreasing expression of a target gene, e.g., MYC than an expression repressor comprising an individual effector moiety.
  • a target gene e.g., MYC than an expression repressor comprising an individual effector moiety.
  • an expression repressor comprises one or more targeting moieties e.g., a Cas domain, TAL effector domain, or Zn Finger domain.
  • the targeting moieties specifically bind two or more different sequences.
  • the two or more Cas 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 Cas domain).
  • an expression repressor comprises a targeting moiety and an effector moiety that are covalently linked, e.g., by a peptide bond.
  • the targeting moiety and the effector moiety are situated on the same polypeptide chain, e.g., connected by one or more peptide bonds and/or a linker.
  • the expression repressor is or comprises a fusion molecule, e.g., comprising the targeting moiety and the effector moiety linked by a peptide bond and/or a linker.
  • the expression repressor comprises a targeting moiety that is disposed N-terminal of an effector moiety on the same polypeptide chain.
  • the expression repressor comprises a targeting moiety that is disposed C-terminal of an effector moiety on the same polypeptide chain.
  • an expression repressor comprises a targeting moiety and an effector moiety that are covalently linked by a non-peptide bond.
  • a targeting moiety is conjugated to an effector moiety by a non-peptide bond.
  • an expression repressor comprises a targeting moiety and a plurality of effector moieties, wherein the targeting moiety and the plurality of effector moieties are covalently linked, e.g., by peptide bonds (e.g., the targeting moiety and plurality of effector moieties are all connected by a series of covalent bonds, although each individual moiety may not share a covalent bond with every other effector moiety).
  • an expression repressor comprises a targeting moiety and an effector moiety that are not covalently linked, e.g., that are non-covalently associated with one another.
  • an expression repressor comprises a targeting moiety that non-covalently binds to an effector moiety or vice versa.
  • an expression repressor comprises a targeting moiety and a plurality of effector moieties, wherein the targeting moiety and at least one effector moiety are not covalently linked, e.g., are non-covalently associated with one another, and wherein the targeting moiety and at least one other effector moiety are covalently linked, e.g., by a peptide bond.
  • an expression repressor as described herein binds (e.g., via a targeting moiety) a genomic sequence element proximal to and/or operably linked to a target gene, e.g., MYC.
  • binding of the expression repressor to the genomic sequence element modulates (e.g., decreases) expression of the target gene, e.g., MYC.
  • binding of an expression repressor comprising an effector moiety that recruits or inhibits recruitment of components of the transcription machinery to the genomic sequence element may modulate (e.g., decrease) expression of the target gene, e.g., MYC.
  • binding of an expression repressor comprising an effector moiety with an enzymatic activity may modulate (e.g., decrease) expression of the target gene, e.g., MYC) through the localized enzymatic activity of the effector moiety.
  • an expression repressor comprising an effector moiety with an enzymatic activity e.g., an epigenetic modifying moiety
  • both binding of an expression repressor to a genomic sequence element and the localized enzymatic activity of an expression repressor may contribute to the resulting modulation (e.g., decrease) in expression of the target gene, e.g., MYC.
  • an expression repressor comprises an effector moiety wherein the effector moiety comprises a protein chosen from MQ1, DNMT1, DNMT3A1, DNMT3A2, DNMT3B1, DNMT3B2, DNMT3B3, DNMT3B4, DNMT3B5, DNMT3B6, DNMT3L, HDAC1, HDAC2, HDAC3, HDAC4, HDAC5, HDAC6, HDAC7, HDAC8, HDAC9, HDAC10, HDAC11, SIRT1, SIRT2, SIRT3, SIRT4, SIRT5, SIRT6, SIRT7, SIRT8, SIRT9, KDM1A (i.euze LSD1), KDM1B (i.e., LSD2), KDM2A, KDM2B, KDM5A, KDM5B, KDM5C, KDM5D, KDM4B, NO66, SETDB1, SETDB2, EHMT2 (i.e., G9A), EHMT1 (i.e., G9A), E
  • an expression repressor comprises a first effector moiety and a second effector moiety , wherein the first effector moiety comprises a protein chosen from MQ1, DNMT1, DNMT3A1, DNMT3A2, DNMT3B1, DNMT3B2, DNMT3B3, DNMT3B4, DNMT3B5, DNMT3B6, DNMT3L, HDAC1, HDAC2, HDAC3, HDAC4, HDAC5, HDAC6, HDAC7, HDAC8, HDAC9, HDAC10, HDAC11, SIRT1, SIRT2, SIRT3, SIRT4, SIRT5, SIRT6, SIRT7, SIRT8, SIRT9, KDM1A (i.e., LSD1), KDM1B (i.e., LSD2), KDM2A, KDM2B, KDM5A, KDM5B, KDM5C, KDM5D, KDM4B, NO66, SETDB1, SETDB2, EHMT2 (i.e., GSD1), K
  • the disclosure provides nucleic acid sequences encoding an expression repressor, an expression repressor system, a targeting moiety and/or an effector moiety as described herein.
  • a skilled artisan is aware that the nucleic acid sequences of RNA are identical to the corresponding DNA sequences, except that typically thymine (T) is replaced by uracil (U).
  • nucleotide sequence when a nucleotide sequence is represented by a DNA sequence (e.g., comprising, A, T, G, C), this disclosure also provides the corresponding RNA sequence (e.g., comprising, A, U, G, C) in which “U” replaces “T.”
  • RNA sequence e.g., comprising, A, U, G, C
  • U replaces “T”
  • Conventional notation is used herein to describe polynucleotide sequences: the left-hand end of a single-stranded polynucleotide sequence is the 5 ’-end; the left-hand direction of a double-stranded polynucleotide sequence is referred to as the 5 '-direction.
  • nucleotide sequences encoding an expression repressor comprising targeting moiety and/or an effector moiety as described herein may be produced, some of which have similarity, e.g., 90%, 95%, 96%, 97%, 98%, or 99% identity to the nucleic acid sequences disclosed herein.
  • codons AGA, AGG, CGA, CGC, CGG, and CGU all encode the amino acid arginine.
  • nucleic acid cohesion encoding an expression repressor comprising a targeting moiety and/or an effector moiety may be part or all of a codon-optimized coding region, optimized according to codon usage in mammals, e.g., humans.
  • a nucleic acid cohesion encoding a targeting moiety and/or an effector moiety is codon optimized for increasing the protein expression and/or increasing the duration of protein expression.
  • a protein produced by the codon optimized nucleic acid sequence is at least 1%, at least 2%, at least 5%, at least 10%, at least 15%, at least 20%, at least 30%, at least 40%, or at least 50% higher compared to levels of the protein when encoded by a nucleic acid sequence that is not codon optimized.
