Methods and compositions for regulating methylation of target genes

Abstract

This disclosure relates to a method for increasing DNA methylation at a site in a region of the genome containing a target gene, using, for example, an expression repressor comprising a DNA target-directed portion that binds to a target sequence in that region and an effector domain that methylates DNA (e.g., a DNA methyltransferase), or a nucleic acid encoding an expression repressor. Systems comprising two or more expression repressors are also disclosed. This composition can be used, for example, to treat conditions related to the expression of a target gene by reducing the expression of the target gene in a cell or subject.

Description

Technical Field

[0001] Cross - reference to related applications This application claims the benefit of U.S. Provisional Patent Application No. 63 / 502,581, filed on May 16, 2023, which is hereby incorporated by reference in its entirety.

Background Art

[0002] The treatment and / or prevention of numerous disease states can be achieved by down - regulating or inhibiting the expression and / or activity of a target gene or its transcriptional or translational products. Such approaches include, for example, introducing precise gene editing or deletions that impair the expression of a target gene or functional gene product by gene editing based on the CRISPR / Cas system. However, there are problems with the manipulation of genomic sequences using such systems because there is a risk of introducing harmful off - target editing. Alternatively, oligonucleotide inhibitors (e.g., antisense or RNA interference techniques) and pharmacological agents (e.g., antibodies or small - molecule antagonists) are used as therapeutic methods to silence or down - regulate the expression and / or activity of a target transcript or its product. The application of such inhibitors generally requires repeating a dosing scheme, which can be quite costly, but there is no risk of off - target mutagenesis (e.g., of the kind that can occur with certain gene - editing techniques), or the complex and cumbersome dosing schedules resulting from the limited half - life of therapeutic agents are not required.

Summary of the Invention

Means for Solving the Problems

[0003] In certain embodiments, the Disclosure provides a method for increasing the DNA methylation of a target gene, comprising administering to a subject a certain dose of a composition comprising an expression repressor or a nucleic acid molecule encoding an expression repressor, wherein the expression repressor comprises a DNA target-directed moiety and a DNA methyltransferase, and the DNA methylation of the target gene increases in the subject for a period of at least 21 days after administration of the dose, provided that the subject has not received any subsequent doses during that period.

[0004] In some embodiments, the Disclosure provides a method for increasing the DNA methylation of a target gene in a subject, comprising administering to the subject a first dose of a composition comprising an expression repressor or a nucleic acid molecule encoding an expression repressor, wherein the expression repressor comprises a DNA target-directed moiety and a DNA methyltransferase, and the DNA methylation of the target gene is increased in the subject for a period of at least 21 days after administration of the first dose. In some embodiments, the subject is not administered subsequent doses during that period.

[0005] In another embodiment, the Specified Information provides a method for reducing the expression of a target gene, comprising administering to a subject a first dose of a composition comprising an expression repressor or a nucleic acid molecule encoding an expression repressor, wherein the expression repressor comprises a DNA target-directing moiety and a DNA methyltransferase, and the expression of the target gene is reduced in the subject for a period of at least 21 days after administration of the first dose.

[0006] In another embodiment, the Specified Provision provides a method for increasing the DNA methylation of a target gene in a cell, comprising contacting the cell with a first dose of a composition comprising an expression repressor or a nucleic acid molecule encoding an expression repressor, wherein the expression repressor comprises a DNA target-directing moiety and a DNA methyltransferase, and the DNA methylation of the target gene is increased for a period of at least 21 days after contact with the first dose.

[0007] In another embodiment, the Specified Provision provides a method for reducing the expression of a target gene in a cell, comprising contacting the cell with a first dose of a composition comprising an expression repressor or a nucleic acid molecule encoding an expression repressor, wherein the expression repressor comprises a DNA target-directing moiety and a DNA methyltransferase, and the expression of the target gene is reduced for a period of at least 21 days after contact with the first dose.

[0008] In another embodiment, this specification provides a method for reducing the expression of a target gene, comprising administering to a subject a certain dose of an expression repressor or a nucleic acid molecule encoding an expression repressor, wherein the expression of the target gene is reduced for a period of at least 21 days after the administration of the dose, provided that the subject has not received a subsequent dose during that period, thereby reducing the expression of the target gene. In some embodiments, the reduction in the expression of the target gene is measured in a tissue sample obtained from the subject compared to a control sample. In some embodiments, the level of one or more biomarkers associated with the target gene is reduced in the tissue sample compared to a control sample.

[0009] In another embodiment, the Specified provides a method for treating a condition associated with dysregulation of a target gene, comprising administering to a subject a certain dose of an expression repressor or a nucleic acid molecule encoding an expression repressor, wherein the expression repressor comprises a DNA target-directing moiety and a DNA methyltransferase, and the expression of the target gene is reduced and / or DNA methylation of the target gene is increased for a period of at least 21 days after the administration of the dose, provided that the subject has not received a subsequent dose during that period, thereby treating the condition.

[0010] In another embodiment, the Specified provides a method for treating a condition associated with dysregulation of a target gene, comprising administering to a subject a first dose of an expression repressor or a nucleic acid molecule encoding an expression repressor, wherein the expression repressor comprises a DNA target-directing moiety and a DNA methyltransferase, and the expression of the target gene is reduced and / or DNA methylation of the target gene is increased for a period of at least 21 days after administration of the first dose, thereby treating the condition.

[0011] In some embodiments of the aforementioned or related aspects, the subject is not administered the following doses during that period.

[0012] In some embodiments of the aforementioned or related aspects, the pathological condition is related to the overexpression of a target gene.

[0013] In some embodiments of the aforementioned or related aspects, DNA methylation of the target gene increases over a period of at least 21 days to 6 months. In some embodiments, DNA methylation of the target gene increases by at least about 1.5 times, 2 times, 3 times, 4 times, 5 times, 10 times, 15 times, 20 times, 30 times, 40 times, or 50 times. In certain embodiments, DNA methylation of the target gene increases by at least 20 times to 50 times.

[0014] In some embodiments of the aforementioned or related aspects, the expression of the target gene is reduced. In some embodiments, the expression of the target gene is reduced by at least 25%.

[0015] In some embodiments of the aforementioned or related aspects, the DNA methyltransferase increases the methylation of at least one CpG dinucleotide in the target gene. In some embodiments, the at least one CpG dinucleotide is located in the promoter of the target gene. In some embodiments, the DNA methyltransferase increases the proportion of methylated CpG dinucleotides in a region of the target gene.

[0016] In some embodiments of the aforementioned or related aspects, the DNA target-directing moiety binds to a region of a target gene. In some embodiments, the DNA target-directing moiety binds to a region of a promoter, anchor sequence, or cis-regulatory element. In some embodiments, the anchor sequence includes a CTCF binding site or a YY1 binding site.

[0017] In some embodiments of the aforementioned or related aspects, the DNA targeting moiety targets the repressor system to an isolated genomic domain (IGD) containing the target gene. In some embodiments, the DNA targeting moiety includes a zinc finger (ZF) domain or a transcription activator-like effector (TALE) domain. In some embodiments, the DNA methyltransferase is MQ1, DNMT1, DNMT3A1, DNMT3A1, DNMT3B1, DNMT3B2, DNMT3B3, DNMT3B4, DNMT3B5, DNMT3B6, DNMT3L, or a functional variant or fragment thereof. In some embodiments, the DNA methyltransferase is MQ1, or a functional variant or fragment thereof.

[0018] In some embodiments of the aforementioned or related aspects, the repressor is a fusion protein comprising a DNA target-directing moiety operably linked to a DNA methyltransferase. In some embodiments, the DNA target-directing moiety is linked to the DNA methyltransferase by a linker. In some embodiments, the linker is a peptide linker. In some embodiments, the peptide linker is a Gly-Ser linker.

[0019] In some embodiments of the aforementioned or related aspects, the target gene is a gene associated with cancer.

[0020] In some embodiments of the aforementioned or related aspects, the target gene is a gene associated with a metabolic disease or disorder.

[0021] In some embodiments of the aforementioned or related aspects, the target gene is a pro-inflammatory gene.

[0022] In some embodiments of the aforementioned or related aspects, the nucleic acid molecule encoding the repressor is administered to a subject or brought into contact with cells. In some embodiments, the nucleic acid molecule is messenger RNA (mRNA) encoding the repressor. In some embodiments, the mRNA includes a 3'UTR, a poly-A tail, a ribosome skipping sequence, or any combination thereof.

