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WO2025064469A1 - Procédés d'évaluation de dosage pour agents de modification épigénétiques - Google Patents

Procédés d'évaluation de dosage pour agents de modification épigénétiques Download PDF

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Publication number
WO2025064469A1
WO2025064469A1 PCT/US2024/047157 US2024047157W WO2025064469A1 WO 2025064469 A1 WO2025064469 A1 WO 2025064469A1 US 2024047157 W US2024047157 W US 2024047157W WO 2025064469 A1 WO2025064469 A1 WO 2025064469A1
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Prior art keywords
dna
level
expression
modifying agent
dose
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Inventor
Williiam SENAPEDIS
Eugine Lee
Defne YARAR
Sangeetha Palakurthi
Justin Chen
Charles O'donnell
Kayleigh GALLAGHER
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Omega Therapeutics Inc
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Omega Therapeutics Inc
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6883—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material
    • C12Q1/6886—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material for cancer
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/106—Pharmacogenomics, i.e. genetic variability in individual responses to drugs and drug metabolism
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/154—Methylation markers
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/158—Expression markers

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.
  • Techniques geared towards modulating gene expression e.g., decrease or increase expression from gene of interest provides a viable alternative approach in treating these diseases.
  • Modulating gene expression can be accomplished by altering methylation levels at promoters and/or regulatory sequences. Tools and methods are needed to determine efficacy of treatments that alter methylation level at genomic loci.
  • the present invention provides a method of treating a subject with an epigenetic modifying agent comprising determining whether at least one of (i) a measured level of DNA methylation of one or more biomarkers, or (ii) a measured level of RNA of the one or more biomarkers in a biological sample obtained from a subject who has received the first dose of epigenetic modifying agent is higher than, less than or equal to a control level; and administering a second dose of the epigenetic modifying agent to the subject.
  • the foregoing methods may further involve measuring (i) the level of DNA methylation of the one or more biomarkers, or (ii) the level of RNA of the one or more biomarkers in the biological sample.
  • the foregoing methods further involve determining whether a measured level of extracellular vesicle RNA in a second biological sample obtained from the subject who has received the second dose of the epigenetic modifying agent is higher than, less than or equal to a control level; and administering a third dose of the epigenetic modifying agent to the subject.
  • the method may further include measuring the level of extracellular vesicle RNA in the second biological sample.
  • the present invention provides a method of assessing the efficacy of a first dose of an epigenetic modifying agent comprising: determining whether at least one of (i) a measured level of DNA methylation of one or more biomarkers, and (ii) a measured level of RNA of one or more biomarkers in a biological sample obtained from a subject who has received the first dose of epigenetic modifying agent is higher than, less than or equal to a control level.
  • the present invention provides a method of treating a subject with an epigenetic modifying agent comprising: a. determining whether at least one of (i) a measured level of DNA methylation of one or more biomarkers, and (ii) a measured level of RNA of the one or more biomarkers in a biological sample obtained from a subject who has received the first dose of epigenetic modifying agent is higher than, less than or equal to a control level; and b. administering a second dose of the epigenetic modifying agent to the subject.
  • the foregoing methods may further involve measuring (i) the level of DNA methylation of the one or more biomarkers, or (ii) the level of RNA of the one or more biomarkers in the biological sample.
  • measuring the level of RNA of the one or more biomarkers comprises measuring the level of extracellular vesicle RNA.
  • the extracellular vesicle RNA is derived from cancer cells.
  • the epigenetic modifying agent achieves therapeutic effect by repressing expression of a gene target.
  • the gene target is MYC, SFRP1, APOB or HNF4a.
  • the epigenetic modifying agent achieves therapeutic effect by enhancing expression of a gene target.
  • the gene target is FOXP3.
  • the level of extracellular vesicle RNA is less than or equal to the control level and the subject is administered a second dose of the epigenetic modifying agent that is higher than the first dose of the epigenetic modifying agent; or (ii) wherein the level of extracellular vesicle RNA is higher than the control level and the subject is administered a second dose of the epigenetic modifying agent that is less than or equal to the first dose of the epigenetic modifying agent.
  • control level is the level of extracellular vesicle RNA prior to administration of the first dose of the epigenetic modifying agent.
  • control level is a standardized level of extracellular vesicle RNA.
  • standardized level of extracellular vesicle RNA is a predetermined level of extracellular vesicle RNA associated with a disease state.
  • the foregoing methods may further involve a. determining whether a measured level of extracellular vesicle RNA in a second biological sample obtained from the subject who has received the second dose of the epigenetic modifying agent is higher than, less than or equal to a control level; and b. administering a third dose of the epigenetic modifying agent to the subject.
  • the foregoing methods may further involve measuring the level of extracellular vesicle RNA in the second biological sample.
  • the epigenetic modifying agent achieves therapeutic effect by repressing expression of a gene target; and wherein (i) the level of extracellular vesicle RNA is higher than the control level and the subject is administered a second dose of the epigenetic modifying agent that is higher than the first dose of the epigenetic modifying agent; or (ii) the level of extracellular vesicle RNA is less than or equal to the control level and the subject is administered a second dose of the epigenetic modifying agent that is less than or equal to the first dose of the epigenetic modifying agent; or (b) wherein the epigenetic modifying agent achieves therapeutic effect by enhancing expression of a gene target, and wherein (i) the level of extracellular vesicle RNA is less than or equal to the control level and the subject is administered a second dose of the epigenetic modifying agent that is higher than the first dose of the epigenetic modifying agent; or (ii) the level of extracellular ves
  • both the level of DNA methylation and the level of extracellular vesicle RNA is measured, either of the same or different biomarkers.
  • the extracellular vesicle is an exosome. In one embodiment, the extracellular vesicle is a microvesicle.
  • the epigenetic modifying agent achieves therapeutic effect by repressing expression of a target gene, wherein repression of expression of the target gene is by methylating DNA.
  • the level of DNA methylation in the biological sample is less than or equal to the control level and wherein the subject is administered a second dose of the epigenetic modifying agent that is higher than the first dose of the epigenetic modifying agent; or (ii) the level of DNA methylation in the biological sample is higher than the control level and wherein the subject is administered a second dose of the epigenetic modifying agent that is less than or equal to the first dose of the epigenetic modifying agent.
  • the epigenetic modifying agent achieves therapeutic effect by enhancing expression of the target gene, wherein the enhancement of expression of the target gene is by demethylating DNA.
  • the level of DNA methylation in the biological sample is higher than the control level and wherein the subject is administered a second dose of the epigenetic modifying agent that is higher than the first dose of the epigenetic modifying agent; or (ii) the level of DNA methylation in the biological sample is less than or equal to the control level and wherein the subject is administered a second dose of the epigenetic modifying agent that is less than or equal to the first dose of the epigenetic modifying agent.
  • the biological sample is selected from the group consisting of blood, cerebrospinal fluid, plasma, pleural fluid, saliva, serum sputum, stool, and urine.
  • the DNA is cell-free DNA.
  • the cell-free DNA is circulating tumor DNA (ctDNA).
  • the cell-free DNA is extracellular vesicle DNA.
  • the extracellular vesicle is an exosome.
  • the extracellular vesicle is a microvesicle.
  • the biological sample is blood.
  • the biological sample is tissue.
  • the tissue sample is a biopsy, optionally, a liquid biopsy.
  • the DNA comprises cellular genomic DNA.
  • measuring the level of methylation comprises quantitative polymerase chain reaction (qPCR), next-generation sequencing, nanopore sequencing, beam emulsion sequencing, sodium bisulfite conversion and sequencing, differential enzymatic cleavage, affinity capture of methylated DNA, or epiallele methylation detection.
  • measuring the level of methylation comprises nanopore sequencing.
  • measuring the level of methylation comprises sodium bisulfite conversion and sequencing.
  • measuring the level of methylation comprises differential enzymatic cleavage.
  • measuring the level of methylation comprises affinity capture of methylated DNA.
  • epiallele methylation detection comprises determining variant epiallele fraction (VEF), wherein the VEF is the level of DNA methylation.
  • VEF variant epiallele fraction
  • the foregoing methods may further involve identifying genomic sequence with methylation at one or more cytosine guanine (CpG) sites in DNA extracted from the biological sample.
  • CpG cytosine guanine
  • the extracted DNA is sequenced using next generation sequencing.
  • the next generation sequencing comprises: 1) fragmenting extracted genomic DNA; and 2) amplifying DNA fragments with oligonucleotides that hybridize to the DNA fragments.
  • the one or more biomarkers comprises 1) a primary biomarker, wherein the primary biomarker is the target gene or DNA sequence located within 1 kb of the target gene; and/or 2) one or more secondary biomarkers, wherein the secondary biomarker is a gene other than the target gene, and wherein the expression and/or methylation status of the secondary biomarker is modified as a result of the epigenetic modifying agent repressing or enhancing expression of the target gene.
  • FIG. 2A depicts methylation levels of total extracellular vesicle DNA collected from serum comparing negative controls (PBS and SNC) to treated samples (ZF17-MQ1 at 1 mg/kg and 0.3 mg/kg).
  • FIG. 9A depicts relative MYC methylation signal from extracellular vesicle DNA 24 and 48 hrs after treatment with MR-30723 encapsulated in a LNP.
  • FIG. 14A depicts Capture-based method selected to enrich signal sufficiently for biomarker identification (TwistBio platform). Captured methylome was compared to whole-genome methyl-seq and showed greater than 2000x coverage efficiency increased with the captured methylome.
  • FIG. 14B depicts DNA methylation levels at evDNA (extracellular vesicle DNA) and cfDNA (cell free DNA) after treatment with epigenetic modifying agent compared to a control.
  • FIG. 15B depicts weight in mice after treatment with MR-30723/LNP over the course of day 0 through day 25 after treatment with epigenetic modifying agent compared to controls.
  • FIG. 16A depicts DNA methylation of genomic DNA after treatment with MR-30723/LNP compared to controls.
  • FIG. 16B depicts estimated reads of circulating tumor DNA (ctDNA) after treatment with MR-30723/LNP compared to controls.
  • FIG. 16C depicts methylation detection assays in evDNA from control animals, saline and non-coding treatments. No methylation was detected in the controls.
  • FIG. 16D depicts methylation detections assays in plasma evDNA and Tumor gDNA from mice treated with MR-30882/LNP.
  • FIG. 17A depicts mean tumor volume over the course of 25 days after treatment with MR- 30723/LNP compared to controls.
  • FIG. 17B depicts mean percent weight change over the course of 25 days after treatment with MR-30723/LNP compared to controls.
  • FIG. 18A depicts mean tumor volume over the course of 25 days after treatment with two doses of MR-30882/LNP compared to controls.
  • FIG. 18B depicts percent variant epiallele fraction (VEF) in MYC promoter by treatment after 14 days (top panel) and after 48 hours (bottom panel).
  • FIG. 18C depicts percent variant epiallele fraction (VEF) for extracellular vesicle DNA after treatment with MR-30882/LNP for 48 hours compared to controls.
  • VEF percent variant epiallele fraction
  • FIG. 18D depicts percent variant epiallele fraction (VEF) for genomic DNA after treatment with MR-30882/LNP for 48 hours compared to controls.
  • FIG. 19A depicts a subcutaneous Hep3B xenograft model was intravenously dosed with MR- 30882/LNP (1 or 2 mg/kg), a non-coding control mRNA (2 mg/kg), or PBS after tumors reached 200 mm3 in size.
  • FIG. 19B depicts percent variant epiallele fraction (VEF) of samples 24 hours post-dose. Sparse DNA methylation was detected at MYC in cfDNA (top panel) in MR-30882/LNP animals compared to tumor gDNA (bottom panel).
  • FIG. 19C depicts percent variant epiallele fraction (VEF) of samples 24 hours post-dose. MYC methylation was mostly absent in cfDNA (top panel) or tumor gDNA (bottom panel) from PBS control treated animals.
  • FIG. 19D depicts enrichment of cfDNA from plasma (whole blood). Plasma was harvested from animals in FIG. 19A and cfDNA was examined for MYC methylation using the minimal hybridization capture panel, providing the necessary enrichment over whole-genome sequencing to detect regions of interest.
  • FIG. 20 depicts percent variant epiallele fraction (VEF) of target enrichment panel. Methylation was detected down to 0.00001% using a synthetic control titration.
  • FIG. 21 depicts percent variant epiallele fraction (VEF) targeted methylation sequencing was performed on a titration series of methylated control gDNA spiked into unmethylated control gDNA (Zymo Research) and analyzed for methylated epialleles at the MYC promoter. MYC promoter methylation was detected in samples containing as low as 0.05% methylated gDNA compared to fully unmethylated control gDNA. This was at the theoretical limit of the number of copies present in the assay.
  • VEF percent variant epiallele fraction
  • FIG. 22A shows results from examination of the MYC methylation panel performance on a biological sample designed to mimic a clinically-derived cfDNA specimen.
  • Targeted methylation sequencing using the MYC methylation panel was able to discriminate between the MR-30882/LNP - treated (EC) sample (bottom panel) and the PBS-treated sample (top panel).
  • FIG. 22B depicts enrichment of cfDNA from plasma (whole blood). Plasma was harvested from animals in FIG. 22A and cfDNA was examined for MYC methylation using the minimal hybridization capture panel, providing the necessary enrichment over whole-genome sequencing to detect regions of interest. DETAILED DESCRIPTION
  • the present invention is based, at least in part, on the finding that dosing of an epigenetic modifying agent can be determined based on assessing the DNA methylation of a biomarker associated with a target gene for which expression is sought to modulated and/or assessing the level of extracellular vesicle (e.g., exosome or microvesicle) RNA associated with one or more biomarkers.
  • the epigenetic modifying agent serves to methylate or demethylate a DNA locus, for example, a promoter, surrounding a gene target, thereby repressing or enhancing expression of the gene target, as desired.
  • the second dose can be modified accordingly.
  • Isolating and sequencing DNA or RNA for levels of methylation for one or more small genomic regions is challenging and requires ultrasensitive detection systems compared to assays detecting methylated DNA across tens of megabases of genomic regions.
  • the epigenetic modifying agent of the present invention can be tissue specific, increasing the scarcity of the specific DNA and/or RNA being captured during the isolation steps and subsequently sequenced for methylation levels.
  • approximately less than 1% of nucleic acids from a standard whole-genome methylation-sequencing assay is cell-free nucleic acids or extracellular vesicle nucleic acids.
  • the invention provides a method of assessing the efficacy of a first dose of an epigenetic modifying agent by a. measuring in a biological sample obtained from a subject who has received the first dose of the epigenetic modifying agent at least one of: the level of DNA methylation of one or more biomarkers, or the level of RNA (e.g., extracellular vesicle (e.g., exosome or microvesicle) RNA) of one or more biomarkers; and b.
  • RNA e.g., extracellular vesicle (e.g., exosome or microvesicle) RNA
  • a method of treating a subject with an epigenetic modifying agent by a. measuring in a biological sample obtained from a subject who has received a first dose of an epigenetic modifying agent at least one of: the level of DNA methylation of one or more biomarkers, and the level of extracellular vesicle (e.g., exosome or microvesicle) RNA of one or more biomarkers; b.
  • extracellular vesicle e.g., exosome or microvesicle
  • the disclosure provides a method of assessing the efficacy of a first dose of an epigenetic modifying agent by: determining whether at least one of (i) a measured level of DNA methylation of one or more biomarkers, or (ii) a measured level of RNA (e.g., extracellular vesicle (e.g., exosome or microvesicle) RNA) of one or more biomarkers in a biological sample obtained from a subject who has received the first dose of epigenetic modifying agent is higher than, less than or equal to a control level.
  • RNA e.g., extracellular vesicle (e.g., exosome or microvesicle) RNA
  • a method of treating a subject with an epigenetic modifying agent by: determining whether at least one of (i) a measured level of DNA methylation of one or more biomarkers, or (ii) a measured level of RNA (e.g., extracellular vesicle (e.g., exosome or microvesicle) RNA) of one or more biomarkers in a biological sample obtained from a subject who has received the first dose of epigenetic modifying agent is higher than, less than or equal to a control level.
  • RNA e.g., extracellular vesicle (e.g., exosome or microvesicle) RNA
  • the method further may involve measuring (i) the level of DNA methylation of the one or more biomarkers, or (ii) the level of RNA (e.g., extracellular vesicle (e.g., exosome or microvesicle) RNA) of the one or more biomarkers in the biological sample.
  • RNA e.g., extracellular vesicle (e.g., exosome or microvesicle) RNA
  • the method further may involve administering a second dose of the epigenetic modifying agent that is higher than the first dose, when the level of DNA methylation in the biological sample is less than or equal to the control level, and the epigenetic modifying agent achieves therapeutic effect by methylating the DNA.
  • the method may involve administering a second dose of the epigenetic modifying agent that is less than or equal to the first dose, when the level of DNA methylation in the biological sample is higher than the control level, and the epigenetic modifying agent achieves therapeutic effect by methylating the DNA.
  • the method may involve administering a second dose of the epigenetic modifying agent that is higher than the first dose, when the level of DNA methylation in the biological sample is higher than the control level, and the epigenetic modifying agent achieves therapeutic effect by demethylating the DNA.
  • the method may involve administering a second dose of the epigenetic modifying agent that is less than or equal to the first dose, when the level of DNA methylation in the biological sample is lower than the control level, and the epigenetic modifying agent achieves therapeutic effect by demethylating the DNA.
  • the method may involve administering a second dose of the epigenetic modifying agent that is higher than the first dose, wherein the level of extracellular vesicle (e.g., exosome or microvesicle) RNA is higher than the control level and the epigenetic modifying agent achieves therapeutic effect by repressing expression of a gene target.
  • the level of extracellular vesicle e.g., exosome or microvesicle
  • the method may involve administering a second dose of the epigenetic modifying agent that is less than or equal to the first dose, wherein the level of extracellular vesicle (e.g., exosome or microvesicle) RNA is less than or equal to the control level and the epigenetic modifying agent achieves therapeutic effect by repressing expression of a gene target.
  • the level of extracellular vesicle e.g., exosome or microvesicle
  • the method may involve administering a second dose of the epigenetic modifying agent that is higher than the first dose, wherein the level of extracellular vesicle (e.g., exosome or microvesicle) RNA is less than or equal to the control level and the epigenetic modifying agent achieves therapeutic effect by enhancing expression of a gene target.
  • the level of extracellular vesicle e.g., exosome or microvesicle
  • the method may involve administering a second dose of the epigenetic modifying agent that is less than or equal to the first dose, wherein the level of extracellular vesicle (e.g., exosome or microvesicle) RNA is higher than the control level and the epigenetic modifying agent achieves therapeutic effect by enhancing expression of a gene target.
  • the level of extracellular vesicle e.g., exosome or microvesicle
  • the method may involve determining whether a measured level of DNA methylation of the one or more biomarkers in a second biological sample obtained from the subject who has received the second dose of the epigenetic modifying agent is higher than, less than or equal to a control level; and administering a third dose of the epigenetic modifying agent to the subject.
  • the method may involve measuring the level of DNA methylation in the second biological sample.
  • the method further may involve administering a third dose of the epigenetic modifying agent that is higher than the first dose or second dose, when the level of DNA methylation in the biological sample is less than or equal to the control level, and the epigenetic modifying agent achieves therapeutic effect by methylating the DNA.
  • the method may involve administering a third dose of the epigenetic modifying agent that is less than or equal to the first dose or second dose, when the level of DNA methylation in the biological sample is higher than the control level, and the epigenetic modifying agent achieves therapeutic effect by methylating the DNA.
  • the method may involve administering a third dose of the epigenetic modifying agent that is higher than the first dose or second dose, when the level of DNA methylation in the biological sample is higher than the control level, and the epigenetic modifying agent achieves therapeutic effect by demethylating the DNA.
  • the method may involve administering a third dose of the epigenetic modifying agent that is less than or equal to the first dose or second dose, when the level of DNA methylation in the biological sample is lower than the control level, and the epigenetic modifying agent achieves therapeutic effect by demethylating the DNA.
  • the method may involve a. determining whether a measured level of extracellular vesicle (e.g., exosome or microvesicle) RNA in a second biological sample obtained from the subject who has received the second dose of the epigenetic modifying agent is higher than, less than or equal to a control level; and b. administering a third dose of the epigenetic modifying agent to the subject.
  • the method may involve measuring the level of extracellular vesicle (e.g., exosome or microvesicle) RNA in the second biological sample.
  • the method may involve administering a third dose of the epigenetic modifying agent that is higher than the first dose or second dose, wherein the level of extracellular vesicle (e.g., exosome or microvesicle) RNA is higher than the control level and the epigenetic modifying agent achieves therapeutic effect by repressing expression of a gene target.
  • the level of extracellular vesicle e.g., exosome or microvesicle
  • the method may involve administering a third dose of the epigenetic modifying agent that is less than or equal to the first dose or second dose, wherein the level of extracellular vesicle (e.g., exosome or microvesicle) RNA is less than or equal to the control level and the epigenetic modifying agent achieves therapeutic effect by repressing expression of a gene target.
  • the level of extracellular vesicle e.g., exosome or microvesicle
  • the method may involve administering a third dose of the epigenetic modifying agent that is higher than the first dose or second dose, wherein the level of extracellular vesicle (e.g., exosome or microvesicle) RNA is less than or equal to the control level and the epigenetic modifying agent achieves therapeutic effect by enhancing expression of a gene target.
  • the level of extracellular vesicle e.g., exosome or microvesicle
  • the method may involve administering a third dose of the epigenetic modifying agent that is less than or equal to the first dose or second dose, wherein the level of extracellular vesicle (e.g., exosome or microvesicle) RNA is higher than the control level and the epigenetic modifying agent achieves therapeutic effect by enhancing expression of a gene target.
  • the level of extracellular vesicle e.g., exosome or microvesicle
  • 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 1 kb 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.).
  • a domain is or comprises a section of a polypeptide.
  • a domain is characterized by a particular structural element (e.g., a particular amino acid sequence or sequence motif, alpha-helix character, beta-sheet character, coiled-coil character, random coil character, etc.), and/or by a particular functional feature (e.g., binding activity, enzymatic activity, folding activity, signaling activity, etc.).
  • 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., DNA methylation or DNA demethylation), when the epigenetic modifying moiety is appropriately localized to a nucleic acid (e.g., by a targeting moiety).
  • 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 DNA demethylase, or a functional fragment of any thereof.
  • the term “epigenetic modifying agent” refers to an agent capable of altering gene expression (e.g., decease or increase expression of target gene).
  • the epigenetic modifying agent comprises an epigenetic modifying moiety.
  • 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.
  • expression enhancer refers to an agent or entity with one or more functionalities that increases expression of a target gene in a cell and that specifically binds to a DNA sequence (e.g., a DNA sequence associated with a target gene or a transcription control element operably linked to a target gene).
  • An expression enhancer 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.
  • an expression enhancing system refers to a plurality of expression enhancers which increase expression of a target gene in a cell.
  • an expression enhancing system comprises a first expression enhancer and a second expression enhancer, wherein the first expression enhancer and second expression enhancer (or nucleic acids encoding the first expression enhancer and second expression enhancer) are present together in a single composition, mixture, or pharmaceutical composition.
  • an expression enhancing system comprises a first expression enhancer and a second expression enhancer, wherein the first expression enhancer and second expression enhancer (or nucleic acids encoding the first expression enhancer and second expression enhancer) are present in separate compositions or pharmaceutical compositions.
  • the first expression enhancer and the second expression enhancer are present in the same cell at the same time. In some embodiments, the first expression enhancer and the second expression enhancer are not present in the same cell at the same time, e.g., they are present sequentially. For example, the first expression enhancer may be present in a cell for a first time period, and then the second expression enhancer 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.
  • an expression enhancer 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, Secreted Frizzled Related Protein 1 (SFRP1), Hepatocyte Nuclear Factor 4-alpha (HNF4a), Forkhead box P3 (FOXP3), and Apolipoprotein B (APOB).
  • MYC Secreted Frizzled Related Protein 1
  • HNF4a Hepatocyte Nuclear Factor 4-alpha
  • FOXP3 Forkhead box P3
  • APOB Apolipoprotein B
  • 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 promoter the promoter operably linked to MYC
  • the 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.
  • the terms “secreted frizzled related protein 1” and “SFRP1,” as used interchangeably herein, refer to the gene as well as the well-known encoded protein that is a Wnt signaling pathway component and, more specifically, a secreted extracellular polypeptide that binds to a Wnt protein.
  • the Wnt proteins control the expression of several genes, including pre-mitotic genes involved in hair growth.