  • Expression repression systems of the present disclosure may comprise two or more expression repressors.
  • an expression repression system comprises 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, or more expression repressors (and optionally no more than 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, or 2).
  • an expression repression system targets two or more different sequences (e.g., a 1 st and 2 nd , 3 rd , 4 th , 5 th , 6 th , 7 th , 8 th , 9 th , 10 th , 11 th , 12 th , and/or further DNA sequence, and optionally no more than a 20 th , 19 th , 18 th , 17 th , 16 th , 15 th , 14 th , 13 th , 12 th , 11 th , 10 th , 9 th , 8 th , 6 th , 5 th , 4 th , 3 rd , or 2 nd sequence).
  • sequences e.g., a 1 st and 2 nd , 3 rd , 4 th , 5 th , 6 th , 7 th , 8 th , 9 th , 10 th , 11
  • an expression repression system comprises a plurality of expression repressors, wherein each member of the plurality of expression repressors does not detectably bind, e.g., does not bind, to another member of the plurality of expression repressors.
  • an expression repression system comprises a first expression repressor and a second expression repressor, wherein the first expression repressor does not detectably bind, e.g., does not bind, to the second expression repressor.
  • an expression repression system of the present disclosure comprises two or more expression repressors, wherein the expression repressors are present together in a composition, pharmaceutical composition, or mixture. In some embodiments, an expression repression system of the present disclosure comprises two or more expression repressors, wherein one or more expression repressors is not admixed with at least one other expression repressor.
  • an expression repression system may comprise a first expression repressor and a second expression repressor, wherein the presence of the first expression repressor in the nucleus of a cell does not overlap with the presence of the second expression repressor in the nucleus of the same cell, wherein the expression repression system achieves a decrease in expression of a MYC gene via the non-overlapping presences of the first and second expression repressors.
  • the expression repression system achieves a greater decrease in expression of a MYC gene in comparison to the decrease in expression of a MYC gene achieved by the first or the second expression repressor alone.
  • the expression repressors of an expression repressor system each comprise a different targeting moiety (e.g., the first, second, third, or further expression repressors each comprise different targeting moieties from one another).
  • an expression repression system may comprise a first expression repressor and a second expression repressor wherein the first expression repressor comprises a first targeting moiety (e.g., a Cas9 domain, TAL effector domain, or Zn Finger domain), and the second expression repressor comprises a second targeting moiety (e.g., a Cas9 domain, TAL effector domain, or Zn Finger domain) different from the first targeting moiety.
  • different can mean 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.
  • different can mean comprising distinct variants of the same type of targeting moiety, e.g., 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).
  • an expression repressor system comprises two or more targeting moieties of the same type, e.g., two or more Cas9 or ZF domains
  • the targeting moieties specifically bind two or more different sequences.
  • 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 repressor system comprises three or more expression repressors and two or more expression repressors comprise the same targeting moiety.
  • an expression repressor system may comprise three expression repressors, wherein the first and second expression repressors both comprise a first targeting moiety and the third expression repressor comprises a second different targeting moiety.
  • an expression repressor system may comprise four expression repressors, wherein the first and second expression repressors both comprise a first targeting moiety and the third and fourth expression repressors comprises a second different targeting moiety.
  • an expression repressor system may comprise five expression repressors, wherein the first and second expression repressors both comprise a first targeting moiety, the third and fourth expression repressors both comprise a second different targeting moiety, and the fifth expression repressor comprises a third different targeting moiety.
  • different can mean comprising different types of -targeting moieties or comprising distinct variants of the same type of targeting moiety.
  • the expression repressors of an expression repressor system each bind to a different DNA sequence (e.g., the first, second, third, or further expression repressors each bind DNA sequences that are different from one another).
  • an expression repression system may comprise a first expression repressor and a second expression repressor wherein the first expression repressor binds to a first DNA sequence, and the second expression repressor binds to a second DNA sequence.
  • different can mean that: there is at least one position that is not identical between the DNA sequence bound by one expression repressor and the DNA sequence bound by another expression repressor, or that there is at least one position present in the DNA sequence bound by one expression repressor that is not present in the DNA sequence bound by another expression repressor.
  • the first DNA sequence may be situated on a first genomic DNA strand and the second DNA sequence may be situated on a second genomic DNA strand. In some embodiments, the first DNA sequence may be situated on the same genomic DNA strand as the second DNA sequence.
  • an expression repressor system comprises three or more expression repressors and two or more expression repressors bind the same DNA sequence.
  • an expression repressor system may comprise three expression repressors, wherein the first and second expression repressors both bind a first DNA sequence, and the third expression repressor binds a second different DNA sequence.
  • an expression repressor system may comprise four expression repressors, wherein the first and second expression repressors both bind a first DNA sequence and the third and fourth expression repressors both bind a second DNA sequence.
  • an expression repressor system may comprise five expression repressors, wherein the first and second expression repressors both bind a first DNA sequence, the third and fourth expression repressors both bind a second DNA sequence, and the fifth expression repressor binds a third DNA sequence.
  • different can mean that there is at least one position that is not identical between the DNA sequence bound by one expression repressor and the DNA sequence bound by another expression repressor, or that there is at least one position present in the DNA sequence bound by one expression repressor that is not present in the DNA sequence bound by another expression repressor.
  • an expression repression system comprises two or more (e.g., two) expression repressors and a plurality (e.g., two) of the expression repressors comprise targeting moieties that bind to different DNA sequences.
  • a first targeting moiety may bind to a first DNA sequence and a second DNA-targeting moiety may bind to a second DNA sequence, wherein the first and the second DNA sequences are different and do not overlap.
  • the first DNA sequence is separated from the second DNA sequence by at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or 100 base pairs (and optionally, no more than 500, 400, 300, 200, 100, 95, 90, 85, 80, 75, 70, 65, 60, 55, or 50 base pairs).
  • the first DNA sequence is separated from the second DNA sequence by no more than 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or 100 base pairs (and optionally, no base pairs, e.g., the first and second sequence are directly adjacent one another).
  • the expression repressors of an expression repressor system each comprise a different effector moiety (e.g., the first, second, third, or further expression repressors each comprise a different effector moiety from one another).
  • an expression repression system may comprise a first expression repressor and a second expression repressor wherein the first expression repressor comprises a first effector moiety (e.g., comprising a DNA methyltransferase or functional fragment thereof), and the second expression repressor comprises a second effector moiety (e.g., comprising a transcription repressor (e.g., KRAB) or functional fragment thereof) different from the first effector moiety.
  • a transcription repressor e.g., KRAB
  • different can mean comprising distinct types of effector moiety.