[0023] In some embodiments of the aforementioned or related aspects, the repressor is a first repressor, and the mRNA comprises a nucleotide sequence encoding a second repressor, which includes a second DNA targeting moiety and an effector domain. In some embodiments, the second repressor is a fusion protein comprising a second DNA targeting moiety operably linked to the effector domain. In some embodiments, the second DNA targeting moiety binds to a region in the target gene different from that of the first repressor. In some embodiments, the effector domain of the second repressor is a histone modifying enzyme selected from DNA methyltransferase or histone methyltransferase, histone deacetylase, and histone demethylase. In some embodiments, the DNA methyltransferase is the same DNA methyltransferase as that of the first repressor. In some embodiments, the DNA methyltransferase is a different DNA methyltransferase from that of the first repressor. In some embodiments, the histone modifying enzyme is histone deacetylase. In some embodiments, the histone deacetylase is 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 thereof. In some embodiments, the histone modifying enzyme is a histone methyltransferase. In some embodiments, the histone methyltransferase is SETDB1, SETDB2, EHMT2, EHMT1, SUV39H1, EZH2, EZH1, SUV39H2, SETD8, SUV420H1, SUV420H2, or a functional variant or fragment thereof. In some embodiments, the effector domain 2 repressor includes a Kruppel-associated box (KRAB) domain or a functional variant or fragment thereof.

[0024] In some embodiments of the foregoing or related aspects, the mRNA comprises a ribosome skipping sequence between a nucleotide sequence encoding a first expression repressor and a nucleotide sequence encoding a second expression repressor.

[0025] In some embodiments of the foregoing or related aspects, the nucleic acid molecule is encapsulated in a lipid nanoparticle. In some embodiments, the lipid nanoparticle comprises an ionizable cationic lipid. In some embodiments, the lipid nanoparticle comprises one or more neutral lipids, ionizable cationic amine-containing lipids, essential cationic amine-containing lipids, biodegradable alkyn lipids, steroids, phospholipids, polyunsaturated lipids, structural lipids, PEG lipids, cholesterol, or polymer conjugate lipids.

[0026] In another aspect, provided herein is a method of increasing DNA methylation of a target gene in a subject, the method comprising administering to the subject a first dose of a composition comprising a lipid nanoparticle comprising an mRNA encoding a fusion protein comprising a DNA targeting moiety linked to a DNA methyltransferase, either with or without a linker, wherein the DNA targeting moiety comprises a ZF domain or a TALE domain, and wherein DNA methylation of the target gene is increased by at least 20-fold over a period of at least 21 days after administration of the first dose in the subject.

[0027] In another aspect, the present disclosure provides a method of increasing DNA methylation of a target gene in a subject, the method comprising administering to the subject a first dose of a composition comprising a lipid nanoparticle comprising an mRNA encoding a fusion protein comprising a DNA targeting moiety linked to a DNA methyltransferase, either with or without a linker, wherein the DNA targeting moiety comprises a ZF domain or a TALE domain, and wherein the percentage of methylated CpG dinucleotides in the promoter region of the target gene is increased by at least 5-fold over a period of at least 21 days after administration of the first dose in the subject.

[0028] In some embodiments of the foregoing or related aspects, the subject is not administered the following dose within that period.

[0029] In some embodiments of the foregoing or related aspects, the DNA methylation of the target gene increases over a period of 21 days to 6 months. In some embodiments, the DNA methylation of the target gene increases by at least about 1.5-fold, 2-fold, 3-fold, 4-fold, 5-fold, 10-fold, 15-fold, 20-fold, 30-fold, 40-fold, or 50-fold. In some embodiments, the DNA methylation of the target gene increases by at least about 20-fold to about 50-fold.

[0030] In some embodiments of the foregoing or related aspects, the expression of the target gene decreases. In some embodiments, the expression of the target gene decreases by at least 25%.

[0031] In some embodiments of the foregoing or related aspects, the DNA methyltransferase increases the methylation of at least one CpG dinucleotide in the target gene. In some embodiments, at least one CpG dinucleotide is in the promoter of the target gene. In some embodiments, the DNA methyltransferase increases the methylation of multiple CpG dinucleotides of the target gene. In some embodiments, the DNA methyltransferase increases the methylation of multiple CpG dinucleotides that are in or proximal to the promoter of the target gene. In some embodiments, the DNA methyltransferase methylates at least about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, or about 100% of the CpG dinucleotides of the target gene that are in or proximal to the promoter of the target gene.

[0032] In some embodiments of the foregoing or related aspects, the DNA targeting moiety binds to a region of a promoter, an anchor sequence, or a cis-regulatory element. In some embodiments, the anchor sequence includes a CTCF binding site or a YY1 binding site.

[0033] In some embodiments of the aforementioned or related aspects, the DNA methyltransferase is MQ1, DNMT1, DNMT3A1, DNMT3A1, DNMT3B1, DNMT3B2, DNMT3B3, DNMT3B4, DNMT3B5, DNMT3B6, DNMT3L, or a functional variant or fragment thereof. In some embodiments, the DNA methyltransferase is MQ1, or a functional variant or fragment thereof. In some embodiments, the linker is a peptide linker, and optionally the peptide linker is a Gly-Ser linker.

[0034] In some embodiments of the aforementioned or related aspects, the target gene is a gene associated with cancer, a gene associated with metabolic disease or disorder, or a pro-inflammatory gene. In some embodiments, the lipid nanoparticles include an ionizable cationic lipid. In some embodiments, the lipid nanoparticles include one or more neutral lipids, ionizable cationic amine-containing lipids, essential cationic amine-containing lipids, biodegradable alkyne (alkyn) lipids, steroids, phospholipids, polyunsaturated lipids, structural lipids, PEG lipids, cholesterol, or polymer-conjugated lipids. [Brief explanation of the drawing]