  • SFRP1 is a Wnt antagonist. In the absence of SFRP1, Wnt can bind to the frizzled receptor, this begins a phosphorylation cascade which de-phosphorylates B-catenin, and frees it from the destruction complex.
  • B-catenin is able to translocate into the nucleus where it activates pro-mitotic genes for hair growth.
  • Decreased expression of the SFRP1 gene has been associated increased expression of pre-mitotic genes and increased hair growth.
  • Expression of the SFRP 1 gene results in sequestering of the Wnt proteins and decreased activation of pre-mitotic genes and has been associated with alopecia (e.g., androgenic alopecia, alopecia areata, traction alopecia, senescent alopecia and cicatricial alopecia).
  • alopecia e.g., androgenic alopecia, alopecia areata, traction alopecia, senescent alopecia and cicatricial alopecia.
  • the nucleotide and amino acid sequence of SFRP1 is known and may be found in, for example, GenBank Accession Nos.
  • NM_003012.5 (NM_003012) and NP_003003.3 (NP_003003), the entire contents of each of which are incorporated herein by reference.
  • the nucleotide sequence of the genomic region of Chromosome 8 which includes the endogenous promoters of SFRP 1 and the SFRP 1 coding sequence is also known and may be found in GenBank Accession No. NC_000008. l l (41261962..41309473) and NC_000008.10 (41119481..41166992).
  • HNF4a locus refers to the portion of the human genome that encodes a HNF4a polypeptide.
  • the HNF4a gene is located on chromosome 20, with transcription regulated by two promoters (P 1 and P2) and alternative splicing variants, resulting in nine distinct isoforms (al- a9).
  • the HNF4a locus is transcriptionally regulated through the use of two distinct promoters that are physically separated by more than 45 kb. Isoforms produced by the activity of the closer promoter are designated P 1 whereas isoforms produced by the second and more distant promoter are designated P2.
  • Isoforms most common in the liver are expressed from promoter 1 (Pl), with isoforms from P2 most commonly found in fetal tissues, and in the adult kidney and small intestine.
  • the nucleotide sequence of the genomic region of Chromosome 20 which includes the endogenous promoters of HNF4a and the HNF4a coding sequence is also known and may be found in GenBank Accession No. NC_000020.10 (42984441. ..43061485).
  • FOXP3 refers to the gene that encodes the well-known FOX protein family member that is a master transcription factor that controls the differentiation of naive T-cells into regulatory T-cells (Tregs).
  • FOX proteins belong to the forkhead/winged-helix family of transcriptional regulators and are believed to exert control via similar DNA binding interactions during transcription.
  • the FOXP3 transcription factor occupies the promoters for genes involved in regulatory T-cell function, and may repress transcription of key genes following stimulation of T cell receptors.
  • IPEX immunodysregulation polyendocrinopathy enteropathy X-linked syndrome
  • FOXP3 immunodysregulation polyendocrinopathy enteropathy X-linked syndrome
  • nucleotide sequence of the genomic region of the X Chromosome in human which includes the endogenous promoters of FOXP3 and the FOXP3 coding sequence, is also known and may be found in, for example, NC_000023. 11 (49250436-49264932).
  • GenBank Accession Nos. NM_014009.4 and NM_001114377.2 There are two common transcript variants for FOXP3 mRNA, the sequences of which can be found in GenBank Accession Nos. NM_014009.4 and NM_001114377.2. The entire contents of each of the foregoing GenBank Accession numbers are incorporated herein by reference as of the date of filing this application.
  • apolipoprotein B refers to the gene that encodes the well-known apolipoprotein of chylomicrons, VLDL, IDL, and LDL particles.
  • the encoded protein is the primary organizing protein component of the particles and is important for the formation of these particles.
  • APOB on the LDL particle also acts as a ligand for LDL receptors in various cells throughout the body. High levels of APOB are related to heart disease. Hypobetalipoproteinemia is a genetic disorder that can be caused by a mutation in the APOB gene, APOB.
  • Mutations in gene APOB 100 can also cause familial hypercholesterolemia, a hereditary (autosomal dominant) form of metabolic disorder hypercholesterolemia.
  • Overproduction of apolipoprotein B can result in lipid- induced endoplasmic reticulum stress and insulin resistance in the liver.
  • the nucleotide and amino acid sequence of APOB is known and may be found in, for example, GenBank Accession Nos.
  • nucleotide sequence of the genomic region of Chromosome 2 in human, or chromosome 12 in mouse which includes the endogenous promoters of APOB and the APOB coding sequence is also known and may be found in: Mouse - mmlO genome build: chrl2:7968110- 8023150, Human - hgl9 genome build: chr2:21160333-21330910.
  • 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. 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 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).
  • 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
  • a nucleic acid comprises one or more modified sugars (e.g., 2'-fluororibose, ribose, 2'-deoxyribose, arabinose, and hexose) as compared with those in natural nucleic acids.
  • a nucleic acid has a nucleotide sequence that encodes a functional gene product such as an RNA or protein.
  • a nucleic acid includes one or more introns.
  • nucleic acids are prepared by one or more of isolation from a natural source, enzymatic synthesis by polymerization based on a complementary template (in vivo or in vitro), reproduction in a recombinant cell or system, and chemical synthesis.
  • a nucleic acid is at least 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 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.
  • next generation sequencing and “NGS” refer to massively parallel sequencing platforms to analyze genome, epigenome and transcriptome.
  • Certain sequencing platforms such as those marketed by Illumina®, Ion TorrentTM, RocheTM, and Life TechnologiesTM, involve solid phase amplification of target polynucleotides of unknown sequence.
  • the genomic DNA is fragmented into smaller lengths of DNA, for example, between 200-1200 nucleotides in length.
  • Solid phase amplification of these polynucleotides is typically performed by first ligating known adapter (such as, an oligonucleotide, primer, or probe) sequences to each end of a target polynucleotide.
  • the double-stranded polynucleotide is then denatured to form a single-stranded template molecule that is immobilized on the solid substrate.
  • the target polynucleotide is selectively captured and isolated (e.g., purified) from the rest of the polynucleotides in the sample.
  • the adapter sequence on the 3 ' end of the template is hybridized to an extension primer, and amplification is performed by extending the primer, thereby amplifying the target polynucleotide.
  • the polynucleotides Prior to sequencing, the polynucleotides can be treated with agents (such as, but not limited to, bisulfite) capable of altering the polynucleotide sequence and/or epigenetic modifications.
  • agents such as, but not limited to, bisulfite
  • bisulfite encompasses all types of bisulfites, such as sodium bisulfite, that are capable of chemically converting a cytosine (C) to a uracil (U) without chemically modifying a methylated cytosine and, therefore, can be used to differentially modify a DNA sequence based on the methylation status of the DNA.
  • cytosine deaminase refers to an enzyme capable of converting an unmodified cytosine to uracil, without chemically modifying a methylated cytosine.
  • the cytosine deaminase is Apolipoprotein B mRNA editing catalytic polypeptide-like (APOBEC).
  • APOBEC proteins belong to a family of deaminase proteins that can catalyze the deamination of cytosine to uracil on single-stranded DNA or/and RNA.
  • the term “adapter” can refer to an oligonucleotide of known sequence, the ligation of which to a target polynucleotide or a target polynucleotide strand of interest enables the generation of amplification-ready products of the target polynucleotide or the target polynucleotide strand of interest.
  • Various adapter designs can be used.
  • Suitable adapter molecules include single or double stranded nucleic acid (DNA or RNA) molecules or derivatives thereof, stem-loop nucleic acid molecules, double stranded molecules comprising one or more single stranded overhangs of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 bases or longer, proteins, peptides, aptamers, organic molecules, small organic molecules, or any adapter molecules known in the art that can be covalently or non-covalently attached, such as for example by ligation, to the double stranded nucleic acid fragments.
  • the adapters can be designed to comprise a double-stranded portion which can be ligated to double-stranded nucleic acid (or double-stranded nucleic acid with overhang) products.
  • oligonucleotide can refer to a polynucleotide chain, typically less than 200 residues long, e.g., between 15 and 100 nucleotides long, but also intended to encompass longer polynucleotide chains. Oligonucleotides can be single- or double-stranded.
  • primer and “oligonucleotide primer” can refer to an oligonucleotide capable of hybridizing to a complementary nucleotide sequence.
  • oligonucleotide can be used interchangeably with the terms “primer,” “adapter,” and “probe.”
  • the term “primer” can refer to an oligonucleotide, generally with a free 3' hydroxyl group, that is capable of hybridizing with a template (such as a target polynucleotide, target DNA, target RNA or a primer extension product) and is also capable of promoting polymerization of a polynucleotide complementary to the template.
  • a primer can contain a non-hybridizing sequence that constitutes a tail of the primer. A primer can still be hybridizing to a target even though its sequences may not fully complementary to the target.
  • hybridization /“hybridizing” and “annealing” can be used interchangeably and can refer to the pairing of complementary nucleic acids.
  • the DNA methylation is measured with a panel of oligonucleotides, that spans a combined length of genomic DNA which is at least 5 kb, at least 10 kb, at least 15 kb, at least 20 kb, at least 25 kb, at least 30 kb, at least 35 kb, at least 40 kb, at least 45 kb, at least 50 kb, at least 55 kb, at least 60 kb, at least 65 kb, at least 70 kb, at least 75 kb, at least 80 kb, at least 85 kb, at least 90 kb, at least 95 kb, at least 100 kb of genomic sequence.
  • the genomic region of interest comprises discontinuous genomic sequence. In some embodiments, the genomic region of interest comprises continuous genomic sequence. [0157] In some embodiments, the DNA methylation is measured on one or more biomarkers. In some embodiments, the DNA methylation is measured on one or more secondary biomarkers. In some embodiments, the DNA methylation is measured on one or more control genomic sequences.
  • the biomarker is the MYC gene.
  • a panel of oligonucleotides or adapters are designed to isolate regions of DNA sequence comprised in the MYC gene, MYC promoter, and/or MYC locus. For example, as shown in Table 1 and 2 below, the panel of oligonucleotides are designed to target the MYC promoter (bold), secondary biomarkers, genomic regions known to be methylated, and genomic regions known to be unmethylated.
  • epigenetic state refers to any structural feature at a molecular level of a nucleic acid (e.g., DNA or RNA) other than the primary nucleotide sequence.
  • a nucleic acid e.g., DNA or RNA
  • the epigenetic state of a genomic DNA may include its secondary or tertiary structure determined or influenced by, e.g., its methylation pattern or its association with cellular proteins.
  • methylation profile “methylation state” or “methylation status,” as used herein to describe the state of methylation of a genomic sequence, refers to the characteristics of a DNA segment at a particular genomic locus relevant to methylation. Such characteristics include, but are not limited to, whether any of the cytosine (C) residues within this DNA sequence are methylated, location of methylated C residue(s), percentage of methylated C at any particular stretch of residues, and allelic differences in methylation due to, e.g., epigenetic inheritance, disease or environmental factors.
  • methylation profile or “methylation status” also refers to the relative or absolute concentration of methylated C or unmethylated C at any particular stretch of residues in a biological sample.
  • cytosine (C) residue(s) within a DNA sequence are methylated it may be referred to as “hypermethylated”; whereas if the cytosine (C) residue(s) within a DNA sequence are not methylated it may be referred to as “hypomethylated”.
  • sequences are said to be “differentially methylated”, and more specifically, when the methylation status differs between different alleles in the same or different samples, the sequences are considered “epialleles”.
  • variable epiallele frequency or “variant epiallele fraction” or “VEF” can be used interchangeably and refer to the calculated frequency of methylated epialleles which pass the threshold parameters as defined at the onset of analysis of NGS data compared to epialleles which do not pass the threshold and are counted as unmethylated at an individual cytosine level.
  • a threshold or parameter used to determine VEF is the percentage of methylation of CpG sites on each fragment sequenced.
  • the genomic sequences are determined to be methylated when a threshold of at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or at least 100% of CpG sites on the sequence are methylated.
  • the threshold is at least 50% methylated CpG sites on the sequence are methylated.
  • determining the VEF comprises determining the fraction of a methylated CpG site which is comprised in a DNA fragment meeting the threshold compared to the fraction of the same CpG site which is comprised in a DNA fragment not meeting the threshold.
  • the algorithm used to determine VEF is epialleleR (for example, see Nikolaienko O, Lonning PE, Knappskog S. epialleleR: an R/Bioconductor package for sensitive allele-specific methylation analysis in NGS data. Gigascience. 2022 Dec 28). Without thresholding, epialleleR produces conventional cytosine reports similar to the ones produced by other tools (e.g., Bismark), which are not as sensitive at reading the level of methylation. In this case, methylation beta value for every genomic location is computed as a ratio of a number of methylated cytosines to the total number of methylated and unmethylated cytosines:
  • VEF C a /( C + T)
  • cell- free polynucleotides may be isolated from a non-cellular fraction of blood (e.g. serum or plasma), from other bodily fluids (e.g. urine), or from non-cellular fractions of other types of samples.
  • circulating tumor DNA or “circulating cancer DNA” refers to the fraction of cell-free DNA (cf DNA) that originates from a tumor.
  • circulating tumor RNA ctRNA
  • circulating cancer RNA refers to the fraction of cell-free RNA (cf RNA) that originates from a tumor.
  • a third class of extracellular vesicles are vesicles produced during apoptosis.
  • the vesicles may be produced by various factors, such as extracellular stimuli, microbial infections, and other disease, such as cancer.
  • Exemplary extracellular vesicles may include but are not limited to exosomes.
  • Exemplary extracellular vesicles may include microvesicles.
  • exosome refers to cell-derived vesicles having a diameter of between about 20- 140 nm, such as between 40 and 120 nm, preferably a diameter of about 50-100 nm, for example, a diameter of about 60 nm, 70 nm, 80 nm, 90 nm, or 100 nm.
  • Extracellular vesicles may be isolated from any suitable biological sample from a mammal, including but not limited to, whole blood, serum, plasma, urine, saliva, breast milk, cerebrospinal fluid, amniotic fluid, ascitic fluid, bone marrow and cultured mammalian cells (e.g. immature dendritic cells (wild-type or immortalized), induced and non-induced pluripotent stem cells, fibroblasts, platelets, immune cells, reticulocytes, tumour cells, mesenchymal stem cells, satellite cells, hematopoietic stem cells, pancreatic stem cells, white and beige preadipocytes and the like).
  • mammalian cells e.g. immature dendritic cells (wild-type or immortalized), induced and non-induced pluripotent stem cells, fibroblasts, platelets, immune cells, reticulocytes, tumour cells, mesenchymal stem cells, satellite cells, hematopoietic stem cells, pancreatic stem cells,
  • 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).
  • the term “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.
  • 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.
  • a delay in the onset of a particular symptom is considered one form of reducing the frequency of that symptom.
  • 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.
  • a nucleic acid having a particular sequence targets a nucleic acid of substantially complementary sequence.
  • target gene refers to 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).
  • the genomic sequence element is proximal to and/or operably linked to a target gene (e.g., MYC).
  • target gene e.g., MYC
  • target sequence refers to a sequence within the target gene which the targeting moiety specifically targets.
  • an epigenetic modifying agent e.g., comprising a DNA binding domain and effector domain targets a genomic locus and binds via the DNA binding domain.
  • 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 an expression activation system, e.g., an expression enhancer, described herein.
  • a therapeutic agent comprises a nucleic acid encoding an expression activation system, e.g., an expression enhancer, described herein.
  • a therapeutic agent comprises a pharmaceutical composition described herein.
  • a therapeutically effective amount means an amount of a substance e.g., a therapeutic agent, composition, and/or formulation) that elicits a desired biological response when administered as part of a therapeutic regimen.
  • a therapeutically effective amount of a substance is an amount that is sufficient, when administered to a subject suffering from or susceptible to a disease, disorder, and/or condition, to treat, diagnose, prevent, and/or delay the onset of the disease, disorder, and/or condition.
  • an effective amount of a substance may vary depending on such factors as desired biological endpoint(s), substance to be delivered, target cell(s) or tissue(s), etc.
  • an effective amount of compound in a formulation to treat a disease, disorder, and/or condition is an amount that alleviates, ameliorates, relieves, inhibits, prevents, delays onset of, reduces severity of and/or reduces incidence of one or more symptoms or features of the disease, disorder, and/or condition.
  • a therapeutically effective amount is administered in a single dose; in some embodiments, multiple unit doses are required to deliver a therapeutically effective amount.
  • the methods of the present invention also comprise measuring the methylation status of one or more biomarkers, e.g., the level of DNA methylation, and/or measuring the level of mRNA of one or more biomarkers in a biological sample obtained from a subject who has received the first dose of the epigenetic modifying agent.
  • DNA methylation in mammalian genomes typically refers to the addition of a methyl group to the 5' carbon of cytosine residues (i.e. 5-methylcytosines) among CpG dinucleotides.
  • DNA methylation may occur in cytosines in other contexts, for example CHG and CHH, where H is adenine, cytosine or thymine. Cytosine methylation may also be in the form of 5- hydroxymethylcytosine.
  • Non-cytosine methylation such as N6-methyladenine, has also been reported.
  • methylation status includes information related to DNA methylation for a region.
  • Information related to DNA methylation can include, but not limited to, a methylation density of CpG sites in a region, a distribution of CpG sites over a contiguous region, a pattern or level of methylation for each individual CpG site within a region that contains more than one CpG site, and non-CpG methylation.
  • methylation level is intended to include the demethylation or methylation of a DNA.
  • a DNA in question can be either methylated or non- or demethylated at least one site thereof. Since this condition is a binary one and thus the demethylation and methylation at a particular position are directly related to one another, the methylation level can be determined either by the demethylation and/or by the methylation at this at least one site. Thus, the normalized DNA methylation level as well as the relative methylation level can be determined via the methylation and/or demethylation of the DNA.
  • Methods of measuring methylation status may include, but are not limited to, massively parallel sequencing (e.g., next-generation sequencing) to determine methylation level, e.g., sequencing by — synthesis, real-time (e.g., single-molecule) sequencing, bead emulsion sequencing, nanopore sequencing, or other sequencing techniques known in the art.
  • a method of measuring the level of methylation can include whole-genome sequencing, e.g., measuring whole genome methylation status from bisulfite or enzymatically treated material with base-pair resolution.
  • Methylation-sensitive restriction enzymes that typically digest unmethylated DNA provide a low cost approach to study DNA methylation. Affinity capture or immunoprecipitation of DNA bound by anti-methylated cytosine antibodies can be used to survey large segments of the genome.
  • the methylation status can be measured by bisulfite sequencing, targeted enzymatic methylation sequencing, reduced representation bisulfite sequencing e.g., utilizing use of restriction enzymes to measure methylation status of high CpG content regions from bisulfite or enzymatically treated material with base-pair resolution, pyrosequencing, polymerase chain reaction (PCR)Zdigestion with restriction endonucleases, methylation-specific PCR, real-time PCR, Southern blot analysis, mass spectrometry, multiplex ligation-dependent probe amplification (MLP A), chromatin immunoprecipitation (ChIP), methylation microarray, high performance liquid chromatography (HPLC), high performance capillary electrophoresis (HPCE), methylation-sensitive single-nucleotide primer extension, methylation-sensitive single-stranded conformational polymorphism, methylation-sensitive restriction endonucleases, ligation mediated PCR,
  • restriction enzymes to measure
  • a method of measuring methylation status can include targeted sequencing e.g., measuring methylation status of pre-selected genomic location from bisulfite or enzymatically treated material with base-pair resolution.
  • the pre-selection (capture) of regions of interest can be done by complementary in vitro synthesized oligonucleotide sequences (either baits, primers or probes).
  • a method for measuring methylation status can include Illumina Methylation Assays e.g., measuring over 850,000 methylation sites quantitatively across a genome at single-nucleotide resolution.
  • Various methylation assay procedures can be used in conjunction with bisulfite treatment to determine methylation status of a target sequence.
  • Such assays can include, among others, methylation-specific restriction enzyme qPCR, sequencing of bisulfite-treated nucleic acid, PCR (e.g., with sequence-specific amplification), Methylation Specific Nuclease-assisted minor-allele enrichment PCR, and methylation-sensitive high resolution melting
  • the target sequence is amplified from a bisulfite-treated DNA sample and a DNA sequencing library is prepared for sequencing according to, e.g., an Illumina protocol or transpose-based Nextera XT protocol.
  • high-throughput and/or next-generation sequencing techniques are used to achieve base-pair level resolution of DNA sequence, permitting analysis of methylation status.
  • Another method, that can be used for methylation detection includes PCR amplification with methylation-specific oligonucleotide primers (MSP methods), e.g., as applied to bisulfite-treated sample (see, e.g., Herman 1992 Proc. Natl. Acad. Sci. USA 93: 9821-9826, which is herein incorporated by reference with respect to methods of determining methylation status).
  • MSP methods methylation-specific oligonucleotide primers
  • Use of methylation-status-specific oligonucleotide primers for amplification of bisulfite-treated DNA allows differentiation between methylated and unmethylated nucleic acids.
  • Oligonucleotide primer pairs for use in MSP methods include at least one oligonucleotide primer capable of hybridizing with sequence that includes a methylation site, e.g., a CpG.
  • An oligonucleotide primer that includes a T residue at a position complementary to a cytosine residue will selectively hybridize to templates in which the cytosine was unmethylated prior to bisulfite treatment, while an oligonucleotide primer that includes a G residue at a position complementary to a cytosine residue will selectively hybridize to templates in which the cytosine was methylated cytosine prior to bisulfite treatment.
  • MSP results can be obtained with or without sequencing amplicons, e.g., using gel electrophoresis.
  • MSP methylation-specific PCR
  • allows for highly sensitive detection detection level of 0.1% of the alleles, with full specificity
  • detection level of 0.1% of the alleles, with full specificity of locus-specific DNA methylation, using PCR amplification of bisulfite-converted DNA.
  • MS-HRM Methylation-Sensitive High Resolution Melting
  • a unique primer design facilitates a high sensitivity of the assays enabling detection of down to 0.1-1% methylated alleles in an unmethylated background.
  • Oligonucleotide primers for MS-HRM assays are designed to be complementary to the methylated allele, and a specific annealing temperature enables these primers to anneal both to the methylated and the unmethylated alleles thereby increasing the sensitivity of the assays.
  • QM-MSP Quantitative Multiplex Methylation-Specific PCR
  • QM-MSP uses methylation specific primers for sensitive quantification of DNA methylation (see, e.g., Fackler 2018 Methods Mol Biol. 1708:473-496, which is herein incorporated by reference with respect to methods of determining methylation status).
  • QM-MSP is a two-step PCR approach, where in the first step, one pair of gene-specific primers (forward and reverse) amplifies the methylated and unmethylated copies of the same gene simultaneously and in multiplex, in one PCR reaction.
  • This methylation-independent amplification step produces amplicons of up to 109 copies per pL after 36 cycles of PCR.
  • the amplicons of the first reaction are quantified with a standard curve using real-time PCR and two independent fluorophores to detect methylated/unmethylated DNA of each gene in the same well (e.g., 6FAM and VIC).
  • a standard curve using real-time PCR and two independent fluorophores to detect methylated/unmethylated DNA of each gene in the same well (e.g., 6FAM and VIC).
  • 6FAM and VIC two independent fluorophores
  • Ms-NaME Methylation Specific Nuclease-assisted Minor-allele Enrichment
  • DSN DNA nuclease specific to double-stranded DNA
  • oligonucleotide probes targeting unmethylated sequences generate local double stranded regions resulting to digestion of unmethylated targets; oligonucleotide probes capable of hybridizing to methylated sequences generate local double-stranded regions that result in digestion of methylated targets, leaving methylated targets intact.
  • oligonucleotide probes can direct DSN activity to multiple targets in bisulfite-treated DNA, simultaneously. Subsequent amplification can enrich nondigested sequences.
  • Ms-NaME can be used, either independently or in combination with other techniques provided herein.
  • Ms-SNuPETM Methylation-sensitive Single Nucleotide Primer Extension
  • Reaction products can be electrophoresed on polyacrylamide gels for visualization and quantitation by phosphor-image analysis.
  • Amplicons can also carry a directly or indirectly detectable labels such as a fluorescent label, radionuclide, or a detachable molecule fragment or other entity having a mass that can be distinguished by mass spectrometry. Detection may be carried out and/or visualized by means of, e.g., matrix assisted laser desorption/ ionization mass spectrometry (MALDI) or using electron spray mass spectrometry (ESI).
  • MALDI matrix assisted laser desorption/ ionization mass spectrometry
  • ESI electron spray mass spectrometry
  • Certain methods that can be used to determine the level of methylation after bisulfite treatment of a sample utilize a first oligonucleotide primer, a second oligonucleotide primer, and an oligonucleotide probe in an amplification-based method.