  • different can mean comprising distinct variants of the same type of effector moiety, e.g., the first effector moiety comprises a first DNA methyltransferase (e.g., having a first site specificity or amino acid sequence) and the second effector moiety comprises a second DNA methyltransferase (e.g., having a second site specificity or amino acid sequence).
  • an expression repressor system comprises a first expression repressor comprising a first effector moiety and a second expression repressor comprising a second effector moiety, wherein the first effector moiety comprises a protein chosen from MQ1, DNMT1, DNMT3A1, DNMT3A2, DNMT3B1, DNMT3B2, DNMT3B3, DNMT3B4, DNMT3B5, DNMT3B6, DNMT3L, HDAC1, HDAC2, HDAC3, HDAC4, HDAC5, HDAC6, HDAC7, HDAC8, HDAC9, HDAC10, HDAC11, SIRT1, SIRT2, SIRT3, SIRT4, SIRT5, SIRT6, SIRT7, SIRT8, SIRT9, KDM1A (i.e., LSD1), KDM1B (i.e., LSD2), KDM2A, KDM2B, KDM5A, KDM5B, KDM5C, KDM5D, KDM4B, NO66
  • the first or second effector moiety comprises a DNA methyltransferase activity (e.g., MQ1, DNMT1, DNMT3A1, DNMT3A2, DNMT3B1, DNMT3B2, DNMT3B3, DNMT3B4, DNMT3B5, DNMT3B6, DNMT3L , or a functional variant or fragment of any thereof
  • the other effector moiety comprises a transcription repressor activity (e.g., KRAB, MeCP2, HP1, RBBP4, REST, FOG1, SUZ12, or a functional variant or fragment of any thereof)
  • the first or second effector moiety comprises a histone methyltransferase activity
  • the other effector moiety comprises a histone deacetylase activity (e.g., HDAC1, HDAC2, HDAC3, HDAC4, HDAC5, HDAC6, HDAC7, HDAC8, HDAC9, HDAC10, HDAC11, SIRT1, SIRT2, SIRT3, SIRT4,
  • the first or second effector moiety comprises a histone methyltransferase activity and the other effector moiety comprises a DNA methyltransferase activity (e.g., MQ1, DNMT1, DNMT3A1, DNMT3A2, DNMT3B1, DNMT3B2, DNMT3B3, DNMT3B4, DNMT3B5, DNMT3B6, DNMT3L, or a functional variant or fragment of any thereof).
  • the first or second effector moiety comprises a DNA methyltransferase activity and the other effector moiety comprises a transcription repressor activity.
  • the first or second effector moiety comprises a histone methyltransferase activity and the other effector moiety comprises a transcription repressor activity (e.g., KRAB, MeCP2, HP1, RBBP4, REST, FOG1, SUZ12, or a functional variant or fragment of any thereof).
  • the first or second effector moiety comprises a transcription repressor activity and the other effector moiety comprises a different transcription repressor activity.
  • the first or second effector moiety comprises a DNA methyltransferase activity and the other effector moiety comprises the same DNA methyltransferase activity.
  • the first or second effector moiety comprises a DNA methyltransferase activity and the other effector moiety comprises a histone deacetylase activity. In some embodiments, the first or second effector moiety comprises a histone demethylase activity and the other effector moiety comprises a DNA methyltransferase activity. In some embodiments, the first or second effector moiety comprises a histone methyltransferase activity and the other effector moiety comprises a DNA demethylase activity. In some embodiments, the first or second effector moiety comprises a histone demethylase activity and the other effector moiety comprises a transcription repressor activity.
  • the first or second effector moiety comprises a histone demethylase activity and the other effector moiety comprises a different histone demethylase activity. In some embodiments, the first or second effector moiety comprises a histone demethylase activity and the other effector moiety comprises the same histone demethylase activity. In some embodiments, the first or second effector moiety comprises a histone deacetylase activity and the other effector moiety comprises a DNA methyltransferase activity. In some embodiments, the first or second effector moiety comprises a histone deacetylase activity and the other effector moiety comprises a DNA demethylase activity.
  • the first or second effector moiety comprises a histone deacetylase activity and the other effector moiety comprises a transcription repressor activity. In some embodiments, the first or second effector moiety comprises a histone deacetylase activity and the other effector moiety comprises a different histone deacetylase activity. In some embodiments, the first or second effector moiety comprises a histone deacetylase activity and the other effector moiety comprises the same histone deacetylase activity. In some embodiments, the first or second effector moiety comprises a DNA methyltransferase activity and the other effector moiety comprises a DNA demethylase activity.
  • the first or second effector moiety comprises a DNA demethylase activity and the other effector moiety comprises a transcription repressor activity. In some embodiments, the first or second effector moiety comprises a DNA methyltransferase activity and the other effector moiety comprises a different DNA methyltransferase activity. In some embodiments, the first or second effector moiety comprises a DNA methyltransferase activity and the other effector moiety comprises the same DNA methyltransferase activity. In some embodiments, the first or second effector moiety comprises a DNA demethylase activity and the other effector moiety comprises a transcription repressor activity.
  • the first or second effector moiety comprises a DNA demethylase activity and the other effector moiety comprises a different DNA demethylase activity. In some embodiments, the first or second effector moiety comprises a DNA demethylase activity and the other effector moiety comprises the same DNA demethylase activity. In some embodiments, the first or second effector moiety comprises a transcription repressor activity and the other effector moiety comprises a different transcription repressor activity. In some embodiments, the first or second effector moiety comprises a transcription repressor activity and the other effector moiety comprises the same transcription repressor activity.
  • an expression repressor system comprises three or more expression repressors and two or more expression repressors comprise the same DNA-targeting moiety.
  • an expression repressor system may comprise three expression repressors, wherein the first and second expression repressors both comprise a first effector moiety and the third expression repressor comprises a second different effector moiety.
  • an expression repressor system may comprise four expression repressors, wherein the first and second expression repressors both comprise a first effector moiety and the third and fourth expression repressors comprises a second different effector moiety.
  • an expression repressor system may comprise five expression repressors, wherein the first and second expression repressors both comprise a first effector moiety, the third and fourth expression repressors both comprise a second different effector moiety, and the fifth expression repressor 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 repressors of an expression repressor system are not covalently associated with each other, e.g., each expression repressor is not covalently associated with any other expression repressor.
  • two or more expression repressors of an expression repressor system are covalently associated with one another.
  • an expression repression system comprises a first expression repressor and a second expression repressor 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 repression system comprises a first expression repressor and a second expression repressor that are connected by a non-peptide bond, e.g., are conjugated to one another.
  • An expression repressor or an expression repressor 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 cleavable to selectively release a moiety (e.g., polypeptide) from a modulating agent, but sufficiently stable to prevent premature cleavage.
  • an expression repression may comprise a linker situated between the targeting moiety and the effector moiety.