[0035] [Figure 1] This document provides a schematic diagram showing the treatment schedule for mice receiving a single intravenous dose (day 0) of LNP-formulated RNA101 mRNA, and the subsequent collection points of liver (days 14, 28, 63, 90, 120, 152, and 180 post-administration) and serum (days 14, 28, 42, 63, 77, 90, 104, 120, 134, 152, 166, and 180 post-administration). Control mice received intravenous injection of PBS. [Figure 2A-2B]Figures 2A-2F provide graphs showing the levels of metabolism-related gene mRNA measured in the liver, as well as the levels of metabolism-related gene protein expression and biomarkers of metabolism-related gene protein products, measured in serum, from mice that were intravenously administered PBS or a certain dose of LNP-formulated RNA101 mRNA according to the treatment schedule shown in Figure 1, at the time indicated. Figure 2A shows the metabolism-related gene mRNA levels normalized by HPRT (housekeeper) and averaged across all subjects as measured by RT-qPCR; Figure 2B shows the normalized metabolism-related gene mRNA levels for individual subjects. Figure 2C shows the averaged serum levels (pg / ml) of metabolism-related genes measured by ELISA across all subjects; Figure 2D shows the serum protein levels (pg / ml) of metabolism-related genes for individual subjects. Figure 2E shows the averaged serum levels (mmol / L) of biomarkers of metabolism-related gene protein products measured by ELISA across all subjects; Figure 2F shows the serum biomarker levels for individual subjects. [Figure 2C-2D] Figures 2A-2F provide graphs showing the levels of metabolism-related gene mRNA measured in the liver, as well as the levels of metabolism-related gene protein expression and biomarkers of metabolism-related gene protein products, measured in serum, from mice that were intravenously administered PBS or a certain dose of LNP-formulated RNA101 mRNA according to the treatment schedule shown in Figure 1, at the time indicated. Figure 2A shows the metabolism-related gene mRNA levels normalized by HPRT (housekeeper) and averaged across all subjects as measured by RT-qPCR; Figure 2B shows the normalized metabolism-related gene mRNA levels for individual subjects. Figure 2C shows the averaged serum levels (pg / ml) of metabolism-related genes measured by ELISA across all subjects; Figure 2D shows the serum protein levels (pg / ml) of metabolism-related genes for individual subjects. Figure 2E shows the averaged serum levels (mmol / L) of biomarkers of metabolism-related gene protein products measured by ELISA across all subjects; Figure 2F shows the serum biomarker levels for individual subjects. [Figure 2E-2F]Figures 2A-2F provide graphs showing the levels of metabolism-related gene mRNA measured in the liver, as well as the levels of metabolism-related gene protein expression and biomarkers of metabolism-related gene protein products, measured in serum, from mice that were intravenously administered PBS or a certain dose of LNP-formulated RNA101 mRNA according to the treatment schedule shown in Figure 1, at the time indicated. Figure 2A shows the metabolism-related gene mRNA levels normalized by HPRT (housekeeper) and averaged across all subjects as measured by RT-qPCR; Figure 2B shows the normalized metabolism-related gene mRNA levels for individual subjects. Figure 2C shows the averaged serum levels (pg / ml) of metabolism-related genes measured by ELISA across all subjects; Figure 2D shows the serum protein levels (pg / ml) of metabolism-related genes for individual subjects. Figure 2E shows the averaged serum levels (mmol / L) of biomarkers of metabolism-related gene protein products measured by ELISA across all subjects; Figure 2F shows the serum biomarker levels for individual subjects. [Figure 3A-3B] Figures 3A-3E provide graphs illustrating the methylation rate of a region of approximately 450 bp containing CpG islets near the promoter of metabolism-related genes, measured in hepatocyte lysates obtained at the time of instruction from mice that received a single dose of LNP-formulated RNA101 (TA-1) intravenously according to the treatment schedule shown in Figure 1. Control mice received intravenous injection of PBS. DNA methylation was quantified by Em-Seq at 14 days (Figures 3A and 3B, respectively) and 28 days (Figures 3C and 3D, respectively) after administration of PBS or TA-1. Figure 3E shows promoter methylation at 14 and 28 days after administration as mean methylation / mouse for each test condition. Figures 3F-3I show promoter methylation at 63, 90, 120, and 152 days after administration as mean methylation / mouse for each test condition. Figures 3E to 3I show that each column corresponds to an individual animal, and each plot represents the average total methylation content of CpG islands in the amplicon. [Figure 3C-3D]Figures 3A-3E provide graphs illustrating the methylation rate of a region of approximately 450 bp containing CpG islets near the promoter of metabolism-related genes, measured in hepatocyte lysates obtained at the time of instruction from mice that received a single dose of LNP-formulated RNA101 (TA-1) intravenously according to the treatment schedule shown in Figure 1. Control mice received intravenous injection of PBS. DNA methylation was quantified by Em-Seq at 14 days (Figures 3A and 3B, respectively) and 28 days (Figures 3C and 3D, respectively) after administration of PBS or TA-1. Figure 3E shows promoter methylation at 14 and 28 days after administration as mean methylation / mouse for each test condition. Figures 3F-3I show promoter methylation at 63, 90, 120, and 152 days after administration as mean methylation / mouse for each test condition. Figures 3E to 3I show that each column corresponds to an individual animal, and each plot represents the average total methylation content of CpG islands in the amplicon. [Figure 3E] Figures 3A-3E provide graphs illustrating the methylation rate of a region of approximately 450 bp containing CpG islets near the promoter of metabolism-related genes, measured in hepatocyte lysates obtained at the time of instruction from mice that received a single dose of LNP-formulated RNA101 (TA-1) intravenously according to the treatment schedule shown in Figure 1. Control mice received intravenous injection of PBS. DNA methylation was quantified by Em-Seq at 14 days (Figures 3A and 3B, respectively) and 28 days (Figures 3C and 3D, respectively) after administration of PBS or TA-1. Figure 3E shows promoter methylation at 14 and 28 days after administration as mean methylation / mouse for each test condition. Figures 3F-3I show promoter methylation at 63, 90, 120, and 152 days after administration as mean methylation / mouse for each test condition. Figures 3E to 3I show that each column corresponds to an individual animal, and each plot represents the average total methylation content of CpG islands in the amplicon. [Figure 3F]Figures 3A-3E provide graphs illustrating the methylation rate of a region of approximately 450 bp containing CpG islets near the promoter of metabolism-related genes, measured in hepatocyte lysates obtained at the time of instruction from mice that received a single dose of LNP-formulated RNA101 (TA-1) intravenously according to the treatment schedule shown in Figure 1. Control mice received intravenous injection of PBS. DNA methylation was quantified by Em-Seq at 14 days (Figures 3A and 3B, respectively) and 28 days (Figures 3C and 3D, respectively) after administration of PBS or TA-1. Figure 3E shows promoter methylation at 14 and 28 days after administration as mean methylation / mouse for each test condition. Figures 3F-3I show promoter methylation at 63, 90, 120, and 152 days after administration as mean methylation / mouse for each test condition. Figures 3E to 3I show that each column corresponds to an individual animal, and each plot represents the average total methylation content of CpG islands in the amplicon. [Figure 3G] Figures 3A-3E provide graphs illustrating the methylation rate of a region of approximately 450 bp containing CpG islets near the promoter of metabolism-related genes, measured in hepatocyte lysates obtained at the time of instruction from mice that received a single dose of LNP-formulated RNA101 (TA-1) intravenously according to the treatment schedule shown in Figure 1. Control mice received intravenous injection of PBS. DNA methylation was quantified by Em-Seq at 14 days (Figures 3A and 3B, respectively) and 28 days (Figures 3C and 3D, respectively) after administration of PBS or TA-1. Figure 3E shows promoter methylation at 14 and 28 days after administration as mean methylation / mouse for each test condition. Figures 3F-3I show promoter methylation at 63, 90, 120, and 152 days after administration as mean methylation / mouse for each test condition. Figures 3E to 3I show that each column corresponds to an individual animal, and each plot represents the average total methylation content of CpG islands in the amplicon. [Figure 3H]Figures 3A-3E provide graphs illustrating the methylation rate of a region of approximately 450 bp containing CpG islets near the promoter of metabolism-related genes, measured in hepatocyte lysates obtained at the time of instruction from mice that received a single dose of LNP-formulated RNA101 (TA-1) intravenously according to the treatment schedule shown in Figure 1. Control mice received intravenous injection of PBS. DNA methylation was quantified by Em-Seq at 14 days (Figures 3A and 3B, respectively) and 28 days (Figures 3C and 3D, respectively) after administration of PBS or TA-1. Figure 3E shows promoter methylation at 14 and 28 days after administration as mean methylation / mouse for each test condition. Figures 3F-3I show promoter methylation at 63, 90, 120, and 152 days after administration as mean methylation / mouse for each test condition. Figures 3E to 3I show that each column corresponds to an individual animal, and each plot represents the average total methylation content of CpG islands in the amplicon. [Figure 3I] Figures 3A-3E provide graphs illustrating the methylation rate of a region of approximately 450 bp containing CpG islets near the promoter of metabolism-related genes, measured in hepatocyte lysates obtained at the time of instruction from mice that received a single dose of LNP-formulated RNA101 (TA-1) intravenously according to the treatment schedule shown in Figure 1. Control mice received intravenous injection of PBS. DNA methylation was quantified by Em-Seq at 14 days (Figures 3A and 3B, respectively) and 28 days (Figures 3C and 3D, respectively) after administration of PBS or TA-1. Figure 3E shows promoter methylation at 14 and 28 days after administration as mean methylation / mouse for each test condition. Figures 3F-3I show promoter methylation at 63, 90, 120, and 152 days after administration as mean methylation / mouse for each test condition. Figures 3E to 3I show that each column corresponds to an individual animal, and each plot represents the average total methylation content of CpG islands in the amplicon. [Figure 4]This paper provides a line graph showing the time course of oncology gene 1 mRNA levels in K-562 cells after treatment with MC3 LNP-formulated RNA102 mRNA. Oncology gene 1 mRNA levels were normalized to ACTB or GAPDH (housekeeper) levels and quantified by RT-qPCR. Control cells were untreated. [Figure 5] This document provides a line graph showing the time-dependent levels of two oncology gene mRNAs in K-562 cells after treatment with MC3 LNP-formulated RNA103 mRNA. The levels of these two oncology gene mRNAs were normalized to ACTB or GAPDH (housekeeper) levels and quantified by RT-qPCR. Control cells were untreated. [Figure 6] This document provides a line graph showing the levels of three oncology gene mRNAs over time in K-562 cells after treatment with MC3 LNP-formulated RNA104 mRNA. The levels of these three oncology gene mRNAs were normalized to ACTB or GAPDH (housekeeper) levels and quantified by RT-qPCR. [Figure 7] This document provides a line graph showing the time course of oncology gene 4 mRNA levels in K-562 cells after treatment with MC3 LNP-formulated RNA105 mRNA. Oncology gene 4 mRNA levels were normalized to ACTB or GAPDH (housekeeper) levels and quantified by RT-qPCR. [Figure 8A] Figure 1 provides a graph illustrating the mean methylation changes in four methylation differential regions (DMRs) in the promoters of metabolism-related genes in mice. [Figure 8B] Figure 1 provides a graph showing the average mRNA expression levels of metabolism-related genes measured in hepatocyte lysates obtained from mice. [Modes for carrying out the invention]

[0036] This disclosure is based, at least in part, on the discovery that DNA methylation at a site of interest is achieved over a prolonged period (e.g., at least 21 days) after administration of a certain dose of an expression repressor described herein or a nucleic acid encoding an expression repressor. Expression repressors found to produce a sustained effect include (i) a DNA target-directed moiety (e.g., ZF, TALE, or dCas9 domain) that binds to a target sequence in a region of the genome containing the target gene; and (ii) a DNA methyltransferase. The sustained DNA methylation effect described herein has also been observed to cause a prolonged reduction in the expression of the target gene.