  • the oligonucleotide primers and probe can be used in a method of real-time polymerase chain reaction (PCR) or droplet digital PCR (ddPCR).
  • the first oligonucleotide primer, the second oligonucleotide primer, and/or the oligonucleotide probe selectively hybridize methylated DNA and/or unmethylated DNA, such that amplification or probe signal indicate methylation status of a sample.
  • Other bisulfite-based methods for detecting methylation status e.g., the presence of level of 5 -methylcytosine
  • Frommer (1992 Proc Natl Acad Sci USA. 1; 89(5): 1827-31 which is herein incorporated by reference with respect to methods of determining methylation status.
  • the amount of total DNA is measured in an aliquot of sample in native (e.g., undigested) form using, e.g., real-time PCR or digital PCR.
  • Amplification technologies can be used alone or in conjunction with other techniques described herein for detection of methylation status. Those of skill in the art, having reviewed the present specification, will understand how to combine various amplification technologies known in the art and/or described herein together with various other technologies for methylation level determination known in the art and/or provided herein.
  • Amplification technologies include, without limitation, PCR, e.g., quantitative PCR (qPCR), real-time PCR, and/or digital PCR.
  • qPCR quantitative PCR
  • qPCR quantitative PCR
  • real-time PCR real-time PCR
  • digital PCR digital PCR.
  • polymerase amplification can multiplex amplification of multiple targets in a single reaction. PCR amplicons are typically 100 to 2000 base pairs in length. In various instances, an amplification technology is sufficient to determine methylations status.
  • 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 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 5 or Table 6. In some embodiments, an expression repressor described herein binds to a target sequence listed in Table 5 or Table 6.
  • Table 5 Exemplary gRNA sequences
  • Table 6 Exemplary gRNA sequences
  • 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 repeat variable di-residues, RVD domain).
  • TAL effectors 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, AvrXalO and AvrBs3.
  • 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 an 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.
  • 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.
  • 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.
  • 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 6.
  • the nucleotide sequences encoding exemplary targeting moieties disclosed herein are listed in Table 9.
  • an expression repressor or system described herein comprises a targeting moiety having a sequence set forth in Table 8, 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 9, or a sequence with at least 70%, 75%, 80%, 85%, 90%, 95%, 98%, or 99% identity thereto.
  • an expression repression or expression enhancer 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, SFRP1, HNF4a , FOXP3, or APOB), 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, SFRP1, HNF4a , FOXP3, or APOB), e.g., a sequence proximal to the anchor sequence in mouse genome.
  • DBD engineered DNA binding domain
  • ZFN Zn finger domain comprising a Zn finger (ZFN) that binds to a target sequence
  • TSS transcription
  • 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 10.
  • the nucleotide sequences encoding exemplary targeting moieties disclosed herein are listed in Table 11.
  • an expression repressor or system described herein comprises a targeting moiety having a sequence set forth in Table 10, 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 11, or a sequence with at least 70%, 75%, 80%, 85%, 90%, 95%, 98%, or 99% identity thereto.
  • 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 W02014/012081) 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 or expression enhancer, 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
  • 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.
  • the effector moiety comprises a DNA methyltransferase, for example, MQ1, DNMT1, DNMT3A1, DNMT3A2, DNMT3B1, DNMT3B2, DNMT3B3, DNMT3B4, DNMT3B5, DNMT3B6, DNMT3L, or a functional variant or fragment of any thereof.
  • the effector moiety comprises a DNA demethylase, for example, DME, DML2, DML3, ROS1, TET1, TET2, TET3FL, TET3s, 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
  • 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.
  • 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
  • SUV39H1 e.g., as according to NP_003164.
  • NM_003173.4 histone demethylase LSD1 (e.g., as according to NP 055828.2 or the protein encoded by NM 015013.4); FOG1 (e.g., the N- terminal residues of FOG1) (e.g., as according to NP_722520.2 or the protein encoded by NM_153813.3); or KAP1 (e.g., as according to NP_005753.1 or the protein encoded by NM_005762.3); a functional fragment or variant 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.
  • 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.
  • DNMT3A e.g., human DNMT3A
  • 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.
  • NM_175867.3 1 or the protein encoded by NM_175867.3; DNMT3A/3L complex, bacterial MQ1 (e.g., as according to CAA35058.1 or P 15840.3); 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.
  • 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, SFRP1, HNF4a , FOXP3, or APOB.
  • a target gene e.g., MYC, SFRP1, HNF4a , FOXP3, or APOB.
  • 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, SFRP1, HNF4a , FOXP3, or APOB 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 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 MQ1, e.g., bacterial MQ1, or a functional variant or fragment thereof.
  • MQ 1 is Mollicutes spiroplasma MQ 1.
  • MQ 1 is Spiroplasma monobiae MQ1.
  • MQ1 is MQ1 from strain ATCC 33825 and/or corresponding to Uniprot ID P 15840.
  • MQ1 comprises an amino acid sequence of SEQ ID NO: 19 (Table 13).
  • MQ1 comprises an amino acid sequence of SEQ ID NO: 87 (Table 13).
  • 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 (Table 13).
  • 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.
  • MQ1 for use in a polypeptide or an expression repressor described herein is a variant, e.g., comprising one or more mutations, relative to a wild type MQ1 (e.g., SEQ ID NO: 19).
  • an MQ1 variant comprises one or more amino acid substitutions, deletions, or insertions relative to a wild type MQ1, e.g., the MQ1 of SEQ ID NO: 19.
  • an MQ 1 variant comprises a K297P substitution.
  • an MQ 1 variant comprises a N299C substitution.
  • an MQ1 variant comprises a E301 Y substitution.
  • an MQ1 variant comprises a Q147L substitution (e.g., and has reduced DNA methyltransferase activity relative to wild type MQ1).
  • an MQ1 variant comprises K297P, N299C, and E301Y substitutions (e.g., and has reduced DNA binding affinity relative to wild type MQ1).
  • an MQ1 variant comprises Q147L, K297P, N299C, and E301Y substitutions (e.g., and has reduced DNA methyltransferase activity and DNA binding affinity relative to wild type MQ1).
  • the polypeptide or the expression repressor is a fusion protein comprising an effector moiety that is or comprises MQ 1 and a targeting moiety is or comprises a zinc finger domain, TAL domain, or CRISPR/Cas domain, a dCas9 domain.
  • 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 MQ1, e.g., bacterial MQ1, or a functional variant or fragment thereof.
  • 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 DNMT1, e.g., human DNMT1, or a functional variant or fragment thereof.
  • DNMT1 is human DNMT1, e.g., corresponding to Gene ID 1786, e.g., corresponding to UniProt ID P26358.2.
  • DNMT1 comprises an amino acid sequence of SEQ ID NO: 20 (Table 12).
  • an effector domain described herein comprises a sequence according to SEQ ID NO: 20 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.
  • DNMT1 is encoded by a nucleotide sequence of SEQ ID NO: 53 (Table 14).
  • a nucleic acid described herein comprises a sequence of SEQ ID NO: 53 (Table 14).
  • NO: 53 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.
  • DNMT1 for use in a polypeptide or an expression repressor described herein is a variant, e.g., comprising one or more mutations, relative to a DNMT sequence of SEQ ID NO: 20.
  • the effector domain comprises one or more amino acid substitutions, deletions, or insertions relative to wild type DNMT1.
  • the polypeptide is a fusion protein comprising a repressor domain that is or comprises DNMT1 and a targeting moiety.
  • the targeting moiety is or comprises a zinc finger domain, TAL domain, or CRISPR/Cas domain, e.g., a dCas9 domain.
  • 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 DNMT1, or a functional variant or fragment thereof.
  • 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 DNMT3a/3L complex, or a functional variant or fragment thereof.
  • the DNMT3a/3L complex fusion construct comprises DNMT3A (e.g., human DNMT3A) (e.g., as according to NP_072046.2 or the protein encoded by NM_022552.4).
  • the DNMT3a/3L complex comprises DNMT3L (e.g., as according to NP_787063. 1 or the protein encoded by NM_175867.3).
  • DNMT3a/3L comprises an amino acid sequence of SEQ ID NO: 21 or SEQ ID NO: 114 (Table 13).
  • an effector domain described herein comprises SEQ ID NO: 21 or SEQ ID NO: 114, 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.
  • DNMT3a/3L is encoded by a nucleotide sequence of SEQ ID NO: 54 (Table 15).
  • a nucleic acid described herein comprises a sequence of SEQ ID NO: 54 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.
  • DNMT3a/3L for use in a polypeptide or an expression repressor described herein is a variant, e.g., comprising one or more mutations, relative to the DNMT3a/3L of SEQ ID NO: 21 or SEQ ID NO: 114.
  • an DNMT3a/3L variant comprises one or more amino acid substitutions, deletions, or insertions relative to SEQ ID NO: 21 or SEQ ID NO: 114.
  • the polypeptide or the expression repressor is a fusion protein comprising an effector moiety that is or comprises DNMT3a/3L and a targeting moiety.
  • the targeting moiety is or comprises a zinc finger domain, TAL domain, or CRISPR/Cas domain e.g., a dCas9 domain.
  • 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 DNMT3a/3L, or a functional variant or fragment thereof.
  • an effector moiety is or comprises a polypeptide. In some embodiments, an effector moiety is or comprises a nucleic acid. In some embodiments, an effector moiety is a chemical, e.g., a chemical that modulates a cytosine I or an adenine(A) (e.g., Na bisulfite, ammonium bisulfite). In some embodiments, an effector moiety has enzymatic activity (e.g., methyl transferase, demethylase, nuclease (e.g., Cas9), or deaminase activity). An effector moiety may be or comprise one or more of a small molecule, a peptide, a nucleic acid, a nanoparticle, an aptamer, or a pharmaco-agent with poor PK/PD.
  • an effector moiety may be or comprise one or more of a small molecule, a peptide, a nucleic acid, a nanop
  • an effector moiety may comprise a peptide ligand, a full-length protein, a protein fragment, an antibody, an antibody fragment, and/or a targeting aptamer.
  • the protein may bind a receptor such as an extracellular receptor, neuropeptide, hormone peptide, peptide drug, toxic peptide, viral or microbial peptide, synthetic peptide, or agonist or antagonist peptide.
  • an effector moiety may comprise antigens, antibodies, antibody fragments such as, e.g. single domain antibodies, ligands, or receptors such as, e.g., glucagon-like peptide- 1 (GLP- 1), GLP-2 receptor 2, cholecystokinin B (CCKB), or somatostatin receptor, peptide therapeutics such as, e.g., those that bind to specific cell surface receptors such as G protein-coupled receptors (GPCRs) or ion channels, synthetic or analog peptides from naturally-bioactive peptides, anti-microbial peptides, poreforming peptides, tumor targeting or cytotoxic peptides, or degradation or self-destruction peptides such as an apoptosis-inducing peptide signal or photosensitizer peptide.
  • GLP-1 glucagon-like peptide- 1
  • CCKB cholecystokinin B
  • Peptide or protein moieties for use in effector moieties as described herein may also include small antigen-binding peptides, e.g., antigen binding antibody or antibody-like fragments, such as, e.g., single chain antibodies, nanobodies (see, e.g., Steeland et al. 2016. Nanobodies as therapeutics: big opportunities for small antibodies. Drug Discov Today: 21(7): 1076-1 13).
  • small antigen binding peptides may bind, e.g., a cytosolic antigen, a nuclear antigen, an intra-organellar antigen.
  • an effector moiety comprises a dominant negative component (e.g., dominant negative moiety), e.g., a protein that recognizes and binds a sequence (e.g., an anchor sequence, e.g., a CTCF binding motif), but with an inactive (e.g., mutated) dimerization domain, e.g., a dimerization domain that is unable to form a functional anchor sequence-mediated conjunction), or binds to a component of a genomic complex (e.g., a transcription factor subunit, etc.) preventing formation of a functional transcription factor, etc.
  • a dominant negative component e.g., dominant negative moiety
  • a protein that recognizes and binds a sequence e.g., an anchor sequence, e.g., a CTCF binding motif
  • an inactive dimerization domain e.g., a dimerization domain that is unable to form a functional anchor sequence-mediated conjunction
  • the Zinc Finger domain of CTCF can be altered so that it binds a specific anchor sequence (by adding zinc fingers that recognize flanking nucleic acids), while the homo-dimerization domain is altered to prevent the interaction between engineered CTCF and endogenous forms of CTCF.
  • a dominant negative component comprises a synthetic nucleating polypeptide with a selected binding affinity for an anchor sequence within a target anchor sequence-mediated conjunction.
  • binding affinity may be at least 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or higher or lower than binding affinity of an endogenous nucleating polypeptide (e.g., CTCF) that associates with a target anchor sequence.
  • a synthetic nucleating polypeptide may have between 30-90%, 30-85%, 30-80%, 30-70%, 50-80%, 50-90% amino acid sequence identity to a corresponding endogenous nucleating polypeptide.
  • a nucleating polypeptide may modulate (e.g., disrupt), such as through competitive binding, e.g., competing with binding of an endogenous nucleating polypeptide to its anchor sequence.
  • an effector moiety comprises an antibody or fragment thereof.
  • target gene e.g., MYC, SFRP1, HNF4a , FOXP3, or APOB
  • expression is altered via use of effector moieties that are or comprise one or more antibodies or fragments thereof.
  • gene expression is altered via use of effector moieties that are or comprise one or more antibodies (or fragments thereof) and dCas9.
  • an antibody or fragment thereof for use in an effector moiety may be monoclonal.
  • An antibody may be a fusion, a chimeric antibody, a non-humanized antibody, a partially or fully humanized antibody, etc.
  • format of antibody(ies) used may be the same or different depending on a given target.
  • an effector moiety comprises a conjunction nucleating molecule, a nucleic acid encoding a conjunction nucleating molecule, or a combination thereof.
  • a conjunction nucleating molecule may be, e.g., CTCF, cohesin, USF1, YY1, TATA-box binding protein associated factor 3 (TAF3), ZNF143 binding motif, or another polypeptide that promotes formation of an anchor sequence-mediated conjunction.
  • a conjunction nucleating molecule may be an endogenous polypeptide or other protein, such as a transcription factor, e.g., autoimmune regulator (AIRE), another factor, e.g., X- inactivation specific transcript (XIST), or an engineered polypeptide that is engineered to recognize a specific DNA sequence of interest, e.g., having a zinc finger, leucine zipper or bHLH domain for sequence recognition.
  • a conjunction nucleating molecule may modulate DNA interactions within or around the anchor sequence-mediated conjunction (e.g., associated with or comprising the genomic sequence element targeted by the targeting moiety). For example, a conjunction nucleating molecule can recruit other factors to an anchor sequence that alters an anchor sequence-mediated conjunction formation or disruption.
  • a conjunction nucleating molecule may also have a dimerization domain for homo- or heterodimerization.
  • One or more conjunction nucleating molecules e.g., endogenous and engineered, may interact to form an anchor sequence-mediated conjunction.
  • a conjunction nucleating molecule is engineered to further include a stabilization domain, e.g., cohesion interaction domain, to stabilize an anchor sequence- mediated conjunction.
  • a conjunction nucleating molecule is engineered to bind a target sequence, e.g., target sequence binding affinity is modulated.
  • a conjunction nucleating molecule is selected or engineered with a selected binding affinity for an anchor sequence within an anchor sequence-mediated conjunction.
  • Conjunction nucleating molecules and their corresponding anchor sequences may be identified through use of cells that harbor inactivating mutations in CTCF and Chromosome Conformation Capture or 3C-based methods, e.g., Hi-C or high-throughput sequencing, to examine topologically associated domains, e.g., topological interactions between distal DNA regions or loci, in the absence of CTCF. Long-range DNA interactions may also be identified. Additional analyses may include ChlA-PET analysis using a bait, such as Cohesin, YY1 or USF1, ZNF143 binding motif, and MS to identify complexes that are associated with a bait.
  • a bait such as Cohesin, YY1 or USF1, ZNF143 binding motif
  • an effector moiety comprises a DNA-binding domain of a protein.
  • a DNA binding domain of an effector moiety enhances or alters targeting of a modulating agent but does not alone achieve complete targeting by a modulating agent (e.g., the targeting moiety is still needed to achieve targeting of the modulating agent).
  • a DNA binding domain enhances targeting of a modulating agent.
  • a DNA binding domain enhances efficacy of a modulating agent.
  • DNA-binding proteins have distinct structural motifs, e.g., that play, a key role in binding DNA, known to those of skill in the art.
  • a DNA-binding domain comprises a helix-tum-helix (HTH) motif, a common DNA recognition motif in repressor proteins.
  • HTH helix-tum-helix
  • Such a motif comprises two helices, one of which recognizes DNA (aka recognition helix) with side chains providing binding specificity.
  • recognition helix a common DNA recognition motif in repressor proteins.
  • Such motifs are commonly used to regulate proteins that are involved in developmental processes. Sometimes more than one protein competes for the same sequence or recognizes the same DNA fragment. Different proteins may differ in their affinity for the same sequence, or DNA conformation, respectively through H-bonds, salt bridges and Van der Waals interactions.
  • a DNA-binding domain comprises a helix-hairpin-helix (HhH) motif.
  • HhH helix-hairpin-helix
  • a DNA-binding domain comprises a helix-loop-helix (HLH) motif.
  • DNA- binding proteins with an HLH structural motif are transcriptional regulatory proteins and are principally related to a wide array of developmental processes.
  • An HLH structural motif is longer, in terms of residues, than HTH or HhH motifs. Many of these proteins interact to form homo- and hetero-dimers.
  • a structural motif is composed of two long helix regions, with an N-terminal helix binding to DNA, while a complex region allows the protein to dimerize.
  • a DNA-binding domain comprises a leucine zipper motif.
  • a dimer binding site with DNA forms a leucine zipper.
  • This motif includes two amphipathic helices, one from each subunit, interacting with each other resulting in a left-handed coiled- coil super secondary structure.
  • a leucine zipper is an interdigitation of regularly spaced leucine residues in one helix with leucines from an adjacent helix.
  • helices involved in leucine zippers exhibit a heptad sequence (abcdefg) with residues a and d being hydrophobic and other residues being hydrophilic.
  • Leucine zipper motifs can mediate either homo- or heterodimer formation.
  • a DNA-binding domain comprises a Zn finger domain, where a Zn ++ ion is coordinated by 2 Cys and 2 His residues.
  • a transcription factor includes a trimer with the stoichiometry (3(3 ‘a.
  • An apparent effect of Zn ++ coordination is stabilization of a small complex structure instead of hydrophobic core residues.
  • Each Zn-finger interacts in a conformationally identical manner with successive triple base pair segments in the major groove of the double helix.
  • Protein-DNA interaction is determined by two factors: (i) H-bonding interaction between a-helix and DNA segment, mostly between Arg residues and Guanine bases, (ii) H-bonding interaction with DNA phosphate backbone, mostly with Arg and His.
  • An alternative Zn-finger motif chelates Zn ++ with 6 Cys.
  • a DNA-binding domain comprises a TATA box binding protein (TBP).
  • TBP was first identified as a component of the class II initiation factor TFIID. These binding proteins participate in transcription by all three nuclear RNA polymerases acting as subunit in each of them. Structure of TBP shows two «/[> structural domains of 89-90 amino acids. The C-terminal or core region of TBP binds with high affinity to a TATA consensus sequence (TATAa/tAa/t, SEQ ID NO: 210) recognizing minor groove determinants and promoting DNA bending. TBP resemble a molecular saddle. The binding side is lined with central 8 strands of a 10-stranded anti-parallel [3- sheet. The upper surface contains four a-helices and binds to various components of transcription machinery.
  • a DNA-binding domain is or comprises a transcription factor.
  • Transcription factors may be modular proteins containing a DNA-binding domain that is responsible for specific recognition of base sequences and one or more effector domains that can activate or repress transcription. TFs interact with chromatin and recruit protein complexes that serve as coactivators or corepressors.
  • an effector moiety comprises one or more RNAs (e.g., gRNA) and dCas9.
  • one or more RNAs is/are targeted to a genomic sequence element via dCas9 and target-specific guide RNA.
  • RNAs used for targeting may be the same or different depending on a given target.
  • An effector moiety may comprise an aptamer, such as an oligonucleotide aptamer or a peptide aptamer. Aptamer moieties are oligonucleotide or peptide aptamers.
  • An effector moiety may comprise an oligonucleotide aptamer.
  • Oligonucleotide aptamers are single-stranded DNA or RNA (ssDNA or ssRNA) molecules that can bind to pre-selected targets including proteins and peptides with high affinity and specificity.
  • Oligonucleotide aptamers are nucleic acid species that may be engineered through repeated rounds of in vitro selection or equivalently, SELEX (systematic evolution of ligands by exponential enrichment) to bind to various molecular targets such as small molecules, proteins, nucleic acids, and even cells, tissues and organisms. Aptamers provide discriminate molecular recognition and can be produced by chemical synthesis. In addition, aptamers possess desirable storage properties, and elicit little or no immunogenicity in therapeutic applications.
  • DNA and RNA aptamers show robust binding affinities for various targets.
  • DNA and RNA aptamers have been selected for t lysozyme, thrombin, human immunodeficiency vims trans-acting responsive element (HIV TAR), hemin, interferon y, vascular endothelial growth factor (VEGF), prostate specific antigen (PSA), dopamine, and the non-classical oncogene, heat shock factor 1 (HSF1).
  • Diagnostic techniques for aptamer-based plasma protein profiling includes aptamer plasma proteomics. This technology will enable future multi-biomarker protein measurements that can aid diagnostic distinction of disease versus healthy states.
  • An effector moiety may comprise a peptide aptamer moiety.
  • Peptide aptamers have one (or more) short variable peptide domains, including peptides having low molecular weight, 12 — 14 Da.
  • Peptide aptamers may be designed to specifically bind to and interfere with protein-protein interactions inside cells.
  • Peptide aptamers are artificial proteins selected or engineered to bind specific target molecules. These proteins include of one or more peptide complexes of variable sequence. They are typically isolated from combinatorial libraries and often subsequently improved by directed mutation or rounds of variable region mutagenesis and selection. In vivo, peptide aptamers can bind cellular protein targets and exert biological effects, including interference with the normal protein interactions of their targeted molecules with other proteins. In particular, a variable peptide aptamer complex attached to a transcription factor binding domain is screened against a target protein attached to a transcription factor activating domain. In vivo binding of a peptide aptamer to its target via this selection strategy is detected as expression of a downstream yeast marker gene.
  • peptide aptamers derivatized with appropriate functional moieties can cause specific post-translational modification of their target proteins or change subcellular localization of the targets.
  • Peptide aptamers can also recognize targets in vitro. They have found use in lieu of antibodies in biosensors and used to detect active isoforms of proteins from populations containing both inactive and active protein forms.
  • tadpoles in which peptide aptamer “heads” are covalently linked to unique sequence double-stranded DNA “tails”, allow quantification of scarce target molecules in mixtures by PCR (using, for example, the quantitative real-time polymerase chain reaction) of their DNA tails.
  • Peptide aptamer selection can be made using different systems, but the most used is currently a yeast two-hybrid system.
  • Peptide aptamers can also be selected from combinatorial peptide libraries constructed by phage display and other surface display technologies such as mRNA display, ribosome display, bacterial display and yeast display. These experimental procedures are also known as biopannings. Among peptides obtained from biopannings, mimotopes can be considered as a kind of peptide aptamers.
  • Peptides panned from combinatorial peptide libraries have been stored in a special database with named MimoDB.
  • An exemplary effector moiety may include, but is not limited to: ubiquitin, bicyclic peptides as ubiquitin ligase inhibitors, transcription factors, DNA and protein modification enzymes such as topoisomerases, topoisomerase inhibitors such as topotecan, DNA methyltransferases such as the DNMT family (e.g., DNMT3A, DNMT3B, DNMT3a/3L, MQ1), protein methyltransferases (e.g., viral lysine methyltransferase (vSET), protein-lysine N- methyltransferase (SMYD2), deaminases (e.g., APOBEC, UG1), histone methyltransferases such as enhancer of zeste homolog 2 (EZH2), PRMT1, histone-lysine- N-methyltransferase (Setdbl), histone methyltransferase (SET2), Vietnamese histone-lysine N
  • a candidate domain may be determined to be suitable for use as an effector moiety by methods known to those of skill in the art.
  • a candidate effector moiety may be tested by assaying whether, when the candidate effector moiety is present in the nucleus of a cell and appropriately localized (e.g., to a target gene or transcription control element operably linked to said target gene, e.g., via a targeting moiety), the candidate effector moiety decreases expression of the target gene in the cell, e.g., decreases the level of RNA transcript encoded by the target gene (e.g., as measured by RNASeq or Northern blot) or decreases the level of protein encoded by the target gene (e.g., as measured by ELISA).