  • the linker may have a sequence of ASGSGGGSGGARD (SEQ ID NO: 137), or ASGSGGGSGG (SEQ ID NO: 138).
  • a system comprising a first and second repressor 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: 137 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: 138 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: 207).
  • 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 thrombinsensitive sequence (e.g., PRS) between the two Cys residues.
  • PRS thrombinsensitive sequence
  • In vitro thrombin treatment of CPRSC results in the cleavage of a thrombin-sensitive 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.
  • In vivo cleavage of linkers in fusions may also be carried out by proteases that are expressed in vivo under certain conditions, in specific cells or tissues, or constrained within certain cellular compartments.
  • the 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 repressor.
  • 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 repressor may be linked using charge-based chemistry, such that a positively charged component of an expression repressor is linked to a negative charge of another component.
  • the present disclosure provides, e.g., expression repressors comprising a 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.
  • Targeting moieties may specifically bind a DNA sequence, e.g., a DNA sequence associated with a target gene, e.g., MYC. Any molecule or compound that specifically binds a DNA sequence may be used as a targeting moiety.
  • a targeting moiety targets, e.g., binds, a component of a genomic complex (e.g., ASMC).
  • a targeting moiety targets, e.g., binds, an expression control sequence (e.g., a promoter or enhancer) operably linked to a target gene.
  • a targeting moiety targets, e.g., binds, a target gene or a part of a target gene.
  • the target of a 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.
  • a targeting moiety binds specifically to one or more target anchor sequences (e.g., within a cell) and not to non-targeted anchor sequences (e.g., within the same cell).
  • a targeting moiety may be or comprise a CRISPR/Cas domain, a TAL effector domain, a Zn finger domain, peptide nucleic acid (PNA) or a nucleic acid molecule.
  • an expression repressor comprises one effector moiety.
  • an expression repressor comprises a plurality of targeting moieties, wherein each targeting moiety does not detectably bind, e.g., does not bind, to another targeting moiety.
  • an expression repression system comprises a plurality of expression repressors, wherein each member of the plurality of expression repressors comprises a targeting moiety, wherein each targeting moiety does not detectably bind, e.g., does not bind, to another targeting moiety.
  • an expression repression system comprises a first expression repressor comprising a first targeting moiety and a second expression repressor comprising a second targeting moiety, wherein the first targeting moiety does not detectably bind, e.g., does not bind, to the second targeting moiety.
  • an expression repression system comprises a first expression repressor comprising a first targeting moiety and a second expression repressor comprising a second targeting moiety, wherein the first targeting moiety does not detectably bind, e.g., does not bind, to another first targeting moiety, and the second targeting moiety does not detectably bind, e.g., does not bind, to another second targeting moiety.
  • a targeting moiety for use in the compositions and methods described herein is functional (e.g., binds to a DNA sequence) in a monomeric, e.g., non-dimeric, state.
  • binding of a targeting moiety to a targeted component decreases binding affinity of the targeted component for another transcription factor, genomic complex component, or genomic sequence element.
  • a targeting moiety binds to its target sequence with a K D 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 K D 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, 0.03, 0.02, 0.01, 0.005, 0.005, 0.002, or
  • a targeting moiety binds to its target sequence with a K D of 0.001 nM to 500 nM, e.g., 0.1 nM to 5 nM, e.g., about 0.5 nM. In some embodiments, a targeting moiety binds to a non-target sequence with a K D 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). In some embodiments, a targeting moiety does not bind to a non-target sequence.
  • a targeting moiety comprises a nucleic acid sequence complementary to a targeted component, e.g., a regulatory element (e.g., promoter or enhancer) of a target gene, e.g., MYC.
  • a targeting moiety comprises a nucleic acid sequence that is at least 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100% complementary to a targeted component.
  • a targeting moiety may be or comprise a CRISPR/Cas domain, a TAL effector domain, a Zn finger domain, or a nucleic acid molecule.
  • the targeting moiety of an expression repressor comprises no more than 100, 90, 80, 70, 60, 50, 40, 30, or 20 nucleotides (and optionally at least 10, 20, 30, 40, 50, 60, 70, 80, or 90 nucleotides).
  • an expression repressor or the effector moiety of a fusion molecule comprises no more than 2000, 1900, 1800, 1700, 1600, 1500, 1400, 1300, 1200, 1100, 1000, 900, 800, 700, 600, 500, 400, 300, 200, or 100 amino acids (and optionally at least 50, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 1100, 1200, 1300, 1400, 1500, 1600, 1700, 1800, or 1900 amino acids).
  • an expression repressor or the effector moiety of a fusion molecule comprises 100- 2000, 100-1900, 100-1800, 100-1700, 100-1600, 100-1500, 100-1400, 100-1300, 100-1200, 100-1100, 100-1000, 100-900, 100-800, 100-700, 100-600, 100-500, 100-400, 100-300, 100-200, 200-2000, 200- 1900, 200-1800, 200-1700, 200-1600, 200-1500, 200-1400, 200-1300, 200-1200, 200-1100, 200-1000, 200-900, 200-800, 200-700, 200-600, 200-500, 200-400, 200-300, 300-2000, 300-1900, 300-1800, 300- 1700, 300-1600, 300-1500, 300-1400, 300-1300, 300-1200, 300-1100, 300-1000, 300-900, 300-800, 300- 700, 300-600, 300-500, 200-400, 200-300, 300-2000, 300-1900, 300-1800, 300
  • nucleic acid may comprise nucleic acid, e.g., one or more nucleic acids.
  • nucleic acid refers to any compound 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 or comprises more than 50% ribonucleotides and is referred to herein as a ribonucleic acid (RNA).
  • a nucleic acid is, comprises, or consists of one or more natural nucleic acid residues. In some embodiments, a nucleic acid is, comprises, or consists of one or more nucleic acid analogs. In some embodiments, a nucleic acid analog differs from a nucleic acid in that it does not utilize a phosphodiester backbone. For example, in some embodiments, 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 invention.
  • 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).
  • a nucleic acid is, comprises, or consists of one or more nucleoside analogs (e.g., 2-aminoadenosine, 2-thiothymidine, inosine, pyrrolo-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)- methylguanine, 2-thiocytidine, methylated bases, intercalated
  • 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.
  • recombinant when used to describe a nucleic acid refers to any nucleic acid that does not naturally occur.
  • 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.
  • nucleic acids may have 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 nucleic acid is partly or wholly single stranded; in some embodiments, a nucleic acid is partly or wholly double stranded. In some embodiments a 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.
  • the targeting moiety comprises or is a nucleic acid sequence, a protein, protein fusion, or a membrane translocating polypeptide.