[0037] As demonstrated herein, when a single dose of mRNA encoding an exemplary repressor comprising (i) a DNA target-directing portion that binds to a target sequence in a genomic region including the promoter of a metabolism-related target gene, and (ii) an effector domain containing DNA methyltransferase, is introduced into cells in vitro or in vivo, DNA methylation adjacent to or located in the promoter (e.g., in CpG-enriched regions in or near the promoter) increases over a long period (at least 28 days) after administration. Furthermore, when administered in vivo, the increase in DNA methylation was associated with a decrease in serum levels of transcriptional and translational products of the metabolism-related target gene for at least 180 days after administration. As further demonstrated herein, when a single dose of mRNA encoding an exemplary repressor, comprising (i) a DNA target-directing portion that binds to a target sequence in a genomic region including the promoter of an oncology target gene, and (ii) an effector domain containing DNA methyltransferase, was introduced into cancer cells, DNA methylation adjacent to or located in the promoter (e.g., in CpG-enriched regions in or near the promoter) increased over a long period after contact (at least 21 days). It was further revealed that the increase in DNA methylation correlated with a decrease in the expression of the transcript of the oncology target gene.

[0038] Accordingly, in some embodiments, the Disclosure provides a method for increasing DNA methylation at a site in a region of the genome containing a target gene of interest, comprising administering to the subject a certain dose of a composition comprising a repressor or a nucleic acid molecule encoding a repressor, wherein the repressor comprises a DNA target-directed portion and a DNA methyltransferase, and the increase in DNA methylation at the site is maintained for a long period after the administration of the dose (e.g., at least 21 days), provided that the subject has not received a subsequent dose during that period. In some embodiments, the Method comprises administering to the subject a first dose of the Composition, wherein the DNA methylation at the site is maintained for a long period after the administration of the first dose (e.g., at least 21 days) until the subject receives a subsequent dose of the Composition.

[0039] In some embodiments, the Disclosure provides expression repressors for use in the methods described herein. As used herein, the term “expression repressor” means a drug that reduces the expression of a target gene in a cell, wherein the drug includes (i) a target-directing function performed by a first portion that specifically binds to a target sequence in a region of the genome containing the target gene; and (ii) an effector function performed by a second portion that, when localized to a site in a region of the genome containing the target gene, has the ability to reduce the expression of the target gene.

[0040] In some embodiments, the target-directed function localizes the effector function of the repressor. In some embodiments, the target-directed function of the repressor is achieved by a DNA target-directed moiety. As used herein, the term “DNA target-directed moiety” refers to a drug or entity that specifically targets, for example, a binding agent, to a target sequence in genomic DNA. In some embodiments, the target-directed function is achieved by the binding of the DNA target-directed moiety to a target sequence in a region of the genome containing the target gene (for example, a target sequence in a transcriptional regulatory element operably linked to the target gene). In some embodiments, the DNA target-directed moiety includes a polypeptide that binds to the target sequence. In some embodiments, the DNA target-directed moiety includes a zinc finger (ZF) domain that binds to the target sequence. In some embodiments, the DNA target-directed moiety includes a transcription activator-like effector (TALE) domain that binds to the target sequence. In some embodiments, the DNA target-directed moiety includes a site-induced nuclease (for example, a catalytically inactive site-induced nuclease) and a guide sequence, the guide sequence being complementary or substantially complementary to the target sequence.

[0041] In some embodiments, the effector function of a repressor increases DNA methylation at a site in a region of the genome containing the target gene. DNA methylation is an epigenetic modification that regulates gene expression by recruiting proteins that promote gene repression and / or inhibiting transcription binding. In eukaryotic DNA, DNA methylation is thought to occur at cytosine bases, and the cytosine base is converted to 5-methylcytosine (5mC) by the DNA methyltransferase (DNMT) enzyme, which transfers a methyl group from S-adenylemethionine (SAM) to the fifth carbon of the cytosine residue. The vast majority of DNA methylation sites in the genome are located in CpG sequences.

[0042] In some embodiments, the effector function, when localized to a region by the target-directed function of an expression repressor, increases DNA methylation at a site in a region of the genome containing the target gene. In some embodiments, increased DNA methylation at the site leads to decreased expression of the target gene. In some embodiments, the effector function is performed by a DNA methyltransferase. In some embodiments, increased DNA methylation at the site serves to recruit inhibitory components that reduce the expression of the target gene by disrupting the endogenous transcription machinery. In some embodiments, increased DNA methylation at the site serves to inactivate, or substantially inactivate, the transcription of the target gene by recruiting one or more co-repressor proteins and / or transcription factors. In some embodiments, increased DNA methylation at the site serves to inhibit the recruitment of transcription factors, thereby reducing the expression of the target gene.

[0043] In some embodiments, a region of the genome containing a target gene is or includes an isolated genomic domain (IGD). As further described herein and as will be understood by those skilled in the art, an IGD is a unit of genomic interval whose boundaries are defined by factors that mechanistically drive functional isolation between gene transcriptional activities. Thus, an IGD is a physical unit that breaks down a chromosome into individual functional segments. For example, in some embodiments, an IGD includes a DNA loop formed by the interaction between two DNA sites linked by homodimerized CTCF and cohesin (see Dowen, et al (2014) Cell 159:374-87). In such an IGD, the occupancy of each of the CTCF and cohesin-linked DNA sites inhibits the interaction of the DNA-binding component on one chromosome of the DNA site with the DNA-binding component on the opposite chromosome. Consequently, the DNA sites occupied by CTCF and cohesin in such a DNA loop act as boundaries for the IGD. In some embodiments, the formation of such a DNA loop promotes (i) enhancer-promoter interactions in which both the enhancer and promoter are located within the loop, (ii) enhancer-promoter interactions in which one of these elements is located within the loop and the other is located outside the loop, or (iii) both (i) and (ii).

[0044] In some embodiments, the disclosure provides an expression repressor for increasing DNA methylation at a site in a region of the genome containing a target gene of a cell, comprising: (i) a target-directing function performed by a DNA target-directing moiety that binds to a target sequence in that region of the genome (e.g., ZF, TALE, or catalytically inactive site-induced nuclease); and (ii) an effector function performed by an effector moiety that, when localized to the region by the target-directing function, introduces DNA methylation at that site (e.g., DNA methyltransferase). In some embodiments, the region of the genome is an IGD containing the target gene, or includes it. In some embodiments, the target sequence is located in a transcriptional regulatory element (e.g., a promoter or enhancer) operably linked to the target gene. In some embodiments, the target sequence is located in or near an enhancer of the target gene. In some embodiments, the target sequence is located in or near a promoter of the target gene. In some embodiments, the target sequence is located in or near a site where the effector function of the expression repressor introduces DNA methylation. In some embodiments, the effector portion increases DNA methylation at a genomic region targeted by the DNA targeting portion when the repressor is localized to that region. In some embodiments, the site is a span of at least about 300, 400, 500, 600, 700, 800, 900, or 1,000 bases (e.g., up to about 2,000, 3,000, 4,000, or 5,000 bases) containing multiple CpG sequences. In some embodiments, the span includes CpG islands. In some embodiments, the effector portion increases DNA methylation at the site by at least about 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100% compared to before localization. In some embodiments, the effector portion increases DNA methylation by at least approximately 1.5 times, approximately 2 times, approximately 3 times, approximately 4 times, or approximately 5 times compared to before localization.In some embodiments, the effector portion increases DNA methylation by at least approximately 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 60, 70, 80, 90, or 100 times compared to before localization. In some embodiments, the effector portion increases DNA methylation by at least approximately 20, 25, 30, 35, or 40 times compared to before localization. In some embodiments, the site includes a CpG island, and after localization, at least approximately 1%, 2%, 3%, 4%, or 5% of the CpG sequence of the CpG island is methylated. In some embodiments, after localization, at least about 5%, about 10%, about 15%, about 20%, about 30%, about 40%, or about 50% of the CpG sequence of the CpG island is methylated. In some embodiments, after localization, about 10% to about 100%, about 10% to about 90%, about 10% to about 80%, about 10% to about 70%, about 10% to about 60%, about 20% to about 80%, about 20% to about 70%, about 20% to about 60%, about 20% to about 50%, or about 20% to about 40% of the CpG sequence of the CpG island is methylated.

[0045] In some embodiments, this disclosure provides nucleic acids encoding the expression repressors described herein. In some embodiments, the nucleic acid is mRNA. In some embodiments, this disclosure provides recombinant expression vectors comprising nucleic acids. In some embodiments, the expression repressor, nucleic acid (e.g., mRNA), or recombinant expression vector is formulated into lipid nanoparticles (LNPs).

[0046] In some embodiments, the Disclosure provides a system comprising two or more of the expression repressors described herein. In some embodiments, the system comprises two, three, four, five, six, seven, eight, nine, or ten of the expression repressors described herein. In some embodiments, the system comprises two or more nucleic acids, each encoding an expression repressor described herein. In some embodiments, the two or more nucleic acids are each mRNA. In some embodiments, the system comprises two or more recombinant expression vectors, each recombinant expression vector comprising a nucleic acid encoding an expression repressor described herein. In some embodiments, the two or more expression repressors, the two or more nucleic acids, or the two or more recombinant expression vectors are formulated into the same LNP or different LNPs.