  • an expression repressor or expression enhancer comprises a plurality of effector moiety, wherein each effector moiety does not detectably bind, e.g., does not bind, to another effector moiety.
  • an expression repression system or expression enhancing system comprises a first expression repressor or expression enhancer comprising a first effector moiety and a second expression repressor or expression enhancer comprising a second effector moiety, wherein the first effector moiety does not detectably bind, e.g., does not bind, to the second effector moiety.
  • an expression repression system or expression enhancing system comprises a plurality of expression repressors or expression enhancers, wherein each member of the plurality of expression repressors or expression enhancers comprises an effector moiety, wherein each effector moiety does not detectably bind, e.g., does not bind, to another effector moiety.
  • an expression repression system or expression enhancing system comprises a first expression repressor or expression enhancer comprising a first effector moiety and a second expression repressor or expression enhancer comprising a second effector moiety, wherein the first effector moiety does not detectably bind, e.g., does not bind, to the second effector moiety.
  • an expression repression system or expression enhancing system comprises a first expression repressor or expression enhancer comprising a first effector moiety and a second expression repressor or expression enhancer comprising a second effector moiety, wherein the first effector moiety does not detectably bind, e.g., does not bind, to another first effector moiety, and the second effector moiety does not detectably bind, e.g., does not bind, to another second effector moiety.
  • an effector moiety for use in the compositions and methods described herein is functional in a monomeric, e.g., non-dimeric, state.
  • an effector moiety is or comprises an epigenetic modifying moiety, e.g., that modulates the two-dimensional structure of chromatin (i.e., that modulate structure of chromatin in a way that would alter its two-dimensional representation).
  • Epigenetic modifying moieties useful in methods and compositions of the present disclosure include agents that affect epigenetic markers, e.g., DNA methylation, histone methylation, histone acetylation, histone sumoylation, histone phosphorylation, and RNA-associated silencing.
  • Exemplary epigenetic enzymes that can be targeted to a genomic sequence element as described herein include DNA methylases (e.g., DNMT3a, DNMT3b, DNMT3a/3L, MQ1), DNA demethylation (e.g., the TET family), histone methyltransferases, histone deacetylase (e.g., HDAC1, HDAC2, HDAC3), sirtuin 1, 2, 3, 4, 5, 6, or 7, lysine-specific histone demethylase 1 (LSD1), histone-lysine-N -methyltransferase (Setdbl), euchromatic histone-lysine N-methyltransferase 2 (G9a), histone-lysine N-methyltransferase (SUV39H1), enhancer of zeste homolog 2 (EZH2), viral lysine methyltransferase (vSET), histone methyltransferase (SET2), and protein-lysine N-methyltrans
  • an expression repressor e.g., comprising an epigenetic modifying moiety, useful herein comprises or is a construct described in Koferle et al. Genome Medicine 7.59 (2015): 1-3 incorporated herein by reference.
  • an expression repressor comprises or is a construct found in Table 1 of Koferle et al., e.g., histone deacetylase, histone methyltransferase, DNA demethylation, or H3K4 and/or H3K9 histone demethylase described in Table 1 (e.g., dCas9-p300, TALE-TET1, ZF-DNMT3A, or TALE-LSD1).
  • an effector moiety comprises a component of a gene editing system e.g, a CRISPR/Cas domain, e.g., a Zn Finger domain, e.g., a TAL effector domain.
  • a CRISPR/Cas domain e.g., a Zn Finger domain, e.g., a TAL effector domain.
  • an epigenetic modifying moiety may comprise a polypeptide (e.g., peptide or protein moiety) linked to a gRNA and a targeted nuclease, e.g., a Cas9, e.g., a wild type Cas9, a nickase Cas9 (e.g., Cas9 D10A), a catalytically inactive Cas9 (dCas9), eSpCas9, Cpfl, C2C1, or C2C3, or a nucleic acid encoding such a nuclease.
  • a Cas9 e.g., a wild type Cas9, a nickase Cas9 (e.g., Cas9 D10A)
  • dCas9 catalytically inactive Cas9
  • eSpCas9 eSpCas9
  • Cpfl C2C1, or C2C3
  • nucleic acid encoding
  • biomarker means any gene, protein, or a fragment derived from that gene, the expression or level of which changes between certain conditions. Where the expression of the gene correlates with a certain condition, the gene is a biomarker for that condition.
  • a biomarker comprises a polynucleotide, such as a DNA of a gene locus, e.g, MYC, SFRP1, HNF4a , FOXP3, or APOB, or RNA transcribed from the biomarker gene.
  • the biomarker is the target gene, e.g., gene that is targeted for modulation, e.g., of expression.
  • the biomarker which is a target gene, is DNA of a gene locus (e.g., MYC, SFRP1, HNF4a , FOXP3, or APOB) or RNA transcribed from the biomarker gene.
  • the target gene as the biomarker is a primary biomarker.
  • the biomarker is a secondary biomarker, whereby expression or DNA modification of the secondary biomarker is indirectly affected by administration of the epigenetic modifying moiety.
  • the methylation status of the secondary biomarker is modified, e.g., increased or decreased, by the epigenetic modifying moiety targeting the target gene.
  • the expression of the secondary biomarker is altered, e.g., repressed or enhanced, by the epigenetic modifying moiety targeting the target gene.
  • the secondary biomarker gene is associated with cancer.
  • the cancer is hepatocellular carcinoma (HCC).
  • the cancer is non-small cell lung cancer (NSCLC).
  • the methylation state of one or more of the secondary biomarker genes listed in Table 16 can be analyzed.
  • the one or more secondary biomarker genes listed in Table 16 can be analyzed for gene expression, e.g., mRNA quantification.
  • HCC Hepatocellular Carcinoma
  • a “biologically active portion of an effector domain” is a portion that maintains function (e.g., completely, partially, minimally) of an effector domain (e.g., a “minimal” or “core” domain).
  • fusion of a dCas9 with all or a portion of one or more effector domains of an epigenetic modifying agent creates a chimeric protein that is linked to the polypeptide and useful in the methods described herein.
  • an epigenetic modifying agent such as a DNA methylase or enzyme with a role in DNA demethylation, e.g., DNMT3a, DNMT3b, DNMT3L, a DNMT inhibitor, combinations thereof, TET family enzymes, protein acetyl transferase or deacetylase, dCas9-DNMT3a/3L, dCas9- DNMT3a/3L/KRAB, dCas9/VP64) creates a chimeric protein that is linked to the polypeptide and useful in the methods described herein.
  • an epigenetic modifying agent such as a DNA methylase or enzyme with a role in DNA demethylation, e.g., DNMT
  • An effector moiety comprising such a chimeric protein is referred to as either a genetic modifying moiety (because of its use of a gene editing system component, Cas9) or an epigenetic modifying moiety (because of its use of an effector domain of an epigenetic modifying agent).
  • a gRNA that specifically targets a target gene.
  • the target gene is an oncogene, a tumor suppressor, or a MYC mis-regulation disorder related gene.
  • the target gene is MYC.
  • the target gene is SFRP1.
  • the target gene is HNF4a.
  • the target gene is FOXP3.
  • the target gene is APOB.
  • technologies provided herein include methods of delivering one or more genetic modifying moieties (e.g., CRISPR system components) described herein to a subject, e.g., to a nucleus of a cell or tissue of a subject, by linking such a moiety to a targeting moiety as part of a fusion molecule.
  • a genetic modifying moieties e.g., CRISPR system components
  • technologies provided herein include methods of delivering one or more genetic modifying moieties (e.g., CRISPR system components) described herein to a subject, e.g., to a nucleus of a cell or tissue of a subject, by encapsulating the one or more genetic modifying moieties (e.g., CRISPR system components) in a lipid nanoparticle.
  • a genetic modifying moieties e.g., CRISPR system components
  • An expression repressor may further comprise one or more additional moieties (e.g., in addition to one or more targeting moieties and one or more effector moieties).
  • an additional moiety is selected from a tagging or monitoring moiety, a cleavable moiety (e.g., a cleavable moiety positioned between a DNA-targeting moiety and an effector moiety or at the N- or C-terminal end of a polypeptide), a small molecule, a membrane translocating polypeptide, or a pharmaco-agent moiety.
  • an expression repressor comprises a targeting moiety comprising dCas9, e.g., an S. aureus dCas9, and an effector moiety comprising MQ1, e.g., bacterial MQ1.
  • the expression repressor is encoded by the nucleic acid sequence of SEQ ID NOs: 68 (e.g., a nucleic acid (e.g., cDNA) encoding the expression repressor).
  • the expression repressor is encoded by the nucleic acid sequence of SEQ ID NOs: 119.
  • the expression repressor is encoded by the nucleic acid sequence of SEQ ID NOs: 209.
  • a nucleic acid described herein comprises a nucleic acid sequence of SEQ ID NO: 68, 1 19,209 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.
  • an expression repressor comprises the amino acid sequence of SEQ ID NOs: 35 or 151.
  • an expression repressor described herein comprises an amino acid sequence of SEQ ID NO: 35, 151, 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.
  • an expression repressor comprises a targeting moiety comprising dCas9, e.g., an S. pyogenes dCas9, and an effector moiety comprising KRAB, e.g., a KRAB domain.
  • the expression repressor is encoded by the nucleic acid sequence of SEQ ID NOs: 67 (e.g., a nucleic acid (e.g., cDNA) encoding the expression repressor).
  • the expression repressor is encoded by the nucleic acid sequence of SEQ ID NOs: 210.
  • a nucleic acid described herein comprises a nucleic acid sequence of SEQ ID NO: 67, 210, 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.
  • an expression repressor comprises the amino acid sequence of SEQ ID NOs: 34 or 150.
  • a nucleic acid described herein comprises an amino acid sequence of SEQ ID NO: 34, 150, 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.
  • an expression repressor comprises a DNA-targeting moiety comprising dCas9, e.g., an S. aureus dCas9, and an effector moiety comprising DNMT1, e.g., human DNMT 1.
  • the expression repressor is encoded by the nucleic acid sequence of SEQ ID NO: 69 (e.g., a nucleic acid (e.g., cDNA) encoding the expression repressor).
  • the expression repressor is encoded by the nucleic acid sequence of SEQ ID NO: 211.
  • a nucleic acid described herein comprises a nucleic acid sequence of SEQ ID NO: 69, 211, 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.
  • an expression repressor comprises the amino acid sequence of SEQ ID NOs: 36, or 152.
  • an expression repressor described herein comprises an amino acid sequence of SEQ ID NO: 36, 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. Table 19
  • an expression repressor comprises a DNA-targeting moiety comprising dCas9, e.g., an S. aureus dCas9, and an effector moiety comprising DNMT13a/3L.
  • the expression repressor is encoded by the nucleic acid sequence of SEQ ID NO: 70 (e.g., a nucleic acid (e.g., cDNA) encoding the expression repressor).
  • the expression repressor is encoded by the nucleic acid sequence of SEQ ID NO: 212.
  • a nucleic acid described herein comprises a nucleic acid sequence of SEQ ID NO: 70, 212, 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.
  • an expression repressor comprises the amino acid sequence of SEQ ID NO: 37 or 153.
  • an expression repressor described herein comprises an amino acid sequence of SEQ ID NO: 37, 153, 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.
  • an expression repressor comprises a targeting moiety comprising a Zn Finger domain, and an effector moiety comprising KRAB, e.g., a KRAB domain.
  • the expression repressors are encoded by a nucleic acid sequence of any of SEQ ID NOs: 55, 56, 57, 58, 59, 60, 189, 193, 194, 195, 213, 214, 215, 216, 217, 218, 219, 220, 221, 222, 223, and 224 (e.g., a nucleic acid (e.g., cDNA) encoding the expression repressor).
  • nucleic acid sequences of these exemplary expression repressors are disclosed in Table 18.
  • a nucleic acid described herein comprises a nucleic acid sequence of any of SEQ ID NOs: 55-60, 189, 193, 194, 195, 213, 214, 215, 216, 217, 218, 219, 220, 221, 222, 223, 224 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.
  • the nucleic acid sequence comprises a poly-A sequence, and in other embodiments, the nucleic acid lacks the poly-A sequence.
  • an expression repressor comprises a targeting moiety comprising a Zn Finger domain (e.g., having an amino acid sequence according to any of SEQ ID NO: 5-10 or 169- 172), and an effector moiety comprising KRAB (e.g., an amino acid sequence SEQ ID NO: 18), e.g., a KRAB domain.
  • an expression repressor described herein comprises an amino sequence of any of SEQ ID NOs: 22, 23, 24, 25, 26, 27, 134, 139-144, 177-180, or 183-186. The protein sequence of these exemplary expression repressors are disclosed in Table 22.
  • an expression repressor described herein comprises an amino acid sequence of any of SEQ ID NOs: 22-27, 134, 139-144, 177-180, 183-186 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.
  • an expression repressor comprises a targeting moiety comprising a Zn Finger domain (e.g., one encoded by a nucleotide sequence of any of SEQ ID NO: 44-49 or 115), and an effector moiety comprising MQ1, e.g., a bacterial MQ1 (e.g., one encoded by a nucleotide sequence of SEQ ID NO: 52).
  • a targeting moiety comprising a Zn Finger domain (e.g., one encoded by a nucleotide sequence of any of SEQ ID NO: 44-49 or 115)
  • an effector moiety comprising MQ1, e.g., a bacterial MQ1 (e.g., one encoded by a nucleotide sequence of SEQ ID NO: 52).
  • the expression repressors are encoded by the nucleic sequence of SEQ ID NOs: 61, 62, 63, 64, 65, 66, 116, 117, 118, 130, 225, 226, 227, 228, 229, 230, or 231.
  • the nucleic acid sequence of these exemplary expression repressors are disclosed in Table 20.
  • a nucleic acid described herein comprises a nucleic acid sequence of any of SEQ ID NO: 61-66, 116-118, 130, 225, 226, 227, 228, 229, 230, 231 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 nucleic acid sequence comprises a poly-A sequence, and in other embodiments, the nucleic acid lacks the poly-A sequence.
  • a nucleic acid described herein comprises a sequence according to any of SEQ ID NO: 61-66, 116-118, 130, 225, 226, 227, 228, 229, 230, or 231 (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), but lacking the 3 ’ poly-A sequence, or comprising a 3 ’ poly-A sequence of a shorter length.
  • an expression repressor comprises a targeting moiety comprising a Zn Finger domain (e.g., comprising an amino acid sequence of any of SEQ ID NO: 11-14), and an effector moiety comprising MQ1, e.g., a bacterial MQ1 (e.g., SEQ ID NO: 19).
  • the expression repressor comprises an amino sequence of any of SEQ ID NOs: 28, 29, 30, 31, 32, ,33, 129, 133 and 145-149. The protein sequence of these exemplary expression repressors are disclosed in Table 24.
  • an expression repressor described herein comprises an amino acid sequence of any of SEQ ID NOs: 28-33, 129, 133, 145-149, 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.
  • an expression repressor comprises a targeting moiety comprising a Zn Finger domain (e.g., having an amino acid sequence of any of SEQ ID NO: 11-14), and an effector moiety comprising MQ1, e.g., a bacterial MQ1 (e.g., SEQ ID NO: 87).
  • a targeting moiety comprising a Zn Finger domain (e.g., having an amino acid sequence of any of SEQ ID NO: 11-14), and an effector moiety comprising MQ1, e.g., a bacterial MQ1 (e.g., SEQ ID NO: 87).
  • an expression repressor comprises a targeting moiety comprising a Zn Finger domain (e.g., one encoded by a nucleotide sequence of any of SEQ ID NO: 166-168, 232, 233, 234, 235, 236), and an effector moiety comprising MQ1, e.g., a bacterial MQ1 (e.g., one encoded by a nucleotide sequence of SEQ ID NO: 52).
  • the expression repressors are encoded by the nucleic sequence of SEQ ID NOs: 157, 158, or 159. The nucleic acid sequence of these exemplary expression repressors are disclosed in Table 25.
  • a nucleic acid described herein comprises a nucleic acid sequence of any of SEQ ID NO: 166-168, 232, 233, 234, 235, 236, 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 nucleic acid sequence comprises a poly-A sequence, and in other embodiments, the nucleic acid lacks the poly-A sequence.
  • a nucleic acid described herein comprises a sequence according to any of SEQ ID NO: 166-168, 232, 233, 234, 235, 236 (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), but lacking the 3’ poly-A sequence, or comprising a 3 ’ poly-A sequence of a shorter length.
  • an expression repressor comprises a targeting moiety comprising a Zn Finger domain (e.g., comprising an amino acid sequence of any of SEQ ID NO: 154-156), and an effector moiety comprising MQ1, e.g., a bacterial MQ1 (e.g., SEQ ID NO: 19).
  • the expression repressor comprises an amino sequence of any of SEQ ID NOs: 160- 165. The protein sequences of these exemplary expression repressors are disclosed in Table 25.
  • an expression repressor described herein comprises an amino acid sequence of any of SEQ ID NOs: 160-165 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 present disclosure provides an expression repressor system comprising a first targeting moiety comprising a first ZF, a first effector moiety comprising a DNA methyltransferase, e.g., MQ1 or a functional fragment thereof, a second targeting moiety comprising a second ZF, and a second effector moiety comprising KRAB, e.g., a KRAB domain.
  • the expression repressor system is encoded by a first nucleic acid encoding the first targeting moiety and first effector moiety, wherein expression is driven by a first promoter or IRES, and a second nucleic acid encoding the second targeting moiety and second effector moiety, wherein expression is driven by a second promoter or IRES.
  • mono-cistronic sequences are used.
  • the nucleic acid encoding the expression repressor system is a multi- cistronic sequence. In some embodiments, the multi-cistronic sequence is a bi-cistronic sequence.
  • the multi-cistronic sequence comprises a sequence encoding the first expression repressor and a sequence encoding the second expression repressor.
  • the multi- cistronic sequence encodes a self-cleavable peptide sequence, e.g., a 2A peptide sequence, e.g., a T2A peptide sequence, a P2A sequence.
  • the multi-cistronic sequence encodes a T2A peptide sequence and a P2A peptide sequence.
  • the multi-cistronic sequence encodes a tandem 2A sequence, e.g., a tPT2A sequence.
  • the multi- cistronic construct encodes, from 5’ to 3’, (i) a first nuclear localization signal, e.g., a SV40 NLS, (ii) a first targeting moiety, e.g., a DNA binding domain, e.g., a zinc finger binding domain, e.g., ZF-9, (iii) a first effector moiety, e.g., a DNA methyltransferase, e.g., MQ1, (iv) a second nuclear localization signal, e.g., a nucleoplasmin NLS, (v) a linker, e.g., a tPT2A linker, (vi) a third nuclear localization signal, e.g., a SV40NLS, (vii) a second targeting moiety, e.g., a DNA binding domain, e.g., a zinc finger binding domain, e.g., ZF-3
  • the bi-cistronic construct further comprises a polyA tail.
  • a single mRNA transcript encoding the first expression repressor, and the second expression repressor are produced, which upon translation gets cleaved, e.g., after the glycine residue within the 2A peptide, to yield the first expression repressor and the second expression repressor as two separate proteins.
  • the first and the second expression repressor are separated by “ribosome-skipping”.
  • the first expression repressor and/ or the second expression repressor retains a fragment of the 2A peptide after ribosome skipping.
  • the expression level of the first and second expression repressor are equal. In some embodiments, the expression level of the first and the second expression repressor are different. In some embodiments, the protein level of the first expression repressor is within 1%, 2%, 5%, or 10% of (greater than or less than) the protein level of the second expression repressor.
  • a system encoded by a bi-cistronic nucleic acid decreases expression of a target gene (e.g., MYC) at least 1%, at least 2%, at least 3%, at least 4%, at least 5%, at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, in a cell, than an otherwise similar system wherein the first and second expression repressor are encoded by mono-cistronic nucleic acids.
  • a target gene e.g., MYC
  • the bi-cistronic sequence encodes an amino acid of SEQ ID NO: 91, 92, 121, 122, 181, 182, 187, 188, 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.
  • an expression repressor system comprises a targeting moiety comprising a Zn Finger domain (e.g., comprising an amino acid sequence of any of SEQ ID NO: 7 or 13), and an effector moiety comprising MQ1, e.g., a bacterial MQ1 (e.g., SEQ ID NO: 19) or KRAB, e.g., a KRAB domain (e.g., SEQ ID NO: 18).
  • the expression repressor comprises an amino sequence of any of SEQ ID NOs: 91, 92, 121, 122, 181, 182, 187, or 188.
  • the protein sequence of these exemplary expression repressor systems are disclosed in Table 24.
  • an expression repressor system described herein comprises an amino acid sequence of any of SEQ ID NOs: 91, 92, 121, 122, 181, 182, 187, or 188, 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 bi-cistronic sequence comprises nucleic acid sequence of SEQ ID NO: 93 or 112 (e.g., a nucleic acid (e.g., cDNA) encoding the expression repressor) or SEQ ID NO: 94 or 113 (e.g., a nucleic acid (e.g., cDNA) encoding the expression repressor).
  • the bi- cistronic sequence comprises nucleic acid sequence of SEQ ID NO: 196 (e.g., a nucleic acid (e.g., cDNA) encoding the expression repressor) or SEQ ID NO: 197 (e.g., a nucleic acid (e.g., cDNA) encoding the expression repressor).
  • the bi-cistronic sequence comprises nucleic acid sequence of SEQ ID NO: 237.
  • the bi-cistronic sequence comprises nucleic acid sequence of SEQ ID NO: 238.
  • the bi- cistronic sequence comprises nucleic acid sequence of SEQ ID NO: 239.
  • the bi-cistronic sequence comprises nucleic acid sequence of SEQ ID NO: 240. In some embodiments, the bi-cistronic sequence comprises nucleic acid sequence of SEQ ID NO: 241. In some embodiments, the bi-cistronic sequence comprises nucleic acid sequence of SEQ ID NO: 242. In some embodiments, the bi-cistronic sequence comprises nucleic acid sequence of SEQ ID NO: 243. In some embodiments, the bi-cistronic sequence comprises nucleic acid sequence of SEQ ID NO: 244. In some embodiments, the bi-cistronic sequence comprises nucleic acid sequence of SEQ ID NO: 245. In some embodiments, the bi-cistronic sequence comprises nucleic acid sequence of SEQ ID NO: 246.
  • a nucleic acid described herein comprises a nucleic acid sequence of SEQ ID NO: 93, 94, 112, 113, 196, 197, 237, 238, 239, 240, 241, 242, 243, 244, 245, 246 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 nucleic acid sequence encoding these exemplary expression repressor systems are disclosed in Table 27.
  • the nucleic acid sequence comprises a poly-A sequence, and in other embodiments, the nucleic acid lacks the poly-A sequence.
  • an expression repressor or expression enhancer comprises a nuclear localization sequence (NLS).
  • the expression repressor or expression enhancer comprises an NLS, e.g., an SV40 NLS at the N-terminus.
  • the expression repressor or expression enhancer comprises an NLS, e.g., a nucleoplasmin NLS at the C- terminus.
  • the expression repressor or expression enhancer comprises a first NLS at the N- terminus and a second NLS at the C-terminus.
  • the first and the second NLS have the same sequence.
  • the first and the second NLS have different sequences.
  • the expression repressor or expression enhancer comprises an SV40 NLS, e.g., the expression repressor or expression enhancer comprises a sequence according to PKKKRK (SEQ ID NO: 135).
  • the N-terminal sequence comprises an NLS and a spacer, e.g., having a sequence according to: MAPKKKRKVGIHGVPAAGSSGS (SEQ ID NO: 88).
  • the expression repressor or expression enhancer comprises a C-terminal sequence comprising one or more of, e.g., any two or all three of: a spacer, a nucleoplasmin nuclear localization sequence and an HA-tag: e g., SGGKRPAATKKAGQAKKKGSYPYDVPDYA (SEQ ID NO: 89).
  • the expression repressor or expression enhancer comprises an epitope tag, e.g., an HA tag: YPYDVPDYA (SEQ ID NO: 90).
  • the expression repressor or expression enhancer may comprise two copies of the epitope tag.
  • an expression repressor or expression enhancer lacks an epitope tag.
  • an expression repressor or expression enhancer described herein comprises a sequence provided herein (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), but lacking the HA tag of SEQ ID NO: 90.
  • a nucleic acid described herein comprises a sequence provided herein (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), but lacking a region encoding the HA tag of SEQ ID NO: 90.