  • the targeting moiety is selected from an exogenous conjunction nucleating molecule, a nucleic acid encoding the conjunction nucleating molecule, or a fusion of a sequence targeting polypeptide and a conjunction nucleating molecule.
  • the conjunction nucleating molecule may be, e.g., CTCF, cohesin, USF1, YY1, TATA-box binding protein associated factor 3 (TAF3), ZNF143 binding motif.
  • a targeting moiety comprises or is a polymer or polymeric moiety, e.g., a polymer of nucleotides (such as an oligonucleotide), a peptide nucleic acid, a peptide-nucleic acid mixmer, a peptide or polypeptide, a polyamide, a carbohydrate, etc.
  • a targeting moiety comprises or is nucleic acid.
  • an effector moiety comprises or is nucleic acid.
  • a nucleic acid that may be included in a moiety may be or comprise DNA, RNA, and/or an artificial or synthetic nucleic acid or nucleic acid analog or mimic.
  • a nucleic acid may be or include one or more of genomic DNA (gDNA), complementary DNA (cDNA), a peptide nucleic acid (PNA), a peptide-nucleic acid mixmer, a peptide- oligonucleotide conjugate, a locked nucleic acid (LNA), a bridged nucleic acid (BNA), a polyamide, a triplex- forming oligonucleotide, an antisense oligonucleotide, tRNA, mRNA, rRNA, miRNA, gRNA, siRNA or other RNAi molecule (e.g., that targets a non-coding RNA as described herein and/or that targets an expression product of a particular gene associated with a targeted genomic complex as described herein), etc.
  • gDNA genomic DNA
  • cDNA complementary DNA
  • PNA peptide nucleic acid
  • LNA locked nucleic acid
  • BNA bridged nucleic acid
  • a polyamide a triple
  • a nucleic acid sequence suitable for use in a modulating agent may include modified oligonucleotides (e.g., chemical modifications, such as modifications that alter backbone linkages, sugar molecules, and/or nucleic acid bases) and/or artificial nucleic acids.
  • modified oligonucleotides e.g., chemical modifications, such as modifications that alter backbone linkages, sugar molecules, and/or nucleic acid bases
  • artificial nucleic acids e.g., chemical modifications, such as modifications that alter backbone linkages, sugar molecules, and/or nucleic acid bases
  • a nucleic acid sequence includes, but is not limited to, 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
  • a nucleic acid may include one or more residues that is not a naturally-occurring DNA or RNA residue, may include one or more linkages that is/are not phosphodiester bonds (e.g., that may be, for example, phosphorothioate bonds, etc.), and/or may include one or more modifications such as, for example, a 2’0 modification such as 2’-OmeP.
  • linkages e.g., that may be, for example, phosphorothioate bonds, etc.
  • modifications such as, for example, a 2’0 modification such as 2’-OmeP.
  • a variety of nucleic acid structures useful in preparing synthetic nucleic acids is known in the art (see, for example, WO20 17/0628621 and W02014/012081) those skilled in the art will appreciate that these may be utilized in accordance with the present disclosure.
  • nucleic acids include, but are not limited to, a nucleic acid that hybridizes to an target gene, e.g., MYC, (e.g., gRNA or antisense ssDNA as described herein elsewhere), a nucleic acid that hybridizes to an exogenous nucleic acid such as a viral DNA or RNA, nucleic acid that hybridizes to an RNA, a nucleic acid that interferes with gene transcription, a nucleic acid that interferes with RNA translation, a nucleic acid that stabilizes RNA or destabilizes RNA such as through targeting for degradation, a nucleic acid that interferes with a DNA or RNA binding factor through interference of its expression or its function, a nucleic acid that is linked to a intracellular protein or protein complex and modulates its function, etc.
  • MYC e.g., gRNA or antisense ssDNA as described herein elsewhere
  • nucleic acid that hybridizes to an exogenous nucleic acid such
  • an expression repressor comprises one or more nucleoside analogs.
  • a nucleic acid sequence may include in addition or as an alternative to one or more natural nucleosides nucleosides, e.g., purines or pyrimidines, e.g., adenine, cytosine, guanine, thymine and uracil, one or more nucleoside analogs.
  • a nucleic acid sequence includes one or more nucleoside analogs.
  • a nucleoside analog may include, 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-dimethylguanine, 2-methyladenine, 2-methylguanine, 3 -methylcytosine, 5- methylcytosine, N6-adenine, 7-methyIguanine, 5-methylaminomethyluracil, 5-methoxyaminomethyl-2- thiour
  • a targeting moiety is or 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. g., a site in the genome that is to be sequence-edited) by sequencespecific, non-coding “guide RNAs” that target single- or double-stranded DNA sequences.
  • target nucleotide sequence e. g., a site in the genome that is to be sequence-edited
  • guide RNAs target single- or double-stranded DNA sequences.
  • Three classes (I-III) of CRISPR systems have been identified.
  • the class II 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. See, e.g., Zetsche et al. (2015) Cell, 163:759 — 771.
  • Cas proteins A variety of CRISPR associated (Cas) genes or proteins can be used in the technologies provided by the present disclosure and the choice of Cas protein will depend upon the particular conditions of the method.
  • Specific examples of Cas proteins include class II systems including Casl, Cas2, Cas3, Cas4, Cas5, Cas6, Casl, Cas8, Cas9, Cas10, Cpfl, C2C1, or C2C3.
  • a Cas protein e.g., a Cas9 protein
  • a particular Cas protein e.g., a particular Cas9 protein, is selected to recognize a particular protospacer-adjacent motif (PAM) sequence.
  • PAM protospacer-adjacent motif
  • 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., a S. pyogenes, or a S. thermophilus), a Francisella (e.g., an F.
  • novicida a Staphylococcus (e.g., an S. aureus), an Acidaminococcus (e.g., an Acidaminococcus sp. BV3L6), a Neisseria (e.g., an N. meningitidis), a Cryptococcus, a Corynebacterium, a Haemophilus, a Eubacterium, a Pasteurella, a Prevotella, a Veillonella, or a Marinobacter.
  • Staphylococcus e.g., an S. aureus
  • an Acidaminococcus e.g., an Acidaminococcus sp. BV3L6
  • Neisseria e.g., an N. meningitidis
  • Cryptococcus e.g., a Corynebacterium, a Haemophilus, a Eubacterium, a Pasteurella, a Prevotella, a Veillon
  • 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. In some embodiments, a Cas protein comprises S542R, K548V, and N552R mutations or analogous substitutions to the amino acids corresponding to said positions.
  • the Cas protein is modified to deactivate the nuclease, e.g., nuclease- deficient Cas.
  • the Cas protein is a Cas9 protein.