[0047] In some embodiments, this disclosure provides nucleic acids encoding two expression repressors described herein. In some embodiments, the nucleic acid is mRNA. In some embodiments, this disclosure provides a recombinant expression vector comprising nucleic acid. In some embodiments, the nucleic acid or recombinant expression vector is formulated into LNPs.

[0048] In some embodiments, the Disclosure provides pharmaceutical compositions comprising an expression repressor described herein, a nucleic acid described herein, a recombinant expression vector described herein, an LNP described herein, or a system described herein, and a pharmaceutically acceptable carrier.

[0049] In some embodiments, the Disclosure provides a method for increasing DNA methylation at a site in a region of the genome containing a target gene of a cell (e.g., a site in an IGD containing a target gene), comprising contacting the cell with a certain dose of an expression repressor, nucleic acid, recombinant expression vector, LNP, system, or pharmaceutical composition described herein. In some embodiments, DNA methylation at the site increases compared to before contact with the expression repressor, nucleic acid, recombinant expression vector, LNP, system, or pharmaceutical composition, or compared to a control cell that has not been contacted therewith. In some embodiments, the increase in DNA methylation at the site is maintained over a long period after contact with the dose. In some embodiments, the increase in DNA methylation at the site is maintained over a long period after contact with the dose until before contact with the cell with the next dose of the expression repressor, nucleic acid, recombinant expression vector, LNP, system, or pharmaceutical composition. In some embodiments, the increase in DNA methylation at the site is maintained for at least about 21 days, about 28 days, about 35 days, about 42 days, or about 49 days after dose contact until the cells are contacted with the next dose of the repressor, nucleic acid, recombinant expression vector, LNP, system, or pharmaceutical composition. In some embodiments, the increase in DNA methylation at the site is maintained for about 3 weeks, about 4 weeks, about 5 weeks, about 6 weeks, about 7 weeks, about 8 weeks, about 9 weeks, about 10 weeks, about 11 weeks, or about 12 weeks or more, about 48 weeks, about 36 weeks, or about 24 weeks or less after dose contact until the cells are contacted with the next dose of the repressor, nucleic acid, recombinant expression vector, LNP, system, or pharmaceutical composition.

[0050] In some embodiments, the Disclosure provides a method for increasing DNA methylation at a site in a region of the genome containing a target gene (for example, a site in an IGD containing the target gene), comprising administering to a subject a certain dose of an expression repressor, nucleic acid, recombinant expression vector, LNP, system, or pharmaceutical composition described herein. In some embodiments, DNA methylation at the site increases compared to before administration of a certain dose of the expression repressor, nucleic acid, recombinant expression vector, LNP, system, or pharmaceutical composition, or compared to a control subject that has not received such administration. In some embodiments, the increase in DNA methylation at the site is maintained over a long period after dose administration. In some embodiments, the increase in DNA methylation at the site is maintained over a long period after dose administration until before the subject receives a subsequent dose of the expression repressor, nucleic acid, recombinant expression vector, LNP, system, or pharmaceutical composition. In some embodiments, the increase in DNA methylation at the site is maintained for at least about 21 days, about 28 days, about 35 days, about 42 days, or about 49 days after dose administration until the subject is administered the next dose of the repressor, nucleic acid, recombinant expression vector, LNP, system, or pharmaceutical composition. In some embodiments, the increase in DNA methylation at the site is maintained for about 3 weeks, about 4 weeks, about 5 weeks, about 6 weeks, about 7 weeks, about 8 weeks, about 9 weeks, about 10 weeks, about 11 weeks, or about 12 weeks or more, about 48 weeks, about 36 weeks, or about 24 weeks or less after dose administration until the subject is administered the next dose of the repressor, nucleic acid, recombinant expression vector, LNP, system, or pharmaceutical composition.

[0051] In some embodiments, the Disclosure provides a method for reducing the expression of a target gene in cells, comprising contacting cells with a certain dose of an expression repressor, nucleic acid, recombinant expression vector, LNP, system, or pharmaceutical composition described herein. In some embodiments, the expression of the target gene is reduced compared to before contact with the expression repressor, nucleic acid, recombinant expression vector, LNP, system, or pharmaceutical composition, or compared to control cells that have not been contacted therewith. In some embodiments, the expression of the target gene is reduced over a long period after contact with the dose. In some embodiments, the expression of the target gene is reduced over a long period after contact with the dose until before contact with the cells with the next dose of the expression repressor, nucleic acid, recombinant expression vector, LNP, system, or pharmaceutical composition. In some embodiments, the reduction in the expression of the target gene is maintained for at least about 21 days, about 28 days, about 35 days, about 42 days, or about 49 days after contact with the dose until before contact with the cells with the next dose of the expression repressor, nucleic acid, recombinant expression vector, LNP, system, or pharmaceutical composition. In some embodiments, the reduction in target gene expression is maintained for approximately 3 weeks, 4 weeks, 5 weeks, 6 weeks, 7 weeks, 8 weeks, 9 weeks, 10 weeks, 11 weeks, or 12 weeks or more, 48 weeks, 36 weeks, or 24 weeks or less, after dose contact until the cells are contacted with the next dose of the repressor, nucleic acid, recombinant expression vector, LNP, system, or pharmaceutical composition.

[0052] In some embodiments, the Disclosure provides a method for reducing the expression of a target gene in a subject, comprising administering to the subject a certain dose of an expression repressor, nucleic acid, recombinant expression vector, LNP, system, or pharmaceutical composition described herein. In some embodiments, the expression of the target gene is reduced compared to before administration of a certain dose of the expression repressor, nucleic acid, recombinant expression vector, LNP, system, or pharmaceutical composition, or compared to a control subject that has not received such administration. In some embodiments, the expression of the target gene is reduced over a long period after dose administration. In some embodiments, the expression of the target gene is reduced over a long period after dose administration until before the subject is administered a subsequent dose of the expression repressor, nucleic acid, recombinant expression vector, LNP, system, or pharmaceutical composition. In some embodiments, the reduction in the expression of the target gene is maintained for at least about 21 days, about 28 days, about 35 days, about 42 days, or about 49 days after dose administration until before the subject is administered a subsequent dose of the expression repressor, nucleic acid, recombinant expression vector, LNP, system, or pharmaceutical composition. In some embodiments, the reduction in target gene expression is maintained for approximately 3 weeks, 4 weeks, 5 weeks, 6 weeks, 7 weeks, 8 weeks, 9 weeks, 10 weeks, 11 weeks, or 12 weeks or more, 48 weeks, 36 weeks, or 24 weeks or less, after dose administration until the subject is administered the next dose of the repressor, nucleic acid, recombinant expression vector, LNP, system, or pharmaceutical composition.

[0053] In some embodiments, the Disclosure provides a method for treating a condition in a subject that requires treatment of a condition related to the expression of a target gene, comprising administering to the subject a certain dose of an expression repressor, nucleic acid, recombinant expression vector, LNP, system, or pharmaceutical composition described herein. In some embodiments, the method reduces the expression of the target gene after administration of the dose, thereby treating the condition. In some embodiments, the reduction in the expression of the target gene is maintained for a long period (e.g., at least 21 days) after administration of the dose until before administration of the next dose of the expression repressor, nucleic acid, recombinant expression vector, LNP, system, or pharmaceutical composition, thereby treating the condition. In some embodiments, the method increases DNA methylation at a site in a region of the genome containing the target gene (e.g., a site in the IGD containing the target gene), thereby reducing the expression of the target gene. In some embodiments, increased DNA methylation at a site is maintained for an extended period (e.g., at least 21 days) after dose administration until the next dose of the repressor, nucleic acid, recombinant expression vector, LNP, system, or pharmaceutical composition is administered, thereby reducing the expression of the target gene.

[0054] In some embodiments, the pathophysiology is a metabolic disorder related to a target gene, for example, the target gene encoding a metabolic enzyme. In some embodiments, the pathophysiology is a cancer related to a target gene, for example, the target gene being an oncogene. In some embodiments, the pathophysiology is an autoimmune disorder. In some embodiments, the pathophysiology is an inflammatory disorder. In some embodiments, the pathophysiology is a neurological disorder.

[0055] Method of Disclosure In some embodiments, the Disclosure provides a method for increasing DNA methylation of a target gene in a cell (e.g., in vitro or in vivo), comprising contacting a cell with a dose of a repressor described herein (e.g., an LNP-formulated repressor) or a nucleic acid encoding a repressor (e.g., an LNP-formulated nucleic acid encoding a repressor), or a repressor system described herein comprising at least one repressor (e.g., at least one LNP-formulated repressor) or at least one nucleic acid encoding a repressor (e.g., an LNP-formulated nucleic acid encoding at least one repressor), wherein DNA methylation of a site in a region of the genome containing the target gene (e.g., a site in the IGD containing the target gene) is increased, and the increase in DNA methylation is maintained at that site for an extended period (e.g., at least 21 days) after contact until the cell is contacted with the next dose of the repressor or repressor system.