  • the expression repressor or expression enhancer comprises a nucleoplasmin NLS, e.g., the expression repressor or expression enhancer comprises a sequence of KRPAATKKAGQAKKK (SEQ ID NO: 136). In some embodiments, the expression repressor or expression enhancer does not comprise an NLS.
  • the expression repressor or expression enhancer does not comprise an epitope tag. In some embodiments the expression repressor or expression enhancer does not comprise an HA tag. In some embodiments, the expression repressor or expression enhancer does not comprise an HA tag sequence according to SEQ ID NO: 90. In some embodiments, the present disclosure provides an expression repressor system or expression enhancing system comprises a self-cleaving peptide. Selfcleaving peptides, first discovered in picomaviruses, are peptides of between 19 to 22 amino acids in length and are usually found between two proteins in some members of the picomavirus family.
  • an expression repressor system or expression enhancing system comprises a selfcleaving peptide, e.g., a 2A self-cleaving peptide.
  • the 2A peptide comprises a single cleavage site, e.g., a 2A peptide, e.g., a P2A, a T2A, a E2A, or a F2A peptide.
  • the self-cleaving peptide e.g., a 2A peptide, comprises two cleavage sites, , e.g., pPT2A, or P2A-T2A-E2A.
  • an expression repressor system or expression enhancing system comprises a self-cleaving peptide comprising a plurality of cleavage sites, e.g., a T2A self-cleaving peptide and a P2A self-cleaving peptide.
  • the 2A peptide gets cleaved after translation.
  • the self-cleaving peptide produces two or more fragments after cleaving.
  • the 2A peptide fragments comprise the sequences of SEQ ID NO: 126-128.
  • the 2A self-cleaving peptide comprises a sequence of SEQ ID NO: 120, 124, 125 or derivative thereof.
  • SEQ ID NO: 95 comprises a sequence of a self-cleaving peptide.
  • a 2A sequence e.g., tPT2A sequence (e.g., according to SEQ ID NO: 124)
  • tPT2A sequence e.g., according to SEQ ID NO: 124
  • a 2 A sequence acts via ribosome-skipping.
  • an mRNA encoding a 2 A sequence may induce ribosome skipping, wherein the ribosome fails to form a peptide bond while translating the 2A region, resulting in a release of the first part of the translation product. The ribosome then produces the second part of the translation product.
  • An expression repressor or expression enhancer or a system of the present disclosure can be used to decrease or increase expression of a target gene, e.g., MYC, SFRP1, HNF4a , FOXP3, or APOB, in a cell.
  • a target gene e.g., MYC, SFRP1, HNF4a , FOXP3, or APOB
  • an expression repressor or expression enhancer or a system 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, SFRP1, HNF4a , FOXP3, or APOB.
  • binding of the expression repressor or expression enhancer or a system to the genomic sequence element modulates (e.g., decreases or increases) expression of the target gene, e.g., MYC, SFRP1, HNF4a , FOXP3, or APOB.
  • binding of an expression repressor or expression enhancer or a system comprising an effector moiety that 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, SFRP1, HNF4a , FOXP3, or APOB.
  • binding of an expression repressor or expression enhancer or a system comprising an effector moiety with an enzymatic activity may modulate (e.g., decrease or increase) expression of the target gene, e.g., MYC, SFRP1, HNF4a , FOXP3, or APOB) through the localized enzymatic activity of the effector moiety.
  • an enzymatic activity e.g., an epigenetic modifying moiety
  • both binding of an expression repressor or expression enhancer or a system to a genomic sequence element and the localized enzymatic activity of an expression repressor or expression enhancer or a system may contribute to the resulting modulation (e.g., decrease or increase) in expression of the target gene, e.g., MYC, SFRP1, HNF4a , FOXP3, or APOB.
  • the target gene e.g., MYC, SFRP1, HNF4a , FOXP3, or APOB.
  • decreasing or increasing expression comprises decreasing or increasing, respectively, the level of RNA, e.g., mRNA, encoded by the target gene e.g., MYC, SFRP1, HNF4a , FOXP3, or APOB.
  • decreasing or increasing expression comprises decreasing or increasing, respectively, the level of a protein encoded by the target gene e.g., MYC, SFRP1, HNF4a , FOXP3, or APOB.
  • decreasing or increasing expression comprises both decreasing or increasing, respectively, the level of mRNA and protein encoded by the target gene e.g., MYC, SFRP1, HNF4a , FOXP3, or APOB.
  • the expression of a target gene in a cell contacted by or comprising the expression repressor or expression enhancer or the expression repression system expression enhancing system disclosed herein is at least 1.05x (i.e., 1.05 times), l.
  • lx 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, lOx, 20x, 30x, 40x, 50x, 60x, 70x, 80x, 90x, or lOOx lower or higher, respectively, than the level of expression of the target gene in a cell not contacted by or comprising the expression repressor or expression enhancer or the expression repression system or expression enhancing system disclosed herein.
  • a target gene e.g., MYC, SFRP1, HNF4a , FOXP3, or APOB may be assayed by methods known to those of skill in the art, including RT-PCR, ELISA, Western blot.
  • Expression level of a target gene, e.g., MYC, SFRP1, HNF4a , FOXP3, or APOB in a subject e.g., a patient, e.g., a patient having a MYC mis-regulation disorder, e.g., a patient having a hepatic disease, a patient having a neoplasia and/or viral or alcohol related hepatic disease, e.g., a patient having a hepatocarcinoma, e.g., a patient having a hepatocarcinoma subtype SI or hepatocarcinoma subtype S2, may be assessed by evaluating blood (e.g., whole blood) levels of the target gene, e.g., MYC, e.g., by the method of either Oglesbee et al.
  • blood e.g., whole blood
  • An expression repressor or expression enhancer or a system of the present disclosure can be used to decrease or increase expression, respectively, of a target gene e.g., MYC, SFRP1, HNF4a , FOXP3, or APOB in a cell for a time period.
  • a target gene e.g., MYC, SFRP1, HNF4a , FOXP3, or APOB
  • the expression of a target gene e.g., MYC, SFRP1, HNF4a , FOXP3, or APOB in a cell contacted by or comprising the expression repressor or expression enhancer or a system is appreciably decreased or increased, respectively, for 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, or 24 hours, or at least 1, 2, 3, 4, 5, 6, 7, 10, 14, or 15 days, or at least 1, 2, 3, 4, or 5 weeks, or at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 months, or at least 1, 2, 3, 4, or 5 years (e.g., indefinitely).
  • the expression of a target gene e.g., MYC, SFRP1, HNF4a , FOXP3, or APOB in a cell contacted by or comprising the expression repressor or the system is appreciably decreased for no more than 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 years.
  • the expression of a target gene e.g., MYC, SFRP1, HNF4a , FOXP3, or APOB in a cell contacted by or comprising the expression repressor or the system is appreciably decreased for at least 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 cell divisions.
  • An expression repressor or expression enhancer or a system of the present disclosure can be used to methylate CpG nucleotides in a target promoter, e.g., MYC, SFRP1, HNF4a , FOXP3, or APOB promoter.
  • a target promoter e.g., MYC, SFRP1, HNF4a , FOXP3, or APOB promoter.
  • the transcriptional changes in MYC, SFRP1, HNF4a , FOXP3, or APOB expression correlates to percentage of CpG methylation.
  • the methylation persists for at least 1 days, at least 2 days, at least 5 days, at least 7 days, at least 10 days, at least 15 days, or at least 20 days post-treatment with an expression repressor or a system disclosed herein.
  • An expression repressor or expression enhancer or a system of the present disclosure can be used to decrease the viability of a cell comprising the target locus, e.g., MYC, SFRP1, HNF4a , FOXP3, or APOB locus.
  • expression repressor or expression enhancer or a system of the present disclosure can be used to decrease the viability of a plurality of cells comprising the target locus, e.g., MYC, SFRP1, HNF4a , FOXP3, or APOB locus.
  • an expression repressor or expression enhancer or a system of the present disclosure can be used to decrease the viability of a plurality of cells comprising infected cells and uninfected cells.
  • an expression repressor or expression enhancer or a system of the present disclosure can be used to decrease the viability of the plurality of infected cells more than it decreases the viability of the plurality of uninfected cells. In some embodiments, an expression repressor or expression enhancer or a system of the present disclosure can he used to decrease the viability of the plurality of infected cells I.05x (i.e., 1.05 times), l.
  • lx 1.15x, 1.2x, 1.25x, 1.3x, 1.35x, 1.4x, 1.45x, 1.5x, 1.6x, 1.7x, 1.8x, 1.9x, 2x, 3x, 4x, 5x, 6x, 7x, 8x, 9x, lOx, 20x, 5Ox, or lOOx more than it decreases the viability of the plurality of uninfected cells.
  • An expression repressor or expression enhancer or a system may comprise a plurality of expression repressors or expression enhancers, where each expression repressor or expression enhancer comprises an effector moiety with a different functionality than the effector moiety of another expression repressor.
  • an expression repression system or expression enhancing system may comprise two expression repressors, where the first expression repressor or expression enhancer comprises a first effector moiety comprising an epigenetic modifying moiety e.g., DNA methyltransferase, e.g., MQ1 and the second or expression enhancer comprises a second effector moiety comprising a transcription repressor, e.g., KRAB.
  • the second expression repressor or expression enhancer does not comprise a second effector moiety.
  • an expression repressor or expression enhancer or a system comprises expression repressors comprising a combination of effector moieties whose functionalities are complementary to one another with regard to inhibiting 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 or expression enhancer or a system comprises a plurality of expression repressors or expression enhancers, wherein each expression repressor or expression enhancer comprises an effector moiety that complements the effector moieties of each other expression repressor or expression enhancer, e.g., each effector moiety decreases or increases, respectively, expression of a target gene, e.g., MYC, SFRP1, HNF4a , FOXP3, or APOB.
  • a target gene e.g., MYC, SFRP1, HNF4a , FOXP3, or APOB.
  • an expression repression system expression repressor or expression enhancer or a system comprises expression repressors or expression enhancers comprising a combination of effector moieties whose functionalities synergize with one another with regards to inhibiting expression of a target gene, e.g., MYC, SFRP1, HNF4a , FOXP3, or APOB.
  • a target gene e.g., MYC, SFRP1, HNF4a , FOXP3, or APOB.
  • epigenetic modifications to a genomic locus may be cumulative, in that multiple repressive epigenetic markers (e.g., multiple different types of epigenetic markers and/or more extensive marking of a given type) individually together reduce expression or increase expression more effectively than individual modifications alone (e.g., producing a greater decrease in expression and/or a longer-lasting decrease in expression).
  • multiple repressive epigenetic markers e.g., multiple different types of epigenetic markers and/or more extensive marking of a given type individually together reduce expression or increase 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 or expression enhancer or a system comprises a plurality of expression repressors or expression enhancers, wherein each expression repressor or expression enhancer comprises an effector moiety that synergizes with the effector moieties of each other expression repressor or expression enhancer, e.g., each effector moiety decreases or increases expression of a target gene, e.g., MYC.
  • an expression repressor or a system modulates (e.g., decreases) expression of a target gene, e.g., MYC by altering one or more epigenetic markers associated with the target gene, e.g., MYC or an expression control sequence operably linked thereto.
  • altering comprises decreasing the level of an epigenetic marker associated with the target gene, e.g., MYC or an expression control sequence operably linked thereto.
  • Epigenetic markers include, but are not limited to, DNA methylation, histone methylation, and histone deacetylation.
  • altering the level of an epigenetic marker decreases or increases the level of the epigenetic marker associated with the target gene, e.g., MYC, SFRP1, HNF4a , FOXP3, or APOB or an expression control sequence operably linked thereto by at least 1.05x (i.e., 1.05 times), l.
  • lx 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, lOx, 20x, 30x, 40x, 50x, 60x, 70x, 80x, 90x, or lOOx lower or higher than the level of the epigenetic marker associated with the target gene, e.g., MYC, SFRP1, HNF4a , FOXP3, or APOB or an expression control sequence operably linked thereto in a cell not contacted by or comprising the expression repressor or expression enhancer or the system.
  • the epigenetic marker associated with the target gene e.g., MYC, SFRP1, HNF4a , FOXP3, or APOB or an expression control sequence
  • the level of an epigenetic marker may be assayed by methods known to those of skill in the art, including whole genome bisulfite sequencing, reduced representation bisulfite sequencing, bisulfite amplicon sequencing, methylation arrays, pyrosequencing, ChlP-seq, or ChlP-qPCR.
  • the changes (e.g., increase or decrease) in epigenetic marker e.g., DNA methylation may be assayed using bisulfite genomic sequencing at precise genomic coordinates according to hgl9 reference genome, e.g., in between chr8: 129188693- 129189048 according to hgl9 reference genome.
  • the changes (e.g., increase or decrease) in epigenetic marker e.g., DNA methylation may be assayed using bisulfite genomic sequencing at a genomic location according to SEQ ID NO: 123.
  • An expression repressor or the system of the present disclosure can be used to alter the level of an epigenetic marker associated with the target gene, e.g., MYC or an expression control sequence operably linked thereto in a cell for a time period.
  • an epigenetic marker associated with the target gene e.g., MYC or an expression control sequence operably linked thereto in a cell for a time period.
  • the level of the epigenetic marker associated with the target gene or an expression control sequence operably linked thereto in a cell contacted by or comprising the expression repressor or the system is appreciably decreased for 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, or 24 hours, or at least 1, 2, 3, 4, 5, 6, 7, 10, or 14 days, or at least 1, 2, 3, 4, or 5 weeks, or at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 months, or at least 1, 2, 3, 4, or 5 years (e.g., indefinitely).
  • the level of an epigenetic marker associated with the target gene e.g., MYC or an expression control sequence operably linked thereto in a cell contacted by or comprising the expression repressor or the system is appreciably decreased for no more than 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 years.
  • an expression repression system or expression enhancing system comprises a first expression repressor or expression enhancer comprising a first effector moiety and a second expression repressor or expression enhancer comprising a second effector moiety wherein the first effector moiety and second effector moiety are different from one another.
  • the first effector moiety is or comprises a first epigenetic modifying moiety (e.g., that increases or decreases a first epigenetic marker) or functional fragment thereof and the second effector moiety is or comprises a second epigenetic modifying moiety (e.g., that increases or decreases a second epigenetic marker) or functional fragment thereof.
  • the first effector moiety is or comprises a DNA methyltransferase or functional fragment thereof and the second effector moiety is or comprises a KRAB or functional fragment thereof.
  • the first effector moiety is or comprises a histone deacetylase or functional fragment thereof and the second effector moiety is or comprises a KRAB or functional fragment thereof.
  • the first effector moiety is or comprises a histone methyltransferase or functional fragment thereof and the second effector moiety n is or comprises a KRAB or functional fragment thereof.
  • the first effector moiety is or comprises a histone demethylase or functional fragment thereof and the second effector moiety is or comprises a KRAB or functional fragment thereof.
  • the first effector moiety is or comprises 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 effector moiety is or comprises KRAB (e.g., a KRAB domain), MeCP2, HP1, RBBP4, REST, FOG1, SUZ12, or a functional fragment of any thereof
  • the second effector moiety is or comprises 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, KDMDM1A (i.e.
  • the first effector moiety is or comprises bacterial MQ1 or a functional variant or fragment thereof
  • the second effector moiety is or comprises KRAB or a functional variant or fragment thereof.
  • the first effector moiety is or comprises DNMT3A or a functional variant or fragment thereof
  • the second effector moiety is or comprises KRAB or a functional variant or fragment thereof.
  • the first effector moiety is or comprises DNMT3B or a functional variant or fragment thereof
  • the second effector moiety is or comprises KRAB or a functional variant or fragment thereof.
  • the first effector moiety is or comprises DNMT3L or a functional variant or fragment thereof
  • the second effector moiety is or comprises KRAB or a functional variant or fragment thereof.
  • the first effector moiety is or comprises DNMT3a/3L complex or a functional variant or fragment thereof
  • the second effector moiety is or comprises KRAB or a functional variant or fragment thereof.
  • Expression repressors, expression enhancers, expression repressor systems, or expression enhancing systems disclosed herein are useful for modulating, e.g., decreasing, expression of a target gene, e.g., MYC, SFRP1, HNF4a , FOXP3, or APOB in cell, e.g., in a subject or patient.
  • a target gene e.g., MYC, SFRP1, HNF4a , FOXP3, or APOB may be any gene known to those of skill in the art.
  • a target gene e.g., MYC, SFRP1, HNF4a , FOXP3, or APOB is associated with a disease or condition in a subject, e.g., a mammal, e.g., a human, bovine, horse, sheep, chicken, rat, mouse, cat, or dog.
  • a target gene may include coding sequences, e.g., exons, and/or non-coding sequences, e.g., introns, 3’UTR, or 5’UTR.
  • a target gene is operably linked to a transcription control element.
  • a targeting moiety suitable for use in an expression repressor or expression enhancer or an expression repressor of system or expression enhancing system described herein may bind, e.g., specifically bind, to any site within a target gene, e.g., MYC, SFRP1, HNF4a , FOXP3, or APOB, transcription control element operably linked to a target gene, e.g., MYC, SFRP1, HNF4a , FOXP3, or APOB to an anchor sequence (e.g., an anchor sequence proximal to a target gene or associated with an anchor sequence-mediated conjunction operably linked to a target gene, e.g., MYC, SFRP1, HNF4a , FOXP3, or APOB (e.g., an anchor sequence-mediated conjunction is operably linked to a target gene if disruption of the conjunction alters expression of the target gene, e.g., MYC, SFRP1, HNF4a , FOXP3,
  • an expression repressor or expression enhancer described herein binds at a site or at a location that is proximal to the site.
  • a targeting moiety may bind to a first site that is proximal to a repressor or 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 e.g., MYC, SFRP1, HNF4a , FOXP3, or APOB
  • 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., MYC, SFRP1, HNF4a , FOXP3, or APOB (e.g., an exon, intron, or splice site within the target gene, e.g., MYC, SFRP1, HNF4a , FOXP3, or APOB), transcription control element, or anchor sequence (and optionally at least 20, 25, 50, 100, 200, or 300 base pairs from the target gene,
  • a targeting moiety binds to a target gene, e.g., MYC, SFRP1, HNF4a , FOXP3, or APOB.
  • a DNA-targeting moiety binds to a site within an exon of a target gene, e.g., MYC, SFRP1, HNF4a , FOXP3, or APOB.
  • a targeting moiety binds to a site within an intron of a target gene, e.g., MYC, SFRP1, HNF4a , FOXP3, or APOB.
  • a targeting moiety binds to a site at the boundary of an exon and an intron, e.g., a splice site, of a target gene, e.g., MYC, SFRP1, HNF4a , FOXP3, or APOB. In some embodiments, a targeting moiety binds to a site within the 5’UTR of a target gene, e.g., MYC, SFRP1, HNF4a , FOXP3, or APOB.
  • a targeting moiety binds to a site within the 3’UTR of a target gene, e.g., MYC, SFRP1, HNF4a , FOXP3, or APOB.
  • Target genes include, but are not limited to the gene encoding MYC, SFRP1, HNF4a , FOXP3, or APOB.
  • a targeting moiety binds to a transcription control element operably linked to a target gene (e.g., MYC, SFRP1, HNF4a , FOXP3, or APOB), e.g., a promoter or enhancer.
  • a targeting moiety binds to a portion of or a site within a promoter operably linked to a target gene, e.g., MYC, SFRP1, HNF4a , FOXP3, or APOB.
  • a targeting moiety binds to the transcription start site of a target gene, e.g., MYC, SFRP1, HNF4a , FOXP3, or APOB.
  • a targeting moiety binds to a portion of or a site within an enhancer operably linked to a target gene, e.g., MYC, SFRP1, HNF4a , FOXP3, or APOB.
  • a genomic complex e.g., ASMC co-localizes two or more genomic sequence elements, wherein the two or more genomic sequence elements include a promoter.
  • a promoter is, typically, a sequence element that initiates transcription of an associated gene. Promoters are typically near the 5’ end of a gene, not far from its transcription start site.
  • RNA polymerase II e.g., TFIID, TFIIE, TFIIH, FUSE, CT-element etc.
  • mediator e.g., TFIID, TFIIE, TFIIH, FUSE, CT-element etc.
  • a promoter includes a sequence element such as TATA, Inr, DPE, or BRE, but those skilled in the art are well aware that such sequences are not necessarily required to define a promoter.
  • a transcription control element is a transcription factor binding site.
  • a targeting moiety binds to a genomic sequence located within a genomic coordinate GRCh37: chr8: 129162465-129212140.
  • a targeting moiety binds to a target sequence comprised by or partially comprised by a genomic sequence element.
  • the genomic sequence element is or comprises an expression control sequence.
  • the genomic sequence element is or comprises the target gene, e.g., MYC, SFRP1, HNF4a , FOXP3, or APOB or a part of the target gene, e.g., MYC, SFRP1, HNF4a , FOXP3, or APOB.
  • a targeting moiety binds to a target sequence that is 10-30, 15-30, 15-25, 18-24, 19-23, 20-23, 21-23, or 22- 23 bases long.
  • the target sequence is 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, or 40 bases long.
  • the genomic sequence element is or comprises an anchor sequence.
  • Each ASMC comprises one or more anchor sequences, e.g., a plurality.
  • anchor sequences can be manipulated or altered to modulate (e.g., disrupt) a naturally occurring genomic complex (e.g., ASMC) or to form a new genomic complex (e.g., ASMC) (e.g., to form a non-naturally occurring genomic complex (e.g., ASMC) with an exogenous or altered anchor sequence).
  • an anchor sequence-mediated conjunction can be disrupted to alter, e.g., inhibit, e.g., decrease expression of a target gene.
  • Such disruptions may modulate gene expression by, e.g., changing topological structure of DNA, e.g., by modulating the ability of a target gene to interact with a transcription control element (e.g., enhancing and silencing/repressive sequences).
  • a transcription control element e.g., enhancing and silencing/repressive sequences
  • a targeting moiety binds to an anchor sequence, e.g., an anchor sequence proximal to a target gene, e.g., MYC, SFRP1, HNF4a , FOXP3, or APOB or associated with an anchor sequence-mediated conjunction (ASMC) operably linked to a target gene, e.g., MYC, SFRP1, HNF4a , FOXP3, or APOB
  • ASMC anchor sequence-mediated conjunction
  • an anchor sequence-mediated conjunction is operably linked to a target gene, e.g., MYC, SFRP1, HNF4a , FOXP3, or APOB if disruption of the conjunction alters expression of the target gene, e.g., MYC, SFRP1, HNF4a , FOXP3, or APOB).
  • an anchor sequence is a genomic sequence element to which a genomic complex component, e.g., nucleating polypeptide binds specifically.
  • binding of a genomic complex component to an anchor sequence nucleates complex formation, e.g., ASMC formation.
  • a targeting moiety binds to a target gene, e.g., MYC, SFRP1, HNF4a , FOXP3, or APOB locus.
  • a locus is generally defined to encompass transcribed region, promoter, and anchor sites of an ASMC comprising a target gene, e.g., MYC, SFRP1, HNF4a , FOXP3, or APOB.
  • a targeting moiety binds to a sequence comprising any one of SEQ ID NOS: 75-86 or 199-206.
  • the first targeting moiety binds to a sequence comprising any one of SEQ ID NOS: 75-86 and the second targeting moiety binds to a sequence comprising any one of SEQ ID NOS: 75-86, wherein the first and the second targeting moiety binds to the same sequence.
  • the first targeting moiety binds to a sequence comprising any one of SEQ ID NOS: 75-86 and the second targeting moiety binds to a sequence comprising any one of SEQ ID NOS: 75-86 wherein the first and the second targeting moiety binds to different sequences.
  • the first targeting moiety binds to a sequence comprising any of SEQ ID NOs: 83, 203, or 206 and the second targeting moiety binds to a sequence comprising SEQ ID NO: 77. In some embodiments, the first targeting moiety binds to a sequence comprising SEQ ID NO: 77 and the second targeting moiety binds to a sequence comprising any of SEQ ID NOs: 83, 203, or 206. In some embodiments, the first targeting moiety binds to a sequence comprising any of SEQ ID NOs: 83, 203, or 206 and the second targeting moiety binds to a sequence comprising any of SEQ ID NOs: 199, 204, or 205.
  • the first targeting moiety binds to a sequence comprising any of SEQ ID NOs: 199, 204, or 205 and the second targeting moiety binds to a sequence comprising any of SEQ ID NOs: 83, 203, or 206. In some embodiments, the first targeting moiety binds to a sequence comprising any of SEQ ID NOs: 83, 203, or 206 and the second targeting moiety binds to a sequence comprising SEQ ID NO: 201 .