  • wild-type Cas9 generates double-strand breaks (DSBs) at specific DNA sequences targeted by a gRNA
  • DSBs double-strand breaks
  • 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.
  • 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
  • a catalytically inactive Cas9 protein comprises a D11A 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 N995A mutation or an analogous substitution to the amino acid corresponding to said position.
  • a catalytically inactive Cas9 protein e.g., dCas9, comprises DI 1 A, H969A, and N995A 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 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, e.g., dCas9 comprises D10A and H557A 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 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, e.g., dCas9 comprises a N863A mutation or an analogous substitution to the amino acid corresponding to said position.
  • 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, comprises a E993A 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 D917A 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 D1255A mutation or an analogous substitution to the amino acid corresponding to said position.
  • a catalytically inactive Cas9 protein e.g., dCas9, comprises D917A, E1006A, and D1255A 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 comprises a D16A 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 H588A 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
  • the disclosure is directed to an expression repressor or a polypeptide comprising one or more (e.g., one) targeting moiety and one or more effector moiety, wherein the one or more 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: 17:
  • the dCas9 is encoded by a nucleic acid sequence of SEQ ID NO: 50:
  • a targeting moiety may comprise a Cas domain comprising or linked (e.g., covalently) to a gRNA.
  • a gRNA is a short synthetic RNA composed of a “scaffold” sequence necessary for Cas-protein binding and a user-defined ⁇ 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.
  • sgRNA single guide RNA
  • sgRNA single guide RNA
  • tracrRNA for binding the nuclease
  • crRNA to guide the nuclease to the sequence targeted for editing
  • Chemically modified sgRNAs have also been demonstrated to be effective for use with Cas proteins; see, for example, Hendel et al. (2015) Nature BiotechnoL, 985 - 991.
  • the exemplary guide RNA sequences are disclosed in Table 2 and Table 13.
  • a gRNA comprises a nucleic acid sequence that is complementary to a DNA sequence associated with a target gene.
  • the DNA sequence is, comprises, or overlaps an expression control element that is operably linked to the target gene.
  • a gRNA comprises a nucleic acid sequence that is at least 90, 95, 99, or 100% complementary to a DNA sequence associated with a target gene.
  • a gRNA for use with a DNA-targeting moiety that comprises a Cas molecule is an sgRNA.
  • a gRNA for use with a CRISPR/Cas domain specifically binds a target sequence associated with CTCF. In some embodiments, a gRNA for use with a CRISPR/Cas domain specifically binds a target sequence associated with the promoter. In some embodiments, the gRNA binds a target sequence listed in Table 2 or Table 13. In some embodiments, an expression repressor described herein binds to a target sequence listed in Table 2 or Table 13. Table 2: Exemplary gRNA sequences
  • an expression repression system comprises a first expression repressor comprising a first DNA-targeting moiety and a second expression repressor comprising a second DNA- targeting moiety, wherein the first DNA-targeting moiety comprises or is a first CRISPR/Cas domain and the second DNA-targeting moiety comprises or is a second CRISPR/Cas domain.
  • the first CRISPR/Cas domain comprises a first CRISPR/Cas protein and first guide RNA
  • the second CRISPR/Cas domain comprises a second CRISPR/Cas protein and a second guide RNA.
  • the first CRISPR/Cas protein does not appreciably bind (e.g., does not bind) the second guide RNA, e.g., binds with a K D of at least 10, 20, 50, 100, 1000, or 10,000 nM
  • the second CRISPR/Cas protein does not appreciably bind (e.g., does not bind) the first guide RNA, e.g., binds with a K D of at least 10, 20, 50, 100, 1000, or 10,000 nM.
  • a DNA-targeting moiety is or comprises a TAL effector 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 repeatvariable di-residues, RVD domain).
  • 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).
  • RVDs and Nucleic Acid Base Specificity Accordingly, it is possible to modify the repeats of a TAL effector to target specific DNA sequences. Further studies have shown that the RVD NK can target G. Target sites of TAL effectors also tend to include a T flanking the 5' base targeted by the first repeat, but the exact mechanism of this recognition is not known. More than 113 TAL effector sequences are known to date. Non-limiting examples of 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. raphani strain strain 756C and Xanthomonas oryzae pv. Oryzicolastrain BLS256 (Bogdanove et al. 2011).
  • Xanthomonas species such as the African strain of Xanthomonas oryzae pv. Oryzae (Yu et al. 2011), Xanthomonas campestris pv. raphani strain strain 756C and Xanthomonas oryzae pv. Oryzicolastrain BLS256 (Bogdanove et al. 2011).
  • 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. 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 (e.g., monomers or modules) and their specific sequence 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. In an embodiment, TAL effector domain of the present disclosure comprises between 8 and 33.5 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 repression system, e.g., the expression repressor comprising the TAL effector domain.
  • TALE binding is inversely correlated with the number of mismatches.
  • the TAL effector domain of a expression repressor 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 smaller the number of TAL effector repeats in the TAL effector domain the smaller the number of mismatches will be tolerated and still allow for the function of the expression repression system, e.g., the expression repressor comprising the TAL effector domain.
  • the binding affinity is thought to depend on the sum of matching repeat-DNA combinations. For example, 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.
  • transcriptional activity is inversely correlated with the length of N-terminus.
  • C-terminus an important element for DNA binding residues within the first 68 amino acids of the Hax 3 sequence was identified. Accordingly, in some embodiments, the first 68 amino acids on the C-terminal side of the TAL effector repeats of the naturally occurring TAL effector is included in the TAL effector domain of an expression repressor of the present disclosure.
  • 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 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 C-terminal side of the TAL effector repeats.
  • a modulating agent comprises a 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., MYC), 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., MYC), e.g., a sequence proximal to the anchor sequence.
  • the TAL effector domain can be engineered to carry epigenetic effector moieties to target sites.
  • 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. Generally, 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. Nos.
  • 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 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. Nos. 5,789,538; 5,925,523; 6,007,988; 6,013,453; 6,410,248; 6,140,466; 6,200,759; and 6,242,568; as well as International Patent Publication Nos. WO 98/37186; WO 98/53057; WO 00/27878; and WO 01/88197 and GB 2,338,237.
  • enhancement of binding specificity for zinc finger proteins has been described, for example, in International Patent Publication No. WO 02/077227.
  • zinc fingers and/or multi-fmgered zinc finger domains may be linked together using any suitable linker sequences, including for example, linkers of 5 or more amino acids in length. See, also, U.S. Pat. Nos. 6,479,626; 6,903,185; and 7,153,949 for exemplary linker sequences 6 or more amino acids in length.
  • the proteins described herein may include any combination of suitable linkers between the individual zinc fingers of the protein.
  • enhancement of binding specificity for zinc finger binding domains has been described, for example, in co-owned International Patent Publication No. WO 02/077227.