[0056] In some embodiments, the Disclosure provides a method for increasing DNA methylation of a target gene in a cell (e.g., in vitro or in vivo), comprising contacting a cell with a dose of a repressor described herein (e.g., an LNP-formulated repressor) or a nucleic acid encoding a repressor (e.g., an LNP-formulated nucleic acid encoding a repressor), or a repressor system described herein comprising at least one repressor (e.g., at least one LNP-formulated repressor) or a nucleic acid encoding at least one repressor (e.g., an LNP-formulated nucleic acid encoding at least one repressor), wherein the proportion of methylated CpG dinucleotides at a site of the target gene is increased, and the proportion of methylated CpG dinucleotides is maintained at that site for an extended period (e.g., at least 21 days) after contact until the cell is contacted with the next dose of the repressor or repressor system.

[0057] In some embodiments, the Disclosure provides a method for reducing the expression of a target gene in a cell (e.g., in vitro or in vivo), comprising contacting a cell with a certain dose of a repressor described herein (e.g., an LNP-formulated repressor) or a nucleic acid encoding a repressor (e.g., an LNP-formulated nucleic acid encoding a repressor), or a repressor system described herein comprising at least one repressor (e.g., at least one LNP-formulated repressor) or a nucleic acid encoding at least one repressor (e.g., an LNP-formulated nucleic acid encoding at least one repressor), wherein DNA methylation at a site in a region of the genome containing the target gene (e.g., a site in the IGD containing the target gene) is increased, and the reduction in the expression of the target gene and / or the increase in DNA methylation at the site is maintained at that site for an extended period (e.g., at least 21 days) after contact until the cell is contacted with the next dose of the repressor or repressor system.

[0058] In some embodiments, the Disclosure provides a method (e.g., in vitro or in vivo) for increasing DNA methylation at a site in a region of the genome containing a target gene in a subject, comprising administering to the subject a certain dose of a repressor described herein (e.g., an LNP-formulated repressor) or a nucleic acid encoding a repressor (e.g., an LNP-formulated nucleic acid encoding a repressor), or a repressor system described herein comprising at least one repressor (e.g., at least one LNP-formulated repressor) or a nucleic acid encoding at least one repressor (e.g., an LNP-formulated nucleic acid encoding at least one repressor), wherein the increase in DNA methylation is maintained at that site for an extended period (e.g., at least 21 days) after the administration of the dose until the subject is administered a subsequent dose of the repressor or repressor system.

[0059] In some embodiments, the Disclosure provides a method for reducing the expression of a target gene (e.g., in vitro or in vivo) by administering to a subject a certain dose of a repressor described herein (e.g., an LNP-formulated repressor) or a nucleic acid encoding a repressor (e.g., an LNP-formulated nucleic acid encoding a repressor), or a repressor system described herein comprising at least one repressor (e.g., at least one LNP-formulated repressor) or at least one nucleic acid encoding a repressor (e.g., an LNP-formulated nucleic acid encoding at least one repressor), wherein DNA methylation at a site in a region of the genome containing the target gene (e.g., a site in the IGD containing the target gene) is increased, and the increase in DNA methylation is maintained at that site for a long period (e.g., at least 21 days) after the administration of the dose until the subject is administered a subsequent dose of the repressor or repressor system.

[0060] In some embodiments, the Disclosure provides a method for treating a condition related to the expression of a target gene (e.g., overexpression, e.g., dysregulation of expression), comprising administering to a subject a certain dose of a suppressor described herein (e.g., an LNP-formulated suppressor) or a nucleic acid encoding a suppressor (e.g., an LNP-formulated nucleic acid encoding a suppressor), or a suppressor system described herein comprising at least one suppressor (e.g., at least one LNP-formulated suppressor) or at least one nucleic acid encoding a suppressor (e.g., an LNP-formulated nucleic acid encoding at least one suppressor), wherein DNA methylation at a site in a region of the genome containing the target gene (e.g., a site in the IGD containing the target gene) is increased, and the increase in DNA methylation and / or decrease in the expression of the target gene at the site is maintained at that site for a long period (e.g., at least 21 days) after administration until the subject is administered the next dose of the suppressor or suppressor system.

[0061] DNA methylation In some embodiments, the disclosure provides a method for increasing DNA methylation at a site in a region of the genome containing a target gene. In some embodiments, increasing DNA methylation includes increasing the proportion of methylated CpG dinucleotides in a region of the genome containing a target gene. In some embodiments, the method results in a decrease in the expression of the target gene.

[0062] Methods for measuring DNA methylation are known in the art, but are not limited to, mass spectrometry, sequencing-based assays such as methylation-specific PCR and bisulfite sequencing, Hpall tiny fragment enrichment by ligation-mediated PCR (HELP) assays, GLAD-PCR assays, ChIP-on-chip assays, restriction enzyme landmark genome scanning, methylated DNA immunoprecipitation, methyl-sensitive Southern blotting, high-resolution melt analysis, and methylation-sensitive single-nucleotide primer extension assays. In some embodiments, methods for measuring DNA methylation of a target gene include the use of DNA methylation microarrays (e.g., Illumina's methylation arrays). Microarray-based methylation analysis techniques are described in Deatherage, et al (2009) Methods Mol Biol 556:117-139; Schumacher, et al (2006) Nucleic Acids Res 34:528-42; and Willhelm-Benartzi, et al (2013) Br J Cancer 109:1394-1402. In some embodiments, the method includes a sequencing-based assay in which genomic DNA is treated before sequencing with a drug that converts cytosine residues to uracil (or another base with different hybridization properties than cytosine) but does not affect 5-methylcytosine residues. Exemplary drugs known in the art include bisulfites, bisulfites, disulfites, and combinations thereof. Thus, DNA treated with bisulfites retains methylated cytosine but not unmethylated cytosine. Next, the processed DNA is subjected to sequencing analysis (see, for example, Campan et al (2009) Methods Mol Biol 507:325-37; Adusumalli, et al (2015) Brief Bioinform 16:369-79).Exemplary sequencing analysis methods are known in the art and include those based on sequencing-by-synthesis or sequencing-by-ligation, as employed by Illumina, Life Technologies, and Roche; or the use of next-generation sequencing platforms based on nanopore sequencing or electron detection, as employed by Ion Torrent technology. In some embodiments, methods for measuring DNA methylation include enzymatic methyl-seq (EM-seq) (see, e.g., Vaisvila et al (2021) Genome Res 31:1280). In EM-seq, 5-methylcytosine (5mC) and 5-hydroxymethylcytosine (i.e., the oxidized product of 5mC; also referred to as 5hmC) are converted using enzymatic reactions (e.g., carried out using TET2 and T4-BGT) to make the unmodified cytosine a product resistant to enzymatic reactions (e.g., carried out using APOBEC3A) that deaminate it by converting it to uracil. Next, the enzyme-treated DNA is amplified by PCR using EM-seq adapter primers and subjected to sequencing analysis, such as Illumina sequencing.

[0063] In some embodiments, the Disclosure provides a method for increasing DNA methylation at a site in a region of the genome of a cell or a population of cells, which includes contacting the cell or population with a certain dose of a repressor described herein or a nucleic acid encoding a repressor, wherein the repressor comprises (i) a DNA target-directed moiety that binds to a target sequence in the region, and (ii) a DNA methyltransferase.

[0064] In some embodiments, the Disclosure provides a method for increasing DNA methylation at a site in a region of the genome containing a target gene of a cell or a population of cells, comprising contacting the cell or a population of cells with a certain dose of a repressor system described herein comprising at least one repressor (e.g., 1, 2, 3, 4, 5 or more repressors described herein) or a nucleic acid encoding at least one repressor, wherein the at least one repressor comprises (i) a DNA target-directed moiety that binds to a target sequence in the region, and (ii) a DNA methyltransferase.

[0065] In some embodiments, DNA methylation at the site increases over a long period after contact. In some embodiments, DNA methylation at the site increases over a long period after contact, until the cells or cell population are exposed to the next dose of the repressor or repressor system.

[0066] In some embodiments, DNA methylation at the site increases over a long period of time after contact until the next dose of the repressor or repressor system is applied to the cells or cell population, where the long period is at least about 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, or 28 days. In some embodiments, the long period is at least about 21 days. In some embodiments, the long period is at least about 28 days.

[0067] In some embodiments, site-specific DNA methylation increases over a long period of time after contact until the next dose of the repressor or repressor system is applied to the cells or cell population, where long period is approximately 10 to 100 days, 20 to 90 days, 20 to 80 days, 20 to 70 days, 20 to 60 days, 25 to 75 days, 25 to 65 days, 30 to 100 days, 30 to 90 days, 30 to 80 days, or 30 to 70 days. In some embodiments, the long period is approximately 21 to 100 days. In some embodiments, the long period is approximately 21 to 200 days. In some embodiments, the long period is approximately 28 to 100 days. In some embodiments, the long period is approximately 28 to 200 days.