  • a nucleic acid encoding the first and second expression repressors or expression enhancers comprises a first region that encodes the first expression repressor or expression enhancer, wherein the first region is upstream of a second region that encodes the second expression repressor or expression enhancer.
  • a nucleic acid encoding the first and second expression repressors or expression enhancers comprises a first region that encodes the first expression repressor or expression enhancer, wherein the first region is downstream of a second region that encodes the second expression repressor or expression enhancers.
  • the first targeting moiety binds to a sequence comprising any one of SEQ ID NOs: 75-86 or 199-206
  • the second targeting moiety e.g., a CRISPR/Cas domain comprising a gRNA
  • a targeting moiety binds to a sequence comprising any one of SEQ ID NOS: 96-110.
  • the first targeting moiety binds to a sequence comprising any one of SEQ ID NOS: 96- 110 and the second targeting moiety binds to a sequence comprising any one of SEQ ID NOS: 96- 110, wherein the first and the second targeting moiety binds to the same sequence. In some embodiments, the first targeting moiety binds to a sequence comprising any one of SEQ ID NOS: 96- 110 and the second targeting moiety binds to a sequence comprising any one of SEQ ID NOS: 96-110 wherein the first and the second targeting moiety binds to different sequences.
  • the first targeting moiety binds to a sequence comprising any one of SEQ ID NOs: 96-110
  • the second targeting moiety e.g., a CRISPR/Cas domain comprising a gRNA
  • the first targeting moiety binds to a sequence comprising any one of the SEQ ID Nos. disclosed in tables 3, 4, or 26, and the second targeting moiety (e.g., a CRISPR/Cas domain comprising a gRNA) binds to a sequence comprising any one of the SEQ ID Nos. disclosed in tables 3, 4, or 26.
  • Exemplary target sequences are disclosed in Table 29.
  • an expression repressor or expression enhancer binds a genomic locus having a sequence set forth herein, e.g., any one of SEQ ID NOS: 1-4, 75-86, 96-110, or 199-206. It is understood that, in many cases, the genomic locus being bound comprises double stranded DNA, and this locus can be described by giving the sequence of its sense strand or its antisense strand. Thus, a gRNA having a given spacer sequence may cause expression repressor or expression enhancer to bind to a particular genomic locus, wherein one strand of the genomic locus has a sequence similar or identical to the spacer sequence, and the other strand of the genomic locus has the complementary sequence.
  • a targeting moiety binds to an anchor sequence, e.g., an anchor sequence proximal to a target gene, e.g., MYC, SFRP1, HNF4a , FOXP3, or APOB or associated with an anchor sequence-mediated conjunction (ASMC) operably linked to a target gene, e.g., MYC, SFRP1, HNF4a , FOXP3, or APOB
  • ASMC anchor sequence-mediated conjunction
  • an anchor sequence-mediated conjunction is operably linked to a target gene, e.g., MYC, SFRP1, HNF4a , FOXP3, or APOB if disruption of the conjunction alters expression of the target gene, e.g., MYC, SFRP1, HNF4a , FOXP3, or APOB
  • an anchor sequence is a genomic sequence element to which a genomic complex component, e.g., nucleating polypeptide binds specifically.
  • binding of a genomic complex component to an anchor sequence nucleates complex formation, e.g., ASMC formation.
  • a targeting moiety binds to a target gene, e.g., MYC, SFRP1, HNF4a , F0XP3, or APOB locus.
  • a locus is generally defined to encompass transcribed region, promoter, and anchor sites of an ASMC comprising a target gene, e.g., MYC, SFRP1, HNF4a , FOXP3, or APOB.
  • a targeting moiety binds to a sequence comprising any one of SEQ ID NOS: 190-192. In some embodiments, the targeting moiety binds to a sequence comprising any one of the SEQ ID Nos. disclosed in Table 30. Exemplary target sequences in mouse genome are disclosed in Table 30.
  • Table 30 Exemplary target sequences in mouse genome
  • an expression repressor or expression enhancer binds a genomic locus having a sequence set forth herein, e.g., any one of SEQ ID NOS: 190-192. It is understood that, in many cases, the genomic locus being bound comprises double stranded DNA, and this locus can be described by giving the sequence of its sense strand or its antisense strand.
  • the anchor sequence-mediated conjunction comprises a loop, such as an intra-chromosomal loop.
  • the anchor sequence-mediated conjunction has a plurality of loops.
  • One or more loops may include a first anchor sequence, a nucleic acid sequence, a transcriptional control sequence, and a second anchor sequence.
  • at least one loop includes, in order, a first anchor sequence, a transcriptional control sequence, and a second anchor sequence, or a first anchor sequence, a nucleic acid sequence, and a second anchor sequence.
  • either one or both of the nucleic acid sequences and the transcriptional control sequence is located within or outside the loop.
  • one or more of the loops comprises a transcriptional control sequence.
  • the anchor sequence-mediated conjunction includes a TATA box, a CAAT box, a GC box, or a CAP site.
  • the anchor sequence-mediated conjunction comprises a plurality of loops, and where the anchor sequence-mediated conjunction comprises at least one of an anchor sequence, a nucleic acid sequence, and a transcriptional control sequence in one or more of the loops.
  • chromatin structure is modified by substituting, adding, or deleting one or more nucleotides within an anchor sequence. In some embodiments, chromatin structure is modified by substituting, adding, or deleting one or more nucleotides within an anchor sequence of an anchor sequence-mediated conjunction. In some embodiments, transcription is inhibited by inclusion of an activating loop or exclusion of a repressive loop. In one such embodiment, the anchor sequence- mediated conjunction excludes a transcriptional control sequence that decreases transcription of the nucleic acid sequence. In some embodiments, transcription is repressed by inclusion of a repressive loop or exclusion of an activating loop. In one such embodiment, the anchor sequence-mediated conjunction includes a transcriptional control sequence that decreases transcription of the nucleic acid sequence.
  • the anchor sequences may be non-contiguous with one another.
  • the first anchor sequence may be separated from the second anchor sequence by about 500bp to about 500Mb, about 750bp to about 200Mb, about Ikb to about 100Mb, about 25kb to about 50Mb, about 50kb to about 1Mb, about lOOkb to about 750kb, about 150kb to about 500kb, or about 175kb to about 500kb.
  • the first anchor sequence is separated from the second anchor sequence by about 500bp, 600bp, 700bp, 800bp, 900bp, Ikb, 5kb, lOkb, 15kb, 20kb, 25kb, 30kb, 35kb, 40kb, 45kb, 50kb, 55kb, 60kb, 65kb, 70kb, 75kb, 80kb, 85kb, 90kb, 95kb, lOOkb, 125kb, 150kb, 175kb, 200kb, 225kb, 250kb, 275kb, 300kb, 350kb, 400kb, 500kb, 600kb, 700kb, 800kb, 900kb, 1Mb, 2Mb, 3Mb, 4Mb, 5Mb, 6Mb, 7Mb, 8Mb, 9Mb, 10Mb, 15Mb, 20Mb, 25Mb, 50Mb, 75Mb, 100Mb
  • the targeting moiety introduces at least one of the following: at least one exogenous anchor sequence; an alteration in at least one conjunction nucleating molecule binding site, such as by altering binding affinity for the conjunction nucleating molecule; a change in an orientation of at least one common nucleotide sequence, such as a CTCF binding motif, YY1 binding motif, ZNF143 binding motif, or other binding motif mentioned herein; and a substitution, addition or deletion in at least one anchor sequence, such as a CTCF binding motif, YY1 binding motif, ZNF143 binding motif, or other binding motif mentioned herein.
  • an anchor sequence comprises a nucleating polypeptide binding motif, e.g., a CTCF-binding motif: N(T/C/G)N(G/A/T)CC(A/T/G)(C/G)(C/T/A)AG(G/A)(G/T)GG(C/A/T)(G/A)(C/G)(C/T/A)(G/A/C), where N is any nucleotide.
  • a CTCF-binding motif N(T/C/G)N(G/A/T)CC(A/T/G)(C/G)(C/T/A)AG(G/A)(G/T)GG(C/A/T)(G/A)(C/G)(C/T/A)(G/A/C), where N is any nucleotide.
  • a CTCF-binding motif may also be in an opposite orientation, e.g., (G/A/C)(C/T/A)(C/G)(G/A)(C/A/T)GG(G/T)(G/A)GA(C/T/A)(C/G)(A/T/G)CC(G/A/T)N(T/C/G)N.
  • an anchor sequence comprises N(T/C/G)N(G/A/T)CC(A/T/G)(C/G)(C/T/A)AG(G/A)(G/T)GG(C/A/T)(G/A)(C/G)(C/T/A)(G/A/C) or (G/A/C)(C/T/A)(C/G)(G/A)(C/A/T)GG(G/T)(G/A)GA(C/T/A)(C/G)(A/T/G)CC(G/A/T)N(T/C/G)N or a sequence at least 75%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% identical to either N(T/C/G)N(G/A/T)CC(A/T/G)(C/G)(C/T)(C/T)(C/T)AG(
  • an anchor sequence comprises a nucleating polypeptide binding motif, e.g., a YY1 -binding motif: CCGCCATNTT, where N is any nucleotide.
  • a YYl-binding motif may also be in an opposite orientation, e.g., AANATGGCGG, where N is any nucleotide.
  • an anchor sequence-mediated conjunction comprises at least a first anchor sequence and a second anchor sequence.
  • a first anchor sequence and a second anchor sequence may each comprise a nucleating polypeptide binding motif, e.g., each comprises a CTCF binding motif.
  • a first anchor sequence and second anchor sequence comprise different sequences, e.g., a first anchor sequence comprises a CTCF binding motif, and a second anchor sequence comprises an anchor sequence other than a CTCF binding motif.
  • each anchor sequence comprises a nucleating polypeptide binding motif and one or more flanking nucleotides on one or both sides of a nucleating polypeptide binding motif.
  • CTCF-binding motifs e.g., contiguous or non-contiguous CTCF binding motifs
  • an ASMC may be present in a genome in any orientation, e.g., in the same orientation (tandem) either 5 ’-3’
  • the two CTCF-binding motifs that comprise N(T/C/G)N(G/A/T)CC(A/T/G)(C/G)(C/T/A)AG(G/A)(G/T)GG(C/A/T)(G/A)(C/T/A)(G/A/C)) or 3 ’-5’
  • the two CTCF-binding motifs comprise (G/A/C)(C/T/A)(C/G)(G/A)(C/A/T)GG(G/T)(G/A)GA(C/T/A)(C/G)(A
  • an anchor sequence comprises a CTCF binding motif associated with a target gene (e.g., MYC), wherein the target gene is associated with a disease, disorder and/or condition, e.g., MYC mis-regulating disorder, e.g., hepatic disorder, (e.g., hepatocarcinoma) or lung cancer.
  • a target gene e.g., MYC
  • MYC mis-regulating disorder e.g., hepatic disorder, (e.g., hepatocarcinoma) or lung cancer.
  • methods of the present disclosure comprise modulating, e.g., disrupting, a genomic complex (e.g., ASMC), e.g., by modifying chromatin structure, by substituting, adding, or deleting one or more nucleotides within an anchor sequence, e.g., a nucleating polypeptide binding motif.
  • a genomic complex e.g., ASMC
  • One or more nucleotides may be specifically targeted, e.g., a targeted alteration, for substitution, addition or deletion within an anchor sequence, e.g., a nucleating polypeptide binding motif.
  • a genomic complex (e.g., ASMC) may be altered by changing an orientation of at least one nucleating polypeptide binding motif.
  • an anchor sequence comprises a nucleating polypeptide binding motif, e.g., CTCF binding motif, and a targeting moiety introduces an alteration in at least one nucleating polypeptide binding motif, e.g., altering binding affinity for a nucleating polypeptide.
  • the target gene e.g., MYC, SFRP1, HNF4a , FOXP3, or APOB has a defined state of expression, e.g., in a diseased state.
  • the target gene e.g., MYC, SFRP1, HNF4a , FOXP3, or APOB may have a high level of expression in a disease cell.
  • expression of the target gene e.g., MYC, SFRP1, HNF4a , FOXP3, or APOB may be decreased.
  • a targeting moiety suitable for use in an expression repressor, expression enhancer or system described herein may bind, e.g., specifically bind, to a site comprising at least 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50 nucleotides or base pairs (and optionally no more 50, 49, 48, 47, 46, 45, 44, 43, 42, 41, 40, 39, 38, 37, 36, 35, 34, 33, 32, 31, 30, 29, 28, 27, 26, 25, 24, 23, 22, 21, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, or 10 nucleotides or base pairs).
  • a DNA- targeting moiety binds to a site comprising 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50 nucleotides or base pairs.
  • Expression repressor, expression enhancer or system of the present disclosure may comprise two or more expression repressors or expression enhancers.
  • the expression repressors or expression enhancers of an expression repressor system or expression enhancing system each comprise a different targeting moiety.
  • an expression repression system or expression enhancer or system comprises a first expression repressor or expression enhancer comprising a targeting moiety that binds a target gene, e.g., an exon, intron, or exon intron boundary (e.g., splice site), and second expression repressor or expression enhancer comprising a targeting moiety that binds the target gene, e.g., an exon, intron, or exon intron boundary (e.g., splice site).
  • a target gene e.g., an exon, intron, or exon intron boundary (e.g., splice site)
  • second expression repressor or expression enhancer comprising a targeting moiety that binds the target gene, e.g., an exon, intron, or exon intron boundary (e.g., splice site).
  • an expression repression system comprises a first expression repressor or expression enhancer comprising a targeting moiety that binds a target gene, e.g., an exon, intron, or exon intron boundary (e.g., splice site), and second expression repressor or expression enhancer comprising a targeting moiety that binds to a transcription control element (e.g., promoter or enhancer) operably linked to the target gene, e.g., MYC, SFRP1, HNF4a , FOXP3, or APOB.
  • a transcription control element e.g., promoter or enhancer
  • an expression repression system comprises a first expression repressor or expression enhancer comprising a targeting moiety that binds to a transcription control element (e.g., promoter or enhancer) operably linked to a target gene, and a second expression repressor or expression enhancer comprising a targeting moiety that binds to a transcription control element (e.g., promoter or enhancer) operably linked to the target gene.
  • a transcription control element e.g., promoter or enhancer
  • a second expression repressor or expression enhancer comprising a targeting moiety that binds to a transcription control element (e.g., promoter or enhancer) operably linked to the target gene.
  • an expression repression system comprises a first expression repressor or expression enhancer comprising a targeting moiety that binds to an anchor sequence proximal to a target gene, e.g., MYC, SFRP1, HNF4a , FOXP3, or APOB or associated with an anchor sequence-mediated conjunction operably linked to a target gene, e.g., MYC, SFRP1, HNF4a , FOXP3, or APOB, and a second expression repressor or expression enhancer comprising a targeting moiety that binds to a transcription control element (e.g., promoter or enhancer) operably linked to the target gene, e.g., MYC, SFRP1, HNF4a , FOXP3, or APOB.
  • a transcription control element e.g., promoter or enhancer
  • an expression repression system comprises a first expression repressor or expression enhancer comprising a targeting moiety that binds to an anchor sequence proximal to a target gene, e.g., MYC, SFRP1, HNF4a , FOXP3, or APOB or associated with an anchor sequence-mediated conjunction operably linked to a target gene, e.g., MYC, SFRP1, HNF4a , FOXP3, or APOB, and a second expression repressor or expression enhancer comprising a targeting moiety that binds to the target gene (e.g., MYC, SFRP1, HNF4a , FOXP3, or APOB), e.g., an exon, intron, or exon intron boundary (e.g., splice site).
  • a target gene e.g., MYC, SFRP1, HNF4a , FOXP3, or APOB
  • a targeting moiety that binds to the
  • an expression repression system comprises a first expression repressor or expression enhancer comprising a targeting moiety that binds to an anchor sequence proximal to a target gene, e.g., MYC, SFRP1, HNF4a , FOXP3, or APOB or associated with an anchor sequence-mediated conjunction operably linked to a target gene, e.g., MYC, SFRP1, HNF4a , FOXP3, or APOB, and a second expression repressor or expression enhancer comprising a targeting moiety that binds to an anchor sequence proximal to the target gene, e.g., MYC, SFRP1, HNF4a , FOXP3, or APOB or associated with an anchor sequence-mediated conjunction operably linked to the target gene, e g., MYC, SFRP1, HNF4a , FOXP3, or APOB.
  • a target gene e.g., MYC, SFRP1, HNF4a
  • an expression repression system or expression enhancing system comprises a first expression repressor or expression enhancer comprising a targeting moiety that binds to a first site, e.g., in a promoter operably linked to a target gene, e.g., MYC, SFRP1, HNF4a , FOXP3, or APOB, and a second expression repressor or expression enhancer comprising a targeting moiety that binds to a second site, e.g., in the promoter operably linked to a target gene, e.g., MYC, SFRP1, HNF4a , FOXP3, or APOB.
  • a target gene e.g., MYC, SFRP1, HNF4a , FOXP3, or APOB
  • the first site and second site may be different and nonoverlapping sites, e.g., the first site and second site do not share any sequence in common.
  • the first site and second site may be different but overlapping sites, e.g., the first site and second site comprise different sequences but share some sequence in common.
  • the target gene is MYC.
  • MYC is located on human chromosome 8.
  • the expression repressor, expression enhancer or system as described herein binds to the transcription start site (TSS) of MYC.
  • the present disclosure is further directed, in part, to pharmaceutical compositions comprising an expression repressor or an expression repression system, e.g., expression repressor(s), described herein, to pharmaceutical compositions comprising nucleic acids encoding the expression repressor or the expression repression system, e.g., expression repressor(s), described herein, and/or to and/or compositions that deliver an expression repressor or an expression repression system, e.g., expression repressor(s), described herein to a cell, tissue, organ, and/or subject.
  • an expression repressor or an expression repression system e.g., expression repressor(s), described herein
  • pharmaceutical compositions comprising nucleic acids encoding the expression repressor or the expression repression system, e.g., expression repressor(s), described herein
  • compositions comprising nucleic acids encoding the expression repressor or the expression repression system,
  • the term “pharmaceutical composition” refers to an active agent (e.g., an expression repressor or nucleic acids of the expression receptor, e.g., an expression repression system, e.g., expression repressor(s) of an expression repressor system, or nucleic acid encoding the same), formulated together with one or more pharmaceutically acceptable carriers (e.g., pharmaceutically acceptable carriers known to those of skill in the art).
  • active agent e.g., an expression repressor or nucleic acids of the expression receptor, e.g., an expression repression system, e.g., expression repressor(s) of an expression repressor system, or nucleic acid encoding the same
  • active agent is present in unit dose amount appropriate for administration in a therapeutic regimen that shows a statistically significant probability of achieving a predetermined therapeutic effect when administered to a relevant population.
  • a pharmaceutical composition comprising an expression repressor of the present disclosure comprises an expression repressor or nucleic acid(s) encoding the same.
  • a pharmaceutical composition comprising an expression repression system of the present disclosure comprises or each of the expression repressors of the expression repression system or nucleic acid(s) encoding the same (e.g., if an expression repression system comprises a first expression repressor and a second expression repressor, the pharmaceutical composition comprises the first and second expression repressor).
  • a pharmaceutical composition comprises less than all of the expression repressors of an expression repression system comprising a plurality of expression repressors.
  • an expression repression system may comprise a first expression repressor and a second expression repressor, and a first pharmaceutical composition may comprise the first expression repressor or nucleic acid encoding the same and a second pharmaceutical composition may comprise the second expression repressor or nucleic acid encoding the same.
  • a pharmaceutical composition may comprise coformulation of one or more expression repressors, or nucleic acid(s) encoding the same.
  • the term “pharmaceutically acceptable” refers to those compounds, materials, compositions, and/or dosage forms which are, within the scope of sound medical judgment, suitable for use in contact with the tissues of human beings and animals without excessive toxicity, irritation, allergic response, or other problem or complication, commensurate with a reasonable benefit/risk ratio.
  • the dosage of the administered agent or composition can vary based on, e.g., the condition being treated, the severity of the disease, the subject’s individual parameters, including age, physiological condition, size and weight, duration of treatment, the type of treatment to be performed (if any), the particular route of administration and similar factors. Thus, the dose administered of the agents described herein can depend on such various parameters.
  • the dosage of an administered composition may also vary depending upon other factors as the subject’s sex, general medical condition, and severity of the disorder to be treated.
  • a dosage of a modulatory agent or combination of modulatory agents disclosed herein that is in the range of from about 1 mg/kg to 6 mg/kg as a single intravenous infusion, although a lower or higher dosage also may be administered as circumstances dictate.
  • the dosage may be repeated as needed, for example, once every day (e.g., for 1-30 days), once every 3 days (e.g., for 1-30 days) once every 5 days (e.g., for 1-30 days), once per week (e.g., for 1-6 weeks or for 2-5 weeks).
  • dosages may include, but are not limited to, 1.0 mg/kg- 6mg/kg, 1.0 mg/kg- 5 mg/kg, 1.0 mg/kg-4 mg/kg, 1.0-3.0mg/kg, 1.5 mg/kg-3.0mg/kg, 1.0 mg/kg - 1.5 mg/kg, 1.5 mg/kg - 3 mg/kg, 3 mg/kg - 4 mg/kg, 4 mg/kg - 5 mg/kg, or 5 mg/kg - 6 mg/kg.
  • the dosage may be administered multiple times, e.g., once, or twice a week, or once every 1 or 2 weeks.
  • the subject is provided with a dosage of a modulatory agent or combination of modulatory agents disclosed herein that is in the range of from about 1 mg/kg to 6 mg/kg as multiple intravenous infusions although a lower or higher dosage also may be administered as circumstances dictate.
  • a dosage of a modulatory agent or combination of modulatory agents disclosed herein that is in the range of from about 1 mg/kg to 6 mg/kg as multiple intravenous infusions although a lower or higher dosage also may be administered as circumstances dictate.
  • Pharmaceutical compositions according to the present disclosure may be delivered in a therapeutically effective amount.
  • a precise therapeutically effective amount is an amount of a composition that will yield the most effective results in terms of efficacy of treatment in a given subject.
  • the present disclosure provides methods of delivering a therapeutic comprising administering a composition as described herein to a subject, wherein a modulating agent is a therapeutic and/or wherein delivery of a therapeutic causes changes in gene expression relative to gene expression in absence of a therapeutic.
  • compositions are/are targeted to specific cells, or one or more specific tissues.
  • one or more compositions is/are targeted to hepatic, epithelial, connective, muscular, reproductive, and/or nervous tissue or cells.
  • a composition is targeted to a cell or tissue of a particular organ system, e.g., cardiovascular system (heart, vasculature); digestive system (esophagus, stomach, liver, gallbladder, pancreas, intestines, colon, rectum and anus); endocrine system (hypothalamus, pituitary gland, pineal body or pineal gland, thyroid, parathyroids, adrenal glands); excretory system (kidneys, ureters, bladder); lymphatic system (lymph, lymph nodes, lymph vessels, tonsils, adenoids, thymus, spleen); integumentary system (skin, hair, nails); muscular system (e.g., skeletal muscle); nervous system (brain, spinal cord, nerves); reproductive system (ovaries, uterus
  • a composition of the present disclosure crosses a blood-brain-barrier, a placental membrane, or a blood-testis barrier.
  • a pharmaceutical composition as provided herein is administered systemically.
  • administration is non-parenteral and a therapeutic is a parenteral therapeutic.
  • compositions provided herein may comprise a pharmaceutical composition administered by a regimen sufficient to alleviate a symptom of a disease, disorder, and/or condition.
  • the present disclosure provides methods of delivering a therapeutic by administering compositions as described herein.
  • compositions e.g., modulating agents, e.g., epigenetic modifying agents
  • modulating agents e.g., epigenetic modifying agents
  • suitable pharmaceutical compositions can be found in International Applications PCT/US2020/052275, PCT/US2020/052119, PCT/US2021/021825, PCT/US2022/036389, PCT/US2017/050553, and PCT/US2021/010059, all incorporated herein by reference in their entireties.
  • Nanoparticles include particles with a dimension (e.g. diameter) between about 1 and about 1000 nanometers, between about 1 and about 500 nanometers in size, between about 1 and about 100 nm, between about 30 nm and about 200 nm, between about 50 nm and about 300 nm, between about 75 nm and about 200 nm, between about 100 nm and about 200 nm, and any range therebetween.
  • a nanoparticle has a composite structure of nanoscale dimensions.
  • nanoparticles are typically spherical although different morphologies are possible depending on the nanoparticle composition.
  • the portion of the nanoparticle contacting an environment external to the nanoparticle is generally identified as the surface of the nanoparticle.