  • 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 fingers recognizes a target DNA sequence comprising 9 or 10 nucleotides. In some embodiments, a Zn finger domain comprising four Zn fingers recognizes a target DNA sequence comprising 12 to 14 nucleotides. In some embodiments, a Zn finger domain comprising six Zn fingers recognizes a target DNA sequence comprising 18 to 21 nucleotides.
  • a 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.
  • 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 repressor comprises a 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., MYC), 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., MYC), e.g., a sequence proximal to the anchor sequence.
  • DBD engineered DNA binding domain
  • ZFN Zn finger domain comprising a Zn finger
  • TSS transcription start site
  • the ZFN can be engineered to carry epigenetic effector molecules to target sites.
  • the targeting moiety comprises a Zn Finger domain that comprises 2, 3, 4, 5, 6, 7, or 8 zinc fingers.
  • the amino acid sequences of exemplary targeting moieties disclosed herein are listed in Table 4.
  • the nucleotide sequences encoding exemplary targeting moieties disclosed herein are listed in Table 5.
  • an expression repressor or system described herein comprises a targeting moiety having a sequence set forth in Table 4, or a sequence with at least 70%, 75%, 80%, 85%, 90%, 95%, 98%, or 99% identity thereto.
  • a nucleic acid described herein comprises a sequence set forth in Table 5, or a sequence with at least 70%, 75%, 80%, 85%, 90%, 95%, 98%, or 99% identity thereto.
  • an expression repression comprises a targeting moiety comprising an engineered
  • DNA binding domain 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., MYC), 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., MYC), e.g., a sequence proximal to the anchor sequence in mouse genome.
  • the ZFN can be engineered to carry epigenetic effector molecules to target sites.
  • the targeting moiety comprises a Zn Finger domain that comprises 2, 3, 4, 5, 6, 7, or 8 zinc fingers.
  • the amino acid sequences of exemplary targeting moieties disclosed herein are listed in Table 14.
  • the nucleotide sequences encoding exemplary targeting moieties disclosed herein are listed in Table 15.
  • an expression repressor or system described herein comprises a targeting moiety having a sequence set forth in Table 14, or a sequence with at least 70%, 75%, 80%, 85%, 90%, 95%, 98%, or 99% identity thereto.
  • a nucleic acid described herein comprises a sequence set forth in Table 15, or a sequence with at least 70%, 75%, 80%, 85%, 90%, 95%, 98%, or 99% identity thereto.
  • a targeting moiety is or comprises a DNA-binding domain from a nuclease.
  • the recognition sequences of homing endonucleases and meganucleases such as I- Scel, I-Ceul, PI-PspI, Pl-Sce, I-SceIV, I-CsmI, I-PanI, I-SceII, I-Ppol, 1-SceIII, I-Crel, I-TevI, I-TevII and I-TevIII are known. See also U.S. Pat. Nos. 5,420,032; 6,833,252; Belfort, et al. (1997) Nucleic Acids Res.
  • a DNA-targeting moiety comprises or is nucleic acid.
  • a nucleic acid that may be included in a DNA-targeting moiety may be or comprise DNA, RNA, and/or an artificial or synthetic nucleic acid or nucleic acid analog or mimic.
  • a nucleic acid may be or include one or more of genomic DNA (gDNA), complementary DNA (cDNA), a peptide nucleic acid (PNA), a peptide- oligonucleotide conjugate, a locked nucleic acid (LNA), a bridged nucleic acid (BNA), a polyamide, a triplex- forming oligonucleotide, an antisense oligonucleotide, tRNA, mRNA, rRNA, miRNA, gRNA, siRNA or other RNAi molecule (e.g., that targets a non-coding RNA as described herein and/or that targets an expression product of a particular gene associated with a targeted genomic complex as described herein), etc.
  • genomic DNA genomic DNA
  • cDNA complementary DNA
  • PNA peptide nucleic acid
  • LNA locked nucleic acid
  • BNA bridged nucleic acid
  • a polyamide a triplex- forming oligonucleotide
  • a nucleic acid may include one or more residues that is not a naturally-occurring DNA or RNA residue, may include one or more linkages that is/are not phosphodiester bonds (e.g., that may be, for example, phosphorothioate bonds, etc.), and/or may include one or more modifications such as, for example, a 2’0 modification such as 2’-OmeP.
  • linkages e.g., that may be, for example, phosphorothioate bonds, etc.
  • modifications such as, for example, a 2’0 modification such as 2’-OmeP.
  • a variety of nucleic acid structures useful in preparing synthetic nucleic acids is known in the art (see, for example, WO2017/0628621 and WO2014/012081) those skilled in the art will appreciate that these may be utilized in accordance with the present disclosure.
  • a nucleic acid suitable for use in an expression repressor, e.g., in the DNA-targeting moiety may include, but is not limited to, DNA, RNA, modified oligonucleotides (e.g., chemical modifications, such as modifications that alter backbone linkages, sugar molecules, and/or nucleic acid bases), and artificial nucleic acids.
  • a nucleic acid includes, but is not limited to, 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
  • a DNA-targeting moiety comprises a nucleic acid with a length from about 15-200, 20-200, 30-200, 40-200, 50-200, 60-200, 70-200, 80-200, 90-200, 100-200, 110-200, 120- 200, 130-200, 140-200, 150-200, 160-200, 170-200, 180-200, 190-200, 215-190, 20-190, 30-190, 40-190, 50-190, 60-190, 70-190, 80-190, 90-190, 100-190, 110-190, 120-190, 130-190, 140-190, 150-190, 160- 190, 170-190, 180-190, 15-180, 20-180, 30-180, 40-180, 50-180, 60-180, 70-180, 80-180, 90-180, 100- 180, 110-180, 120-180, 130-180, 140-180, 150-180, 160-180, 170-180, 15-170, 20-170, 30-170, 40-1
  • expression repressors of the present disclosure comprise one or more effector moieties.
  • an effector moiety when used as part of an expressor repressor or an expression repression system described herein, decreases expression of a target gene in a cell.
  • the effector moiety has functionality unrelated to the binding of the targeting moiety.
  • effector moieties may target, e.g., bind, a genomic sequence element or genomic complex component proximal to the genomic sequence element targeted by the targeting moiety or recruit a transcription factor.
  • an effector moiety may comprise an enzymatic activity, e.g., a genetic modification functionality.
  • an effector moiety comprises an epigenetic modifying moiety.
  • an effector moiety comprises a DNA modifying functionality, e.g., a DNA methyltransferase.
  • an effector moiety is or comprises a protein chosen from MQ1, DNMT1, DNMT3A1, DNMT3A2, DNMT3B1, DNMT3B2, DNMT3B3, DNMT3B4, DNMT3B5, DNMT3B6, DNMT3L, or a functional variant or fragment of any thereof.