[0068] In some embodiments, DNA methylation at the site increases over a long period of time after contact until the next dose of the repressor or repressor system is applied to the cells or cell population, where the long period is at least about 1 week, about 2 weeks, about 3 weeks, about 4 weeks, about 5 weeks, about 6 weeks, about 7 weeks, about 8 weeks, about 9 weeks, about 10 weeks, about 11 weeks, or about 12 weeks. In some embodiments, the long period is at least about 3 weeks. In some embodiments, the long period is at least about 4 weeks.

[0069] In some embodiments, site-specific DNA methylation increases over a long period of time after contact until the next dose of the repressor or repressor system is applied to the cells or cell population, where long period is approximately 1 to 48 weeks, approximately 1 to 36 weeks, approximately 1 to 24 weeks, approximately 1 to 12 weeks, approximately 2 to 48 weeks, approximately 2 to 36 weeks, approximately 2 to 24 weeks, approximately 2 to 12 weeks, approximately 3 to 48 weeks, approximately 3 to 36 weeks, approximately 3 to 24 weeks, approximately 3 to 12 weeks, approximately 4 to 48 weeks, approximately 4 to 36 weeks, approximately 4 to 24 weeks, or approximately 4 to 12 weeks. In some embodiments, long period is at least approximately 3 to 12 weeks. In some embodiments, long period is at least approximately 3 to 24 weeks. In some embodiments, long period is at least approximately 3 to 48 weeks. In some embodiments, the long term is at least about 4 weeks to about 12 weeks. In some embodiments, the long term is at least about 4 weeks to about 24 weeks. In some embodiments, the long term is at least about 4 weeks to about 48 weeks.

[0070] In some embodiments, DNA methylation at the site increases over a long period of time after contact, until the next dose of the repressor or repressor system is applied to the cells or cell population, where the long period is at least 21 days.

[0071] In some embodiments, the increase in DNA methylation is maintained at the site for a long period after contact. In some embodiments, the increase in DNA methylation at the site is maintained for a long period after contact until the cell or cell population is exposed to the next dose of the repressor or repressor system.

[0072] In some embodiments, the increase in DNA methylation is maintained at the site for a prolonged period after contact until the next dose of the repressor or repressor system is applied to the cell or cell population, where prolonged period is at least about 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, or 28 days. In some embodiments, prolonged period is about 10 to about 100 days, about 20 to about 90 days, about 20 to about 80 days, about 20 to about 70 days, about 20 to about 60 days, about 25 to about 75 days, about 25 to about 65 days, about 30 to about 100 days, about 30 to about 90 days, about 30 to about 80 days, or about 30 to about 70 days.

[0073] In some embodiments, the increase in DNA methylation is maintained at the site for an extended period after contact until the next dose of the repressor or repressor system is applied to the cell or cell population, where extended period is at least about 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 7 weeks, 8 weeks, 9 weeks, 10 weeks, 11 weeks, or 12 weeks.

[0074] In some embodiments, the increase in DNA methylation is maintained at the site for an extended period after contact until the next dose of the repressor or repressor system is applied to the cell or cell population, where extended period is approximately 1 to 48 weeks, 1 to 36 weeks, 1 to 24 weeks, 1 to 12 weeks, 2 to 48 weeks, 2 to 36 weeks, 2 to 24 weeks, 2 to 12 weeks, 3 to 48 weeks, 3 to 36 weeks, 3 to 24 weeks, 3 to 12 weeks, 4 to 48 weeks, 4 to 36 weeks, 4 to 24 weeks, or 4 to 12 weeks.

[0075] In some embodiments, the increase in DNA methylation is maintained at the site for a prolonged period after contact until the next dose of the repressor or repressor system is applied to the cell or cell population, where prolonged period is at least 21 days.

[0076] In some embodiments, DNA methylation at the site increases compared to before contact, or compared to control cells or a control population of cells that have not been in contact with the repressor or repressor system.

[0077] In some embodiments, this method reduces the expression of the target gene in cells or cell populations. In some embodiments, the level of expression of the target gene in cells or cell populations is reduced compared to the level before contact, or compared to the level of control cells or cell populations (e.g., cells or cell populations not in contact with the repressor or repressor system).

[0078] In some embodiments, the Disclosure provides a method for increasing DNA methylation at a site in a region of a genome containing a target gene of interest, comprising administering to the subject a certain dose of an expression repressor described herein, or a nucleic acid encoding an expression repressor, wherein the expression repressor comprises (i) a DNA target-directed moiety that binds to a target sequence in the region, and (ii) a DNA methyltransferase.

[0079] In some embodiments, the Disclosure provides a method for increasing DNA methylation at a site in a region of a genome containing a target gene of interest, comprising administering to the subject a certain dose (e.g., a first dose) of a repressor system described herein comprising at least one repressor (e.g., 1, 2, 3, 4, 5 or more repressors described herein) or a nucleic acid encoding at least one repressor, wherein the at least one repressor comprises (i) a DNA target-directed moiety that binds to a target sequence in the region, and (ii) a DNA methyltransferase.

[0080] In some embodiments, DNA methylation at a site increases compared to before administration or compared to a control group.

[0081] In some embodiments, DNA methylation at the site increases over a long period after administration. In some embodiments, DNA methylation at the site increases over a long period after administration, until the subject receives the next dose of the inhibitor or inhibitor system.

[0082] In some embodiments, site-specific DNA methylation increases over a long period following administration until the subject receives the next dose of the inhibitor or inhibitor system, where the long period is at least about 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, or 28 days. In some embodiments, the long period is about 10 to about 100 days, about 20 to about 90 days, about 20 to about 80 days, about 20 to about 70 days, about 20 to about 60 days, about 25 to about 75 days, about 25 to about 65 days, about 30 to about 100 days, about 30 to about 90 days, about 30 to about 80 days, or about 30 to about 70 days.

[0083] In some embodiments, DNA methylation at the site increases over a long period of time after administration until the subject receives the next dose of the inhibitor or inhibitor system, where long period is at least about 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 7 weeks, 8 weeks, 9 weeks, 10 weeks, 11 weeks, or 12 weeks.

[0084] In some embodiments, site-specific DNA methylation increases over a long period after administration until the subject receives the next dose of the inhibitor or inhibitor system, where long period is approximately 1 to 48 weeks, 1 to 36 weeks, 1 to 24 weeks, 1 to 12 weeks, 2 to 48 weeks, 2 to 36 weeks, 2 to 24 weeks, 2 to 12 weeks, 3 to 48 weeks, 3 to 36 weeks, 3 to 24 weeks, 3 to 12 weeks, 4 to 48 weeks, 4 to 36 weeks, 4 to 24 weeks, or 4 to 12 weeks.

[0085] In some embodiments, DNA methylation at the site increases over a long period after administration until the subject receives the next dose of the inhibitory factor or inhibitory factor system, where the long period is at least 21 days.

[0086] In some embodiments, this method reduces the expression of the target gene in the target (e.g., the target tissue or target cell population). In some embodiments, the level of expression of the target gene is reduced compared to the level before administration or compared to the level of a control that has not received the dose.

[0087] In some embodiments, this method increases DNA methylation at sites in the IGD containing the target gene.

[0088] In some quantifiable cases, this method increases DNA methylation at sites in the IGD containing the target gene, where the sites have spans of at least approximately 300, 400, 500, 600, 700, 800, 900, 1,000, 1,100, 1,200, 1,300, 1,400, 1,500, 1,600, 1,700, 1,800, 1,900, or 2,000 bases.

[0089] In some embodiments, this method increases DNA methylation at sites in the IGD containing the target gene, where the sites are spans of at least approximately 300, 400, 500, 600, 700, 800, 900, 1,000, 1,100, 1,200, 1,300, 1,400, 1,500, 1,600, 1,700, 1,800, 1,900, or 2,000 bases, and the sites contain multiple CpG sequences.

[0090] In some embodiments, the method increases DNA methylation at sites in the IGD containing the target gene, where the sites are spans of at least approximately 300, 400, 500, 600, 700, 800, 900, 1,000, 1,100, 1,200, 1,300, 1,400, 1,500, 1,600, 1,700, 1,800, 1,900, or 2,000 bases, and the sites contain CpG sequence frequencies higher than the average CpG sequence frequency in the whole genome or a control region of the genome. In some embodiments, the sites contain CpG islands.