  • nanoparticles have a greatest dimension ranging between 25 nm and 200 nm.
  • Nanoparticles as described herein comprise delivery systems that may be provided in any form, including but not limited to solid, semi-solid, emulsion, or colloidal nanoparticles.
  • a nanoparticle delivery system may include but not limited to lipid-based systems, liposomes, micelles, micro-vesicles, extracellular vesicles, or gene gun.
  • the nanoparticle is a lipid nanoparticle (LNP).
  • the LNP is a particle that comprises a plurality of lipid molecules physically associated with each other by intermolecular forces.
  • LNP formulations can be found in International Applications PCT/US2020/052275, PCT/US2020/052119, PCT/US2021/021825, PCT/US2022/036389, PCT/US2017/050553, and PCT/US2021/010059, all incorporated herein by reference in their entireties.
  • Methods and compositions provided herein may comprise a pharmaceutical composition administered by a regimen sufficient to alleviate a symptom of a disease, disorder, and/or condition.
  • the present disclosure provides methods of delivering a therapeutic by administering compositions as described herein.
  • the present disclosure is further directed to uses of the expression repressors, expression enhancers or systems disclosed herein.
  • such provided technologies may be used to achieve modulation, e.g., repression, of target gene, e.g., MYC, SFRP1, HNF4a , FOXP3, or APOB expression and, for example, enable control of target gene, e.g., MYC, SFRP1, HNF4a , FOXP3, or APOB activity, delivery, and penetrance, e.g., in a cell.
  • a cell is a mammalian, e.g., human, cell.
  • a cell is a somatic cell. In some embodiments, a cell is a primary cell. For example, in some embodiments, a cell is a mammalian somatic cell. In some embodiments, a mammalian somatic cell is a primary cell. In some embodiments, a mammalian somatic cell is a non-embryonic cell.
  • the present disclosure is further directed, in part, to a method of modulating, e.g., decreasing or increasing, expression of a target gene, e.g., MYC, SFRP1, HNF4a , FOXP3, or APOB, comprising providing an expression repressor (or a nucleic acid encoding the same, or pharmaceutical composition comprising said expression repressor nucleic acid), expression enhancer (or a nucleic acid encoding the same, or pharmaceutical composition comprising said expression enhancer nucleic acid) or an expression repression system described herein (or a nucleic acid encoding the same, or pharmaceutical composition comprising said expression repression system or nucleic acid), or expression enhancing system described herein (or a nucleic acid encoding the same, or pharmaceutical composition comprising said expression enhancing system or nucleic acid), and contacting the target gene e.g., MYC, SFRP1, HNF4a , FOXP3, or APOB, and/or
  • modulating comprises modulation of transcription of a target gene, e.g., MYC, SFRP1, HNF4a , FOXP3, or APOB as compared with a reference value (e.g., a control level), e.g., transcription of a target gene, e.g., MYC in absence of the expression repressor, expression enhancers, expression repressor system, or expression enhancing system.
  • a reference value e.g., a control level
  • the method of modulating, e.g., decreasing or increasing, expression of a target gene e.g., MYC, SFRP1, HNF4a , FOXP3, or APOB are used ex vivo, e.g., on a cell from a subject, e.g., a mammalian subject, e.g., a human subject.
  • the method of modulating, e.g., decreasing or increasing, expression of a target gene, e.g., MYC, SFRP1, HNF4a , FOXP3, or APOB are used in vivo, e.g., on a mammalian subject, e.g., a human subject.
  • the method of modulating, e.g., decreasing or increasing, expression of a target gene e.g., MYC, SFRP1, HNF4a , FOXP3, or APOB are used in vitro, e.g., on a cell or cell line described herein.
  • the present disclosure is further directed, in part to a method of treating a condition associated with mis-regulation, e.g., over-expression or under-expression of a target gene, e.g., MYC, SFRP1, HNF4a , FOXP3, or APOB in a subject, comprising administering to the subject an expression repressor (or a nucleic acid encoding the same, or pharmaceutical composition comprising said expression repressor nucleic acid), expression enhancer (or a nucleic acid encoding the same, or pharmaceutical composition comprising said expression enhancer nucleic acid) or an expression repression system described herein (or a nucleic acid encoding the same, or pharmaceutical composition comprising said expression repression system or nucleic acid), or expression enhancing system described herein (or a nucleic acid encoding the same, or pharmaceutical composition comprising said expression enhancing system or nucleic acid).
  • a target gene e.g., MYC, SFRP1, HNF4a
  • Methods and compositions as provided herein may treat a condition associated with overexpression or mis-regulation of a target gene, e.g., MYC, SFRP1, HNF4a , FOXP3, or APOB by stably or transiently altering (e.g., decreasing or increasing) transcription of a target gene, e.g., MYC, SFRP1, HNF4a , FOXP3, or APOB.
  • a target gene e.g., MYC, SFRP1, HNF4a , FOXP3, or APOB.
  • such a modulation persists for at least about 1 hour to about 30 days, or at least about 2 hours, 6 hours, 12 hours, 18 hours, 24 hours, 2 days, 3, days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 11 days, 12 days, 13 days, 14 days, 15 days, 16 days, 17 days, 18 days, 19 days, 20 days, 21 days, 22 days, 23 days, 24 days, 25 days, 26 days, 27 days, 28 days, 29 days, 30 days, or longer or any time therebetween.
  • such a modulation persists for 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, or 24 hours, or at least 1, 2, 3, 4, 5, 6, or 7 days, or at least 1, 2, 3, 4, or 5 weeks, or at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 months, or at least 1, 2, 3, 4, or 5 years (e.g., permanently or indefinitely).
  • such a modulation persists for no more than 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 years.
  • a method or composition provided herein may decrease expression of a target gene, e.g., MYC, SFRP1, HNF4a , FOXP3, or APOB in a cell by at least 10, 20, 30, 40, 50, 60, 70, 80, 90, or 100% (and optionally up to 100%) relative to expression of the target gene in a cell not contacted by the composition or treated with the method.
  • a target gene e.g., MYC, SFRP1, HNF4a , FOXP3, or APOB
  • a method provided herein may modulate, e.g., decrease or increase, expression of a target gene, e.g., MYC, SFRP1, HNF4a , FOXP3, or APOB by disrupting a genomic complex, e.g., an anchor sequence-mediated conjunction, associated with said target gene.
  • a target gene e.g., MYC, SFRP1, HNF4a , FOXP3, or APOB
  • a genomic complex e.g., an anchor sequence-mediated conjunction
  • a gene that is associated with an anchor sequence-mediated conjunction may be at least partially within a conjunction (that is, situated sequence-wise between a first and second anchor sequences), or it may be external to a conjunction in that it is not situated sequence-wise between a first and second anchor sequences, but is located on the same chromosome and in sufficient proximity to at least a first or a second anchor sequence such that its expression can be modulated by controlling the topology of the anchor sequence-mediated conjunction.
  • distance in three-dimensional space between two elements e.g., between the gene and the anchor sequence- mediated conjunction
  • distance in three-dimensional space between two elements may, in some embodiments, be more relevant than distance in terms of base pairs.
  • an external but associated gene is located within 2 Mb, within 1.9 Mb, within 1.8 Mb, within 1.7 Mb, within 1.6 Mb, within 1.5 Mb, within 1.4 Mb, with 1.3 Mb, within 1.3 Mb, within 1.2 Mb, within 1.1 Mb, within 1 Mb, within 900 kb, within 800 kb, within 700 kb, within 500 kb, within 400 kb, within 300 kb, within 200 kb, within 100 kb, within 50 kb, within 20 kb, within 10 kb, or within 5 kb of the first or second anchor sequence.
  • modulating expression of a gene comprises altering accessibility of a transcriptional control sequence to a gene, e.g., MYC, SFRP1, HNF4a , FOXP3, or APOB.
  • a transcriptional control sequence whether internal or external to an anchor sequence-mediated conjunction, can be an enhancing sequence or a silencing (or repressive) sequence.
  • such provided technologies may be used to treat a gene mis-regulation disorder e.g., MYC, SFRP1, HNF4a, FOXP3, or APOB gene mis-regulation disorder e.g., a symptom associated with a MYC, SFRP1, HNF4a, FOXP3, or APOB gene mis-regulation in a subject, e.g., a patient, in need thereof.
  • a gene mis-regulation disorder e.g., MYC, SFRP1, HNF4a, FOXP3, or APOB gene mis-regulation
  • a subject e.g., a patient, in need thereof.
  • the disorder is associated with MYC mis-regulation, e.g., MYC overexpression.
  • the disorder is associated with AFP mis-regulation, e.g., AFP overexpression.
  • the disorder is associated with SFRP1 mis-regulation, e.g., SFRP1 overexpression.
  • the disorder is associated with HNF4a mis-regulation, e.g., HNF4a overexpression.
  • the disorder is associated with FOXP3 mis-regulation, e.g., FOXP3 under-expression.
  • the disorder is associated with APOB mis-regulation, e.g., APOB under- expression.
  • such provided technologies may be used to methylate the promoter of a target gene, e.g., MYC, SFRP1, HNF4a , FOXP3, or APOB, to treat a gene mis-regulation disorder e.g., MYC, SFRP1, HNF4a , FOXP3, or APOB gene mis-regulation disorder, e.g., a symptom associated with a MYC, SFRP1, HNF4a , FOXP3, or APOB gene mis-regulation in a subject, e.g., a patient, in need thereof.
  • a target gene e.g., MYC, SFRP1, HNF4a , FOXP3, or APOB
  • such provided technologies may selectively affect the viability of a cell which aberrantly expresses a polypeptide encoded by a target gene, e g., MYC, SFRP1, HNF4a , FOXP3, or APOB.
  • a target gene e g., MYC, SFRP1, HNF4a , FOXP3, or APOB.
  • such provided technologies may be used to treat a hepatic disorder or a disorder e.g. a symptom associated with a hepatic disorder in a subject, e.g., a patient, in need thereof.
  • such provided technologies may be used to treat a pulmonary disorder or a disorder e.g. a symptom associated with a hepatic disorder in a subject, e.g., a patient, in need thereof.
  • such provided technologies may be used to treat a neoplasia disorder e.g. a disorder or, a symptom associated with a neoplasia disorder in a subject, e.g., a patient, in need thereof.
  • such provided technologies may be used to treat a viral infection related disorder e.g. a disorder or a symptom associated with viral infection related disorder in a subject, e.g., a patient, in need thereof.
  • a viral infection related disorder e.g. a disorder or a symptom associated with viral infection related disorder
  • such provided technologies may be used to treat an alcohol misuse related disorder e.g. a disorder or a symptom associated with an alcohol misuse related disorder in a subject, e.g., a patient, in need thereof.
  • such provided technologies may be used to treat a neoplasia disorder associated with a viral infection or alcohol misuse, e.g., a disorder or a symptom associated with a neoplasia disorder that is associated with a viral infection or alcohol misuse in a subject, e.g., a patient, in need thereof.
  • a neoplasia disorder associated with a viral infection or alcohol misuse e.g., a disorder or a symptom associated with a neoplasia disorder that is associated with a viral infection or alcohol misuse in a subject, e.g., a patient, in need thereof.
  • the condition treated is neoplasia. In some embodiments, the condition treated is tumorigenesis. In some embodiments, the condition treated is cancer. In some embodiments, the cancer is associated with poor prognosis. In some embodiments, the cancer is associated with MYC misregulation, e.g., MYC overexpression. In some embodiments, the cancer is associated with AFP misregulation, e.g., AFP overexpression. In some embodiments, the cancer is a breast, a hepatic, a colorectal, a lung, a pancreatic, a gastric, and/or a uterine cancer. In some embodiments, the cancer is associated with an infection, e.g., viral, e.g., bacterial. In some embodiments, the cancer is associated with alcohol abuse. In some embodiments, the cancer is hepatocarcinoma.
  • the cancer cells are lung cancer cells, gastric, gastrointestinal, colorectal, pancreatic or hepatic cancer cells.
  • the cancer is hepatocellular carcinoma (HCC), Fibrolamellar Hepatocellular Carcinoma (FHCC), cholangiocarcinoma, Angiosarcoma, secondary liver cancer, non-small cell lung cancer (NSCLC), adenocarcinoma, small cell lung cancer (SCLC), large cell (undifferentiated) carcinoma, triple negative breast cancer, gastric adenocarcinoma, endometrial carcinoma, or pancreatic carcinoma.
  • HCC hepatocellular carcinoma
  • FHCC Fibrolamellar Hepatocellular Carcinoma
  • FHCC Fibrolamellar Hepatocellular Carcinoma
  • Angiosarcoma secondary liver cancer
  • NSCLC non-small cell lung cancer
  • SCLC small cell lung cancer
  • large cell (undifferentiated) carcinoma triple negative breast cancer
  • the condition treated is a hepatic disease. In some embodiments the condition treated is associated with MYC mis-regulation, e.g., MYC overexpression. In some embodiments the condition treated is a chronic disease. In some embodiments the condition treated is a chronic liver disease. In some embodiments the condition treated is a viral infection. In some embodiments, the condition treated is an alcohol misuse associated disorder.
  • the condition treated is a pulmonary disease.
  • the condition treated is associated with MYC mis-regulation, e.g., MYC overexpression.
  • the condition treated is a chronic disease.
  • the condition treated is a chronic pulmonary disease.
  • such provided technologies may be used to treat or reduce lung cancer growth, metastasis, drug resistance, and/or cancer stem cell (CSC) maintenance.
  • the condition treated is a carcinoma, e.g., non-small cell lung cancer (NSCLC).
  • the chronic pulmonary disease is associated with tobacco misuse.
  • the cancer hepatocarcinoma subtype SI HCC SI
  • hepatocarcinoma subtype S2 HCC S2
  • hepatocarcinoma subtype S3 HCC S2
  • the HCC subtype is associated with MYC overexpression.
  • the cancer is HCC SI or HCC S2.
  • the cancer subtype is associated with aggressive tumor and poor clinical outcome.
  • the disclosure provides a treatment regimen that may be devised for the subject on the basis of the HCC subtype in the subject, e.g., a personalized approach to tailor the aggressiveness of treatment based on HCC subtype on a subject.
  • the disclosure provides a method of treatment using the expression repressors or expression repressor systems disclosed herein, the method comprising, identifying the HCC subtype in a patient and determine a dosage and administration schedule of said expression repressors and/or expression repressor systems based on the HCC subtype identification.
  • Methods are described herein to deliver agents, or a composition as disclosed herein to a subject for treatment of a disorder such that the subject suffers minimal side effects or systemic toxicity in comparison to chemotherapy treatment.
  • the subject does not experience any significant side effects typically associated with chemotherapy, when treated with the agents and/or compositions described herein.
  • the subject does not experience a significant side effect including but not limited to alopecia, nausea, vomiting, poor appetite, soreness, neutropenia, anemia, thrombocytopenia, dizziness, fatigue, constipation, oral ulcers, itchy skin, peeling, nerve and muscle damage, auditory changes, weight loss, diarrhea, immunosuppression, bruising, heart damage, bleeding, liver damage, kidney damage, edema, mouth and throat sores, infertility, fibrosis, epilation, moist desquamation, mucosal dryness, vertigo and encephalopathy when treated with the agents and/or compositions described herein.
  • alopecia nausea, vomiting, poor appetite, soreness, neutropenia, anemia, thrombocytopenia, dizziness, fatigue, constipation, oral ulcers, itchy skin, peeling, nerve and muscle damage
  • auditory changes weight loss, diarrhea, immunosuppression, bruising, heart damage, bleeding, liver damage, kidney damage, edema,
  • the subject does not show a significant loss of body weight when treated with the agents and/or compositions described herein.
  • the agents and compositions described herein can be administered to a subject, e.g., a mammal, e.g., in vivo, to treat or prevent a variety of disorders as described herein. This includes disorders involving cells characterized by altered expression patterns of MYC.
  • the present disclosure is further directed, in part, to a method of epigenetically modifying a target gene, a transcription control element operably linked to a target gene, or an anchor sequence (e.g., an anchor sequence proximal to a target gene or associated with an anchor sequence-mediated conjunction operably linked to a target gene), the method comprising providing an expression repressor or expression enhancer (or nucleic acid encoding the same ) or an expression repression system or expression enhancing system (e.g., expression repressor(s) or expression enhancer(s)), or nucleic acid encoding the same or pharmaceutical composition comprising said an expression repressor or expression enhancer (or nucleic acid encoding the same ) or an expression repression system or expression enhancing system (e.g., expression repressor(s) or expression enhancer(s)); and contacting the target gene or a transcription control element operably linked to the target gene with the expression repressor, expression enhancers, expression repress
  • a method of epigenetically modifying a target gene e.g., MYC, SFRP1, HNF4a , FOXP3, or APOB or a transcription control element operably linked to a target gene, e.g., MYC, SFRP1, HNF4a , FOXP3, or APOB may decrease or increase the level of the epigenetic modification by at least 10, 20, 30, 40, 50, 60, 70, 80, 90, or 100% (and optionally up to 100%) relative to the level of the epigenetic modification at that site in a cell not contacted by the composition or treated with the method.
  • a method of epigenetically modifying a target gene e.g., MYC, SFRP1, HNF4a , FOXP3, or APOB or a transcription control element operably linked to a target gene, e.g., MYC may increase the level of the epigenetic modification by at least 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 120, 140, 160, 180, 200, 300, 400, 500, 600, 700, 800, 900, or 1000% (and optionally up to 200, 300, 400, 500, 600, 700, 800, 900, 1000, or 2000%) relative to the level of the epigenetic modification at that site in a cell not contacted by the composition or treated with the method.
  • epigenetic modification of a target gene e.g., MYC, SFRP1, HNF4a , FOXP3, or APOB or a transcription control element operably linked to a target gene, e.g., MYC, SFRP1, HNF4a , FOXP3, or APOB may modify the level of expression of the target gene, e.g., MYC, SFRP1, HNF4a , FOXP3, or APOB, e.g., as described herein.
  • an epigenetic modification produced by a method described herein persists for at least about 1 hour to about 30 days, or at least about 2 hours, 6 hours, 12 hours, 18 hours, 24 hours, 2 days, 3, days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 11 days, 12 days, 13 days, 14 days, 15 days, 16 days, 17 days, 18 days, 19 days, 20 days, 21 days, 22 days, 23 days, 24 days, 25 days, 26 days, 27 days, 28 days, 29 days, 30 days, or longer or any time therebetween.
  • such a modulation persists for 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, or 24 hours, or at least 1, 2, 3, 4, 5, 6, or 7 days, or at least 1, 2, 3, 4, or 5 weeks, or at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 months, or at least 1, 2, 3, 4, or 5 years (e.g., indefinitely).
  • such a modulation persists for no more than 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 years.
  • an expression repressor, expression enhancers, expression repressor system, or expression enhancing system for use in a method of epigenetically modifying a target gene, e.g., MYC, SFRP1, HNF4a , FOXP3, or APOB or a transcription control element operably linked to a target gene, e.g., MYC, SFRP1, HNF4a , FOXP3, or APOB comprises an expression repressor comprising an effector moiety that is or comprises an epigenetic modifying moiety.
  • a effector moiety may be or comprise an epigenetic modifying moiety with DNA methyltransferase activity, and an endogenous or naturally occurring target sequence (e.g. a target gene, e.g., MYC, SFRP1, HNF4a , FOXP3, or APOB or transcription control element) may be altered to increase its methylation (e.g., decreasing interaction of a transcription factor with a portion of target gene, e.g., MYC, SFRP1, HNF4a , FOXP3, or APOB or transcription control element, decreasing binding of a nucleating protein to an anchor sequence, and/or disrupting or preventing an anchor sequence-mediated conjunction), or may be altered to decrease its methylation (e.g., increasing interaction of a transcription factor with a portion of a target gene, e.g., MYC, SFRP1, HNF4a , FOXP3, or APOB or transcription control element, increasing binding of a nucleating
  • a target gene
  • Example 1 Mouse syngeneic Hepal.6 model treated with muOEC (ZF17-MQ1) showed reduction of tumor burden and methylation of circulating DNA extracted from mouse serum.
  • mice were injected intravenously (IV) with PBS Q5D, negative control mRNA in MC3 at 1 mg/kg once every 5 days for 4 times (Q5Dx4), ZF-MQ1 in MC3 at 1 mg/kg or 0.3 mg/kg once every 10 days (Q10D).
  • IV intravenously
  • mice in MC3 at 1 mg/kg once every 5 days for 4 times
  • ZF-MQ1 in MC3 at 1 mg/kg or 0.3 mg/kg once every 10 days
  • Q10D 0.3 mg/kg once every 10 days
  • ExoEasy kit (Qiagen) was used to first isolate exosome from mouse serum, while a Qiagen minElute Vacuum kit was then used to isolate DNA. Isolated DNA was then bisulfite converted using EZ DNA Methylation kit.
  • Quantitative methylation PCR was used to determine on-target methylation of the MYC promoter by ZF17-MQ1. Converted DNA from serum was then amplified using PCR primers specific to methylated MYC promoter DNA. This signal was quantified using the A ACT relative to an ACTIN housekeeping control and a positive 100% methylated control. This study found significant increase in methylation signal in the serum from the animals treated with following treatment with ZF17-MQ1 at 1 mg/kg Q10D (FIGs. 2A and 2B).
  • RNA was then converted to cDNA with RT Lunascript.
  • the cDNA was then analyzed through AACT qPCR with a MYC (target) and GAPDH (reference) probe.
  • MYC target
  • GAPDH reference
  • Cellular supernatant was subject to a hard centrifugation to remove cells and debris and extracellular vesicles were isolated using the Qiagen exoRNeasy kit, which allows direct isolation of extracellular vesicle RNA from supernatant. Isolated extracellular vesicle RNA was then converted to cDNA with RT Lunascript.
  • the cDNA was then analyzed through AACT qPCR with a MYC (target) and GAPDH (reference) probe.
  • FIG. 3A cellular RNA
  • FIG. 3B Extracellular vesicle RNA shows -50% downregulation of MYC mRNA following treatment with MR-30723 as compared to untreated controls.
  • RNA was then converted to cDNA with RT Lunascript.
  • the cDNA was then analyzed through AACT qPCR with a MYC (target) and GAPDH (reference) probe.
  • MYC target
  • GAPDH reference
  • Cellular supernatant was subject to a 0.8 micron filter and extracellular vesicles were isolated using the Qiagen exoRNeasy kit, which allows direct isolation of extracellular vesicle RNA from the supernatant. Isolated extracellular vesicle RNA was then converted to cDNA with RT Lunascript.
  • the cDNA was then analyzed through AACT qPCR with a MYC (target) and GAPDH (reference) probe.
  • the objective of this study is to determine if in vitro and in vivo treatment of MR-30723 encapsulated in a LNP induces DNA methylation changes at the MYC genomic locus in culture SKHEP1 cells or tumors extracted from Hep3B subcutaneous xenografts.
  • Genomic DNA was extracted from SKHEP1 cells or Hep3B xenograft tumors ex vivo after treatment with MR-30723 encapsulated in a LNP (Acuitas Lot FM-1462A) or long non-coding mRNA (negative control; MR- 30627-2; Acuitas lot FM-1571A) in vivo.
  • Genomic DNA was subject to bisulfite conversion and amplified. DNA methylation of the MYC gene locus was quantified using quantitative methylation specific PCR (qMSP) with primer and probe designed to bisulfite converted methylated DNA.
  • qMSP quantitative methylation specific PCR
  • This study utilized qMSP to determine if DNA methylation at the MYC genomic locus is achieved after MR-30723 encapsulated in a LNP treatment.
  • Hep3B tumors were extracted from animals previously dosed with MR-30723 encapsulated in a LNP and lysed in Qiagen ATL buffer and proteinase K using a magnetic bead and SPEX tissue processor to homogenize the material. Lysate from these tumors or SKHEP1 cells cultured in vitro was then subjected to Qiagen DNeasy protocol and DNA was extracted according to manufacturer’s protocol. Total DNA was bisulfite converted using ZYMO EZ DNA methylation kit. Converted DNA was amplified using a custom design methylated MYC specific PCR assay. CT values for amplification were compared to control converted methylated DNA to determine if methylation could be detected in tumors.
  • Genomic DNA from both SKHEP1 cells treated in vitro and Hep3B tumors treated in vivo with MR-30723 encapsulated in a LNP showed positive amplification of the methylated DNA in the MYC genomic locus as measured by the qMSP assay as compared to methylated, bisulfite converted positive control DNA (FIG. 5A). This indicates that MR-30723 encapsulated in a LNP induced DNA methylation of the MYC gene locus in Hep3B xenograft tumors after in vivo and in vitro treatment. Amplification of DNA using methylation specific assay was detected for all treatment groups of MR- 30723 encapsulated in a LNP, while no amplification was detected in animals treated with PBS or negative control mRNA(FIG. 5B).