  • an effector moiety comprises a transcription repressor.
  • the transcription repressor blocks recruitment of a factor that stimulates or promotes transcription, e.g., of the target gene.
  • the transcription repressor recruits a factor that inhibits transcription, e.g., of the target gene.
  • an effector moiety, e.g., transcription repressor is or comprises a protein chosen from KRAB, MeCP2, HP1, RBBP4, REST, FOG1, SUZ12, or a functional variant or fragment of any thereof.
  • an effector moiety promotes epigenetic modification, e.g., directly or indirectly.
  • an effector moiety can indirectly promote epigenetic modification by recruiting an endogenous protein that epigenetically modifies the chromatin.
  • An effector moiety can directly promote epigenetic modification by catalyzing epigenetic modification, wherein the effector moiety comprises enzymatic activity and directly places an epigenetic mark on the chromatin.
  • an effector moiety comprises a histone modifying functionality, e.g., a histone methyltransferase, histone demethylase, or histone deacetylase activity.
  • a effector moiety is or comprises a protein chosen from KDM1A (i.e., LSD1), KDM1B (i.e., LSD2), KDM2A, KDM2B, KDM5A, KDM5B, KDM5C, KDM5D, KDM4B, NO66, or a functional variant or fragment of any thereof.
  • a effector moiety is or comprises a protein chosen from HDAC1, HDAC2, HDAC3, HDAC4, HDAC5, HDAC6, HDAC7, HDAC8, HDAC9, HDAC10, HDAC11, SIRT1, SIRT2, SIRT3, SIRT4, SIRT5, SIRT6, SIRT7, SIRT8, SIRT9, or a functional variant or fragment of any thereof.
  • an effector moiety comprises a protein having a functionality described herein.
  • an effector moiety is or comprises a protein selected from: KRAB (e.g., as according to NP_056209.2 or the protein encoded by NM_015394.5); a SET domain (e.g., the SET domain of: SETDB1 (e.g., as according to NP 001353347.1 or the protein encoded by NM_001366418.1); EZH2 (e.g., as according to NP-004447.2 or the protein encoded by NM_004456.5); G9A (e.g., as according to NP_001350618.1 or the protein encoded by NM_001363689.1); or SUV39H1 (e.g., as according to NP_003164.1 or the protein encoded by NM_003173.4)); histone demethylase LSD1 (e.g., as according to NP 0558), a protein having a
  • a effector moiety is or comprises a protein selected from: DNMT3A (e.g., human DNMT3A) (e.g., as according to NP_072046.2 or the protein encoded by NM_022552.4); DNMT3B (e.g., as according to NP 008823.1 or the protein encoded by NM_006892.4); DNMT3L (e.g., as according to NP_787063.1 or the protein encoded by NM_175867.3); DNMT3A/3L complex, bacterial MQ1 (e.g., as according to CAA35058.1 or P15840.3); a functional fragment of any thereof, or a polypeptide with a sequence that has at least 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% identity to any of the above-referenced sequences.
  • DNMT3A e.g., human DNMT3A
  • the disclosure is directed to an expression repressor or a polypeptide comprising one or more (e.g., one) targeting moiety and one or more effector moiety, wherein the one or more effector moiety is or comprises Krueppel-associated box (KRAB) e.g., as according to NP_056209.2 or the protein encoded by NM_015394.5 or a functional variant or fragment thereof.
  • KRAB is a synthetic KRAB construct.
  • KRAB comprises an amino acid sequence of SEQ ID NO: 18:
  • the KRAB effector moiety is encoded by a nucleotide sequence of SEQ ID NO: 51.
  • a nucleotide sequence described herein comprises a sequence of SEQ ID NO: 51 or a sequence with at least 80, 85, 90, 95, 99, or 100% identity thereto, or having no more than 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 positions of difference thereto.
  • KRAB for use in a polypeptide or an expression repressor described herein is a variant, e.g., comprising one or more mutations, relative to the KRAB sequence of SEQ ID NO: 18.
  • an KRAB variant comprises one or more amino acid substitutions, deletions, or insertions relative to SEQ ID NO: 18.
  • the polypeptide or the expression repressor is a fusion protein comprising a effector moiety that is or comprises KRAB and a DNA-targeting moiety.
  • the targeting moiety is or comprises a zinc finger domain, TAL domain, or CRISPR/Cas domain, e.g., comprising a CRISPR/Cas protein, e.g., a dCas9 protein.
  • the polypeptide or the expression repressor comprises an additional moiety described herein.
  • the polypeptide or the expression repressor decreases expression of a target gene, e.g., MYC.
  • the polypeptide or the expression repressor may be used in methods of modulating, e.g., decreasing, gene expression, methods of treating a condition, or methods of epigenetically modifying a target gene, e.g., MYC or transcription control element described herein, e.g., in place of an expression repression system.
  • an expression repression system comprises two or more (e.g., two, three, or four) expression repressors, wherein the first expression repressor comprises an effector moiety comprising the KRAB sequence of SEQ ID NO: 18, or a functional variant or fragment thereof.
  • the disclosure is directed to a expression repressor or a polypeptide comprising one or more (e.g., one) targeting moiety and one or more effector moiety, wherein the one or more effector moiety is or comprises MQ1, e.g., bacterial MQ1, or a functional variant or fragment thereof.
  • MQ1 is Mollicutes spiroplasma MQ1.
  • MQ1 is Spiroplasma monobiae MQ1.
  • MQ1 is MQ1 from strain ATCC 33825 and/or corresponding to Uniprot ID P15840.
  • MQ1 comprises an amino acid sequence of SEQ ID NO: 19.
  • MQ1 comprises an amino acid sequence of SEQ ID NO: 87.
  • an effector domain described herein comprises SEQ ID NO: 19 or 87, or a sequence with at least 80, 85, 90, 95, 99, or 100% identity thereto, or having no more than 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 positions of difference thereto.
  • MQ1 is encoded by a nucleotide sequence of SEQ ID NO: 52 or 132.
  • a nucleic acid described herein comprises a sequence of SEQ ID NO: 52, 132 or a sequence with at least 80, 85, 90, 95, 99, or 100% identity thereto, or having no more than 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 positions of difference thereto.

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CA3205133A CA3205133A1 (en) 2020-12-15 2021-12-15 Compositions and methods for modulating myc expression
CN202180093730.9A CN116829175A (zh) 2020-12-15 2021-12-15 用于调节myc表达的组合物和方法
JP2023536049A JP2023553490A (ja) 2020-12-15 2021-12-15 Ｍｙｃ発現を調節するための組成物及び方法
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