[0091] In certain embodiments, the Disclosure provides a method for increasing DNA methylation at a site in a region of the genome containing a target gene of a cell or a population of cells, comprising contacting the cell or population with a certain dose of a repressor described herein or a nucleic acid encoding a repressor, wherein the repressor comprises (i) a DNA target-directed moiety that binds to a target sequence in the region, and (ii) a DNA methyltransferase, the region of the genome being an IGD containing the target gene, and the site being a CpG The CpG islands are spans of at least approximately 300, 400, 500, 600, 700, 800, 900, 1,000, 1,100, 1,200, 1,300, 1,400, 1,500, 1,600, 1,700, 1,800, 1,900, or 2,000 bases, and the average methylated CpG sequence rate in the CpG islands increases compared to before contact or compared to control cells or cell populations. In some embodiments, the location of the CpG islands (i.e., genomic coordinates relative to the reference genome) is identified using the UCSC genome browser. In some embodiments, the target sequence is located in or adjacent to the CpG island. In some embodiments, the target sequence does not exceed approximately 500 to 1,000 bases upstream or downstream from the CpG island. In some embodiments, the average methylated CpG sequence rate in CpG islands is measured using EM-seq in test cells or cell populations (i.e., cells or populations exposed to an expression repressor or nucleic acid) compared to control cells or cell populations (e.g., cells or populations not exposed to an expression repressor or nucleic acid). In some embodiments, performing EM-seq involves amplifying a region of about 300-500 base pairs, including a CpG island or a portion thereof, using, for example, PCR. In some embodiments, the amplified region is sequenced using, for example, next-generation sequencing with Illumina, and the methylated CpG sequence rate in the amplified region is determined as the average of all sequence reads. In some embodiments, the average methylated CpG sequence rate of the amplified region obtained from test cells or cell populations is compared to control cells or cell populations.In some embodiments, the increase in DNA methylation is presented as a multiple of the average methylated CpG sequence rate in the amplified region between test cells or cell populations and control cells or cell populations.

[0092] In some embodiments, the method increases DNA methylation of the CpG sequence at that site compared to before contact or administration. In some embodiments, the method results in DNA methylation of at least about 20%, about 30%, about 40%, or about 50% of the CpG sequence at that site. In some embodiments, the method results in DNA methylation of about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, or about 100% of the CpG sequence at that site. In some embodiments, the method results in DNA methylation of the CpG sequence at that site being at least about 5 times, about 10 times, about 15 times, about 20 times, about 25 times, about 30 times, about 35 times, about 35 times, about 40 times, about 50 times, about 60 times, about 70 times, about 80 times, about 90 times, or about 100 times higher than before contact or administration. In some embodiments, this method increases the frequency of methylated CpG sequences at that site by approximately 5 to 50 times compared to before contact or administration.

[0093] In some embodiments, this method increases DNA methylation at a site in the IGD containing the target gene, where the site is a span of at least about 500 base pairs and contains about 50 to 250 CpG sequences, with multiple CpG sequences being methylated.

[0094] In some embodiments, this method increases DNA methylation at a site in the IGD containing the target gene, where the site has a span of at least about 600 base pairs and contains about 50 to 300 CpG sequences, with multiple CpG sequences being methylated.

[0095] In some embodiments, this method increases DNA methylation at a site in the IGD containing the target gene, where the site has a span of at least about 700 base pairs and contains about 50 to 350 CpG sequences, with multiple CpG sequences being methylated.

[0096] In some embodiments, this method increases DNA methylation at a site in the IGD containing the target gene, where the site has a span of at least about 800 base pairs and contains about 50 to 400 CpG sequences, with multiple CpG sequences being methylated.

[0097] In some embodiments, this method increases DNA methylation at a site in the IGD containing the target gene, where the site has a span of at least about 900 base pairs and contains about 50 to 450 CpG sequences, with multiple CpG sequences being methylated.

[0098] In some embodiments, this method increases DNA methylation at a site in the IGD containing the target gene, where the site is at least a span of about 1,000 bases and contains about 50 to 500 CpG sequences, with multiple CpG sequences being methylated.

[0099] In some embodiments, this method increases DNA methylation at sites in the IGD containing the target gene, where the sites are spans of at least approximately 300, 400, 500, 600, 700, 800, 900, or 1,000 bases, and the spans contain CpG islands, in which multiple CpG sequences are methylated.

[0100] In some embodiments, the site is located in or near the promoter of the target gene. In some embodiments, the site is located in or near the enhancer of the target gene. In some embodiments, the site is located in the target gene. In some embodiments, the site is located in the non-coding region of the target gene. In some embodiments, the site is located in the coding region of the target gene.

[0101] Regulation of gene expression In some embodiments, the Disclosure provides a method for reducing the expression of a target gene in cells or a population of cells, comprising contacting the cells or population with a certain dose of an expression repressor described herein, or a nucleic acid encoding an expression repressor, wherein the expression repressor comprises (i) a DNA target-directed moiety that binds to a target sequence in a region, and (ii) a DNA methyltransferase.

[0102] Methods for measuring the expression of a target gene are known in the art. In some embodiments, the expression of a target gene is measured in a cell culture contacted with a certain dose of the repressor or repressor system described herein, or in a tissue sample obtained from a subject administered a certain dose of the repressor or repressor system described herein. In some embodiments, the tissue sample is a fresh, frozen, and / or preserved organ, biopsy, and / or aspirate obtained from the subject. In some embodiments, the tissue sample is blood or any blood component (e.g., plasma) taken from the subject. In some embodiments, the method includes quantifying the level of an RNA transcript encoded by the target gene. Exemplary methods for measuring the level of an RNA transcript include, but are not limited to, Northern blotting, RNA-seq, RT-PCR, real-time RT-PCR, competitive RT-PCR, and nucleic acid microarrays. In some embodiments, the method includes quantifying the level of a protein product encoded by the target gene. Examples of methods for measuring protein expression include, but are not limited to, quantitative immunofluorescence, flow cytometry, Western blotting, ELISA, tissue immunostaining, immunoprecipitation, mass spectrometry, and immunohistochemistry.

[0103] In some embodiments, the Disclosure provides a method for reducing the expression of a target gene in a cell or cell population, comprising contacting the cell or cell population with at least one repressor (e.g., 1, 2, 3, 4, 5 or more repressors described herein), or a repressor system described herein comprising a nucleic acid encoding at least one repressor, wherein the at least one repressor comprises (i) a DNA target-directed moiety that binds to a target sequence in a region, and (ii) a DNA methyltransferase.

[0104] In some embodiments, the expression of the target gene is reduced compared to before contact, or compared to control cells or a control population that have not been in contact with the repressor or repressor system. In some embodiments, the expression of the target gene is reduced over a long period after contact. In some embodiments, the expression of the target gene is reduced over a long period after contact until the cells or cell population are exposed to the next dose of the repressor or repressor system.

[0105] In some embodiments, the expression of the target gene is reduced for a prolonged period after contact until the next dose of the repressor or repressor system is applied to the cells or cell population, where prolonged period is at least about 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, or 28 days. In some embodiments, prolonged period is at least about 21 days. In some embodiments, prolonged period is at least about 28 days.

[0106] In some embodiments, the expression of the target gene is reduced for a long period after contact until the next dose of the repressor or repressor system is applied to the cells or cell population, where the long period is approximately 10 to 100 days, 20 to 90 days, 20 to 80 days, 20 to 70 days, 20 to 60 days, 25 to 75 days, 25 to 65 days, 30 to 100 days, 30 to 90 days, 30 to 80 days, or 30 to 70 days. In some embodiments, the long period is approximately 21 to 100 days. In some embodiments, the long period is approximately 21 to 200 days. In some embodiments, the long period is approximately 28 to 100 days. In some embodiments, the long period is approximately 28 to 200 days.

[0107] In some embodiments, the expression of the target gene is reduced for a prolonged period after contact until the next dose of the repressor or repressor system is applied to the cells or cell population, where prolonged period is at least about 1 week, about 2 weeks, about 3 weeks, about 4 weeks, about 5 weeks, about 6 weeks, about 7 weeks, about 8 weeks, about 9 weeks, about 10 weeks, about 11 weeks, or about 12 weeks. In some embodiments, prolonged period is at least about 3 weeks. In some embodiments, prolonged period is at least about 4 weeks.

[0108] In some embodiments, the expression of the target gene is reduced for a long period after contact until the next dose of the repressor or repressor system is applied to the cells or cell population, where long period is approximately 1 to 48 weeks, approximately 1 to 36 weeks, approximately 1 to 24 weeks, approximately 1 to 12 weeks, approximately 2 to 48 weeks, approximately 2 to 36 weeks, approximately 2 to 24 weeks, approximately 2 to 12 weeks, approximately 3 to 48 weeks, approximately 3 to 36 weeks, approximately 3 to 24 weeks, approximately 3 to 12 weeks, approximately 4 to 48 weeks, approximately 4 to 36 weeks, approximately 4 to 24 weeks, or approximately 4 to 12 weeks. In some embodiments, long period is at least approximately 3 to 12 weeks. In some embodiments, long period is at least approximately 3 to 24 weeks. In some embodiments, long period is at least approximately 3 to 48 weeks. In some embodiments, the long term is at least about 4 weeks to about 12 weeks. In some embodiments, the long term is at least about 4 weeks to about 24 weeks. In some embodiments, the long