  • Example 4 Isolated extracellular vesicle DNA from cell supernatant
  • extracellular vesicle DNA was isolated from cell supernatant. Extracellular vesicles were first harvested from cellular supernatant using the Qiagen ExoEasy kit. These extracellular vesicles were then subjected to the qiAMP MinElute viral DNA isolation kit. DNA was then run through tapestation to analyze concentration and fragment size. This analysis showed that the extracellular vesicle DNA protocol was successful and yielded DNA with average fragment size of 300 BP, consistent with expected results (FIG. 6).
  • extracellular vesicle DNA was isolated from mouse serum. Extracellular vesicles were first harvested from serum using the Qiagen ExoEasy kit. These extracellular vesicles were then subjected to the qiAMP MinElute viral DNA isolation kit. DNA was then run through tapestation to analyze concentration and fragment size. This analysis showed that the extracellular vesicle DNA protocol was successful and yielded DNA with average fragment size on 200 BP, consistent with expected result (FIG. 7).
  • Example 5 MYC Methylation signal from extracellular vesicle DNA
  • qMSP was utilized to determine if MYC methylation could be detected following MR-30723 encapsulated in a LNP treatment in both cellular genomic DNA and in DNA isolated from extracellular vesicles.
  • Hep3B cells were plated in 10 cm dishes at 2 million cells per plate and treated with 1 ug/mL MR-30723 for 48 hours. Data was compared to cells treated with a non-coding mRNA negative control (SNC). After 48 hours of treatment cells were lysed for cellular genomic DNA isolation and cell supernatant was harvested for extracellular vesicle DNA isolation. Genomic DNA was isolated using the Qiagen DNeasy kit.
  • Extracellular vesicles were isolated from supernatant using the Qiagen exoeasy kit and DNA was isolated from these extracellular vesicles using the qiAMP MinElute viral DNA isolation kit. Both sources of DNA were bisulfite converted using ZYMO EZ DNA methylation kit. Converted DNA was amplified using a custom design methylated MYC specific PCR assay. CT values for amplification were compared to control converted methylated DNA to determine if methylation could be detected.
  • qMSP was utilized to determine if MYC methylation could be detected following MR-30723 encapsulated in a LNP treatment in both cellular genomic DNA and in DNA isolated from extracellular vesicles.
  • Hep3B cells were plated in 10 cm dishes at 2 million cells per plate and treated with 0.5 or 0.05 ug/mL MR-30723 encapsulated in a LNPP for 24 or 48 hours. Data was compared to cells treated with a non-coding mRNA negative control (SNC). After treatment cells were lysed for cellular genomic DNA isolation and cell supernatant was harvested for extracellular vesicle DNA isolation. Genomic DNA was isolated using the Qiagen DNeasy kit.
  • Extracellular vesicles were isolated from supernatant using the Qiagen exoeasy kit and DNA was isolated from these extracellular vesicles using the qiAMP minElute viral DNA isolation kit. Both sources of DNA were bisulfite converted using ZYMO EZ DNA methylation kit. Converted DNA was amplified using a custom design methylated MYC specific PCR assay. CT values for amplification were compared to control converted methylated DNA to determine if methylation could be detected.
  • Converted DNA was amplified using a custom design methylated MYC specific PCR assay. CT values for amplification were compared to control converted methylated DNA to determine if methylation could be detected (FIGs. 10A and 10B).
  • QuantiGeneTM Singleplex bDNA assay was used to measure the exogenous mRNA MR- 32380, which encodes an epigenetic modifying agent, in plasma from female nude mice (Jackson Laboratories, strain code 0007850). The luminescent signals from OEC-treated mice reached the maximum detection limit of the assay. Plasma samples derived from the mice were titrated to get the signal within the assay range and quantify the results.
  • MR-32380 RNA in plasma samples was diluted 1:54, 1: 100, 1:300, 1: 1000, or 1: 10000. Samples were quantified using standard curve of formulated OEC spiked in mouse plasma. Measurements of 3 mice from group 2 (1.2 mg/kg and collected 24h after dosing) were compared with 3 PBS-treated mice.
  • Lipid nanoparticles were diluted 80 ug/mL SSOP -MR-32380-2 LNP to 2.06E-03 ug/mL in mouse plasma.
  • Plasma samples were diluted in mouse plasma. Dilution of OEC-treated samples 1:54, 1: 100, 1:300, 1: 1000, and 1: 10000.
  • QuantiGeneTM Sample Processing Kit were used to prepare the blood samples. On day 1 of the QGS assay, the standard and sample lysates were incubated with MQ 1 probe mix in capture plate overnight. On day 2, the plate was read to quantify signal.
  • results show that the branched DNA assay can be used to detect exogenous mRNA administered to subjects.
  • the results also suggest that the assay may be useful for monitoring circulating levels of therapeutic mRNAs for evaluation of dosing regimens in, e.g., human subjects. For example, measurement of exogenous mRNA levels may be used for determining whether adjustments to one or more of the dose, frequency, and route of administration are necessary to achieve optimal therapeutic benefit.
  • the epigenetic modifying agent (OEC) is being evaluated in a first-in-human Phase 1/2 clinical trial as a monotherapy and in combination with standard of care (SoC) agents for patients with HCC and other solid tumors known for association with the MYC oncogene (NCT05497453).
  • SoC standard of care
  • Part 1 dose escalation uses a classic 3+3 design to explore ascending doses of the epigenetic modifying agent to identify dose limiting toxicities, maximum tolerated dose, safety and tolerability, recommended dose for expansion, and preliminary antitumor activity in patients with HCC and other solid tumors.
  • Biomarkers of pharmacodynamic (PD) activity and tumor response via blood and tissue are also being investigated.
  • a targeted NGS-based approach was applied, with MYC locus cell-free DNA captured and processed using standard molecular techniques to quantify methylation at high resolution.
  • Highly-methylated DNA fragments are categorized and statistics applied to measure on- target methylation before and during treatment with the epigenetic modifying agent.
  • Hepatocellular carcinoma (HCC) tumor cells were induced in female nude mice, which were inoculated subcutaneously in the left flank with 1 x 10 7 HCC cells. Treatment was initiated when the tumors reached a mean volume of 150 mm 3 . Mice were allocated into groups such that mean tumor volume in each group was within similar range. Mice were treated with vehicle, or test articles. Test articles were given via intravenous injection (IV) or oral gavage (PO). Animal weights and conditions were recorded daily. Tumors were measured on Mondays, Wednesdays, and Fridays by measuring each tumor in two dimensions, first by measuring the longest dimension (“length”), and then the dimension perpendicular to this (“width”). Tumor volumes were calculated using the standard formula: (L x W2)/2. The mean tumor volume and standard error of the mean was calculated for each group at each time point.
  • IV intravenous injection
  • PO oral gavage
  • Genomic DNA was extracted from cells derived from in vitro cultures or tissue samples, and extracellular vesicle DNA (evDNA) or cell-free DNA (cfDNA) were extracted from plasma samples using the appropriate Qiagen® extraction kit(s).
  • gDNA was sheared on the PIXUL acoustic sonicator (Active Motif) to an average fragment size of 300 bp (Pulse 5 N; PRF 10.00 kHz; Process Time 120:00 min; Burst Rate 20.00 Hz).
  • cfDNA and evDNA were used directly into the library preparation as they exist in an already fragmented state.
  • Methylation enrichment NGS libraries were prepared according the manufacturer’s protocol for Targeted Methylation Sequencing (Twist Biosciences). Briefly, NGS adapters were ligated onto end-prepped DNA fragments, and the fragments were enzymatically converted via TET oxidation and APOBEC deamination (NEB). After 9 cycles of PCR amplification and dual-indexing, 187.5 ng of each library was combined into 8-plex pools and dried in a centrifuge vacuum concentrator (Eppendorf). Libraries were rehydrated with a MYC Methylation probe panel (or the Twist Total MethylomeTM panel) combined with blocking and enhancer reagents. Libraries were hybridized overnight (> 16h) at 60 C.
  • Circulating tumor MYC fragments were rare overall, indicating the necessity for target enrichment for efficient detection.
  • Target enrichment with the Twist Total MethylomeTM panel using cf- and evDNA was preformed and 2000X coverage increase with less sequencing depth was obtained, while identifying low-level (5-20%) methylation at the MYC promoter (FIGs. 14A and 14B). Since the preclinical studies were mice bearing human tumors, our human-specific target enrichment panel followed by human-specific computational alignment, these methylation events were considered to be derived from circulating tumor DNA (ctDNA).
  • TGI Tumor growth inhibitions
  • Hep 3B tumor model in mice were treated with three doses of MR-30882LNP then collected at different time points to measure mean tumor volume (FIG. 17A) and mean percent weight change (FIG. 17B) to assess PK/PD.
  • Triangles along x-axis indicate time points (days 0, 5, and 10) that the mice were dosed (FIG. 17A and 17B).
  • mice were treated with two doses of MR- 30882/LNP on days 0 and 5 as indicated by triangles along the x-axis and collected at the same time points (post-dosing) (FIG. 18A).
  • the MYC promoter methylation distribution was determined after 48 hours and 14 days using percent VEF analysis (FIG. 18B).
  • EVDNA refers to extracellular DNA from a liquid biopsy
  • GDNA genomic DNA derived from a tumor.
  • the MYC Methylation Panel was used to identify MYC promoter methylation in evDNA and genomic DNA derived from these animals (FIGs. 18C and 18D).
  • DNA Methylation was assessed using EM-conversion (NEB) followed by NGS library preparation, using Twist Bioscience’s NGS Methylation Detection System for target enrichment where indicated.
  • the enrichment panel spanned a total of 51.5 kb, including both the MYC promoter and gene body as well as promoter CpG islands from control genes.
  • Epiallele detection measured as the variant epiallele fraction (VEF) was performed using the EpiAlleleR package3 after Bismark mapping to identify methylated MYC molecules as opposed to averaging per-CpG rates over a region.
  • MYC gene expression was assessed via qPCR.
  • VEF Variant epiallele fraction
  • variable epiallele represents a group of epialleles (i.e., individual methylation patterns) with similar methylation properties that is defined by thresholding; therefore, VEF effectively represents the frequency of this group of epialleles passing the threshold at the level of individual cytosines or extended genomic regions.
  • mice were treated with one dose of MR-30723/LNP at day 0 and collected at the same time points (post-dosing) (FIG. 19A).
  • cfDNA was extracted (as opposed to evDNA), and the MYC Methylation Panel was used to identify percent VEF and MYC promoter methylation (FIGs. 19B and 19C).
  • Ultramer ssDNA oligonucleotides (4nmol) were ordered to represent the postconverted sequence based on the fully methylated or fully unmethylated state of chr8: 127,735,839- 127,735,972 (hg38). These contained sequencing-ready adapters to facilitate NGS library preparation.
  • Targeted methylation sequencing was performed on a titration series of methylated control gDNA spiked into unmethylated control gDNA (Zymo Research) and analyzed for methylated epialleles at the MYC promoter. 37.5 ng of total gDNA was used as input into library preparation and hybridization onto the MYC Methylation Panel. MYC promoter methylation was detected in samples containing as low as 0.05% methylated gDNA compared to fully unmethylated control gDNA (FIG. 21). This was at the theoretical limit of the number of copies present in the assay.
  • a Hep3B HCC xenograft model was set up as in Examples 11-13.
  • MR-30882/LNP was dosed at 1 mg/kg or PBS Q5D until day 25. Animals were sacrificed when tumors reached 2000 mm3.
  • cfDNA was extracted from a pool of 3 mice from the PBS and MR-30882/LNP -treated groups. Separately, healthy human plasma was obtained from a commercial vendor, and cfDNA was extracted. cfDNA was extracted from 2 individuals, pooled, and redistributed to two individual samples. To mimic the amount of ctDNA in the background of cfDNA in a clinical sample, the PBS- treated and MR-30882/LNP -treated xenograft cfDNA samples were spiked into the human cfDNA pools.
  • Targeted methylation sequencing using the MYC methylation panel was able to discriminate between the MR-30882/LNP -treated (EC) sample and the PBS-treated sample (FIG. 22A).
  • the MYC Methylation Panel provides the necessary enrichment over whole-genome sequencing to detect regions of interest. (FIG. 22B).

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Abstract

La présente invention est basée, au moins en partie, sur la découverte selon laquelle le dosage d'un agent de modification épigénétique peut être déterminé sur la base de l'évaluation de la méthylation de l'ADN d'un biomarqueur associé à un gène cible pour lequel l'expression est nécessaire pour moduler et/ou évaluer le niveau d'ARN de vésicule extracellulaire associé à un ou plusieurs biomarqueurs.
PCT/US2024/047157 2023-09-18 2024-09-18 Procédés d'évaluation de dosage pour agents de modification épigénétiques Pending WO2025064469A1 (fr)

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Citations (20)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US6453242B1 (en) 1999-01-12 2002-09-17 Sangamo Biosciences, Inc. Selection of sites for targeting by zinc finger proteins and methods of designing zinc finger proteins to bind to preselected sites
US6503717B2 (en) 1999-12-06 2003-01-07 Sangamo Biosciences, Inc. Methods of using randomized libraries of zinc finger proteins for the identification of gene function
US6534261B1 (en) 1999-01-12 2003-03-18 Sangamo Biosciences, Inc. Regulation of endogenous gene expression in cells using zinc finger proteins
US6599692B1 (en) 1999-09-14 2003-07-29 Sangamo Bioscience, Inc. Functional genomics using zinc finger proteins
US6689558B2 (en) 2000-02-08 2004-02-10 Sangamo Biosciences, Inc. Cells for drug discovery
US6794136B1 (en) 2000-11-20 2004-09-21 Sangamo Biosciences, Inc. Iterative optimization in the design of binding proteins
US20050064474A1 (en) 2003-08-08 2005-03-24 Sangamo Biosciences, Inc. Methods and compositions for targeted cleavage and recombination
US20050267061A1 (en) 2004-04-08 2005-12-01 Sangamo Biosciences, Inc. Methods and compositions for treating neuropathic and neurodegenerative conditions
US7030215B2 (en) 1999-03-24 2006-04-18 Sangamo Biosciences, Inc. Position dependent recognition of GNN nucleotide triplets by zinc fingers
US7067317B2 (en) 2000-12-07 2006-06-27 Sangamo Biosciences, Inc. Regulation of angiogenesis with zinc finger proteins
US7070934B2 (en) 1999-01-12 2006-07-04 Sangamo Biosciences, Inc. Ligand-controlled regulation of endogenous gene expression
US7253273B2 (en) 2004-04-08 2007-08-07 Sangamo Biosciences, Inc. Treatment of neuropathic pain with zinc finger proteins
US7262054B2 (en) 2002-01-22 2007-08-28 Sangamo Biosciences, Inc. Zinc finger proteins for DNA binding and gene regulation in plants
US20070218528A1 (en) 2004-02-05 2007-09-20 Sangamo Biosciences, Inc. Methods and compositions for targeted cleavage and recombination
US7361635B2 (en) 2002-08-29 2008-04-22 Sangamo Biosciences, Inc. Simultaneous modulation of multiple genes
WO2014012081A2 (fr) 2012-07-13 2014-01-16 Ontorii, Inc. Contrôle chiral
WO2021061815A1 (fr) * 2019-09-23 2021-04-01 Omega Therapeutics, Inc. Compositions et procédés de modulation de l'expression génique du facteur nucléaire hépatocytaire 4-alpha (hnf4α)
WO2022067033A1 (fr) * 2020-09-24 2022-03-31 Flagship Pioneering Innovations V, Inc. Compositions et procédés d'inhibition de l'expression génique
WO2022132195A2 (fr) * 2020-12-15 2022-06-23 Flagship Pioneering Innovations V, Inc. Compositions et méthodes de modulation de l'expression de myc
WO2023283359A2 (fr) * 2021-07-07 2023-01-12 Omega Therapeutics, Inc. Compositions et procédés de modulation de l'expression génique de la protéine 1 du récepteur frizzled secrété (sfrp1)

Patent Citations (20)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US6534261B1 (en) 1999-01-12 2003-03-18 Sangamo Biosciences, Inc. Regulation of endogenous gene expression in cells using zinc finger proteins
US6453242B1 (en) 1999-01-12 2002-09-17 Sangamo Biosciences, Inc. Selection of sites for targeting by zinc finger proteins and methods of designing zinc finger proteins to bind to preselected sites
US7070934B2 (en) 1999-01-12 2006-07-04 Sangamo Biosciences, Inc. Ligand-controlled regulation of endogenous gene expression
US7030215B2 (en) 1999-03-24 2006-04-18 Sangamo Biosciences, Inc. Position dependent recognition of GNN nucleotide triplets by zinc fingers
US6599692B1 (en) 1999-09-14 2003-07-29 Sangamo Bioscience, Inc. Functional genomics using zinc finger proteins
US6503717B2 (en) 1999-12-06 2003-01-07 Sangamo Biosciences, Inc. Methods of using randomized libraries of zinc finger proteins for the identification of gene function
US6689558B2 (en) 2000-02-08 2004-02-10 Sangamo Biosciences, Inc. Cells for drug discovery
US6794136B1 (en) 2000-11-20 2004-09-21 Sangamo Biosciences, Inc. Iterative optimization in the design of binding proteins
US7067317B2 (en) 2000-12-07 2006-06-27 Sangamo Biosciences, Inc. Regulation of angiogenesis with zinc finger proteins
US7262054B2 (en) 2002-01-22 2007-08-28 Sangamo Biosciences, Inc. Zinc finger proteins for DNA binding and gene regulation in plants
US7361635B2 (en) 2002-08-29 2008-04-22 Sangamo Biosciences, Inc. Simultaneous modulation of multiple genes
US20050064474A1 (en) 2003-08-08 2005-03-24 Sangamo Biosciences, Inc. Methods and compositions for targeted cleavage and recombination
US20070218528A1 (en) 2004-02-05 2007-09-20 Sangamo Biosciences, Inc. Methods and compositions for targeted cleavage and recombination
US20050267061A1 (en) 2004-04-08 2005-12-01 Sangamo Biosciences, Inc. Methods and compositions for treating neuropathic and neurodegenerative conditions
US7253273B2 (en) 2004-04-08 2007-08-07 Sangamo Biosciences, Inc. Treatment of neuropathic pain with zinc finger proteins
WO2014012081A2 (fr) 2012-07-13 2014-01-16 Ontorii, Inc. Contrôle chiral
WO2021061815A1 (fr) * 2019-09-23 2021-04-01 Omega Therapeutics, Inc. Compositions et procédés de modulation de l'expression génique du facteur nucléaire hépatocytaire 4-alpha (hnf4α)
WO2022067033A1 (fr) * 2020-09-24 2022-03-31 Flagship Pioneering Innovations V, Inc. Compositions et procédés d'inhibition de l'expression génique
WO2022132195A2 (fr) * 2020-12-15 2022-06-23 Flagship Pioneering Innovations V, Inc. Compositions et méthodes de modulation de l'expression de myc
WO2023283359A2 (fr) * 2021-07-07 2023-01-12 Omega Therapeutics, Inc. Compositions et procédés de modulation de l'expression génique de la protéine 1 du récepteur frizzled secrété (sfrp1)

Non-Patent Citations (29)

* Cited by examiner, † Cited by third party
Title
"GenBank", Database accession no. NM 00 1114377.2
"NCBI", Database accession no. NM - 002467
BEERLI, NATURE BIOTECHNOL., vol. 20, 2002, pages 135 - 141
BUCKLEY DAVID N ET AL: "Targeted DNA methylation from cell-free DNA using hybridization probe capture", NAR GENOMICS AND BIOINFORMATICS, vol. 4, no. 4, 31 December 2022 (2022-12-31), XP093214328, ISSN: 2631-9268, Retrieved from the Internet <URL:https://academic.oup.com/nargab/article/4/4/lqac099/6965981?login=true> DOI: 10.1093/nargab/lqac099 *
CHOO ET AL., CURR. OPIN. STRUCT. BIOL., vol. 10, 2000, pages 411 - 416
CHROMY ET AL., J. PROTEOME RES, vol. 3, 2004, pages 1120 - 1127
DE GROOTE ET AL., NUC. ACIDS RES., 2012, pages 1 - 18
DELPHINE LISSA ET AL: "Methylation analyses in liquid biopsy", TRANSLATIONAL LUNG CANCER RESEARCH, vol. 5, no. 5, 1 October 2016 (2016-10-01), Hong Kong, pages 492 - 504, XP055723704, ISSN: 2218-6751, DOI: 10.21037/tlcr.2016.10.03 *
DEUTSCH ET AL., J NEUROL NEUROSURG PSYCHIATRY, vol. 85, no. 9, September 2014 (2014-09-01), pages 994 - 1002
EMINAGA ET AL.: "Quantification of microRNA Expression with Next-Generation Sequencing", CURRENT PROTOCOLS IN MOLECULAR BIOLOGY, vol. 103, 2013, pages 1 - 14
FROMMER, PROC NATL ACAD SCI USA, vol. 89, no. 5, 1992, pages 1827 - 31
GONZALGO, NAT PROTOC, vol. 2, no. 8, 2007, pages 1931 - 6
HENDEL, NATURE BIOTECHNOLOGY, 2015, pages 985 - 991
HERMAN, PROC. NATL. ACAD. SCI. USA, vol. 93, 1992, pages 9821 - 9826
HUSSMANN, METHODS MOL BIOL, vol. 1708, 2018, pages 497 - 513
ISALAN ET AL., NATURE BIOTECHNOL, vol. 19, 2001, pages 656 - 660
KOFERLE ET AL., GENOME MEDICINE, vol. 7, no. 59, 2015, pages 1 - 3
LEI YONG ET AL: "DNA methylation and de-methylation using hybrid site-targeting proteins", GENOME BIOLOGY, vol. 19, no. 1, 6 November 2018 (2018-11-06), XP093036053, Retrieved from the Internet <URL:http://link.springer.com/article/10.1186/s13059-018-1566-2/fulltext.html> DOI: 10.1186/s13059-018-1566-2 *
LEI YONG ET AL: "Targeted DNA methylation in vivo using an engineered dCas9-MQ1 fusion protein", NATURE COMMUNICATIONS, vol. 8, no. 1, 11 July 2017 (2017-07-11), UK, XP093197851, ISSN: 2041-1723, Retrieved from the Internet <URL:https://www.nature.com/articles/ncomms16026> DOI: 10.1038/ncomms16026 *
LIU, NUCLEIC ACIDS RES., vol. 45, no. 6, 2017, pages e39
NIKOLAIENKO OLØNNING PEKNAPPSKOG S: "epialleleR: an R/Bioconductor package for sensitive allele-specific methylation analysis in NGS data", GIGASCIENCE, 28 December 2022 (2022-12-28)
OGLESBEE ET AL., CLIN CHEM, vol. 59, no. 10, October 2013 (2013-10-01), pages 1461 - 9
PABO ET AL., ANN. REV. BIOCHEM., vol. 70, 2001, pages 313 - 340
PISITKUN ET AL., PROC NATL ACAD SCI USA, vol. 101, 2004, pages 13368 - 13373
SEGAL ET AL., CURR. OPIN. BIOTECHNOL., vol. 12, 2001, pages 632 - 637
SENAPEDIS WILLIAM ET AL: "Abstract P-307: Modulation of the MYC oncogene using programmable epigenetic mRNA therapeutics as a novel therapy for hepatocellular carcinoma", 30 June 2023 (2023-06-30), XP093236807, Retrieved from the Internet <URL:https://omegatherapeutics.com/wp-content/uploads/2023/06/Modulation-of-the-MYC-Oncogene-Using-Programmable-mRNA-Therapeutics-as-a-Novel-Therapy-for-Hepatocellular-Carcinoma.pdf> *
STEELAND ET AL.: "Nanobodies as therapeutics: big opportunities for small antibodies", DRUG DISCOV TODAY, vol. 21, no. 7, 2016, pages 1076 - 113, XP093161503, DOI: 10.1016/j.drudis.2016.04.003
TAYLOR FEEHLEY ET AL: "Drugging the epigenome in the age of precision medicine", CLINICAL EPIGENETICS, BIOMED CENTRAL LTD, LONDON, UK, vol. 15, no. 1, 11 January 2023 (2023-01-11), pages 1 - 13, XP021313184, DOI: 10.1186/S13148-022-01419-Z *
ZHANG ET AL., MOL CELL PROTEOMICS, vol. 4, 2005, pages 144 - 155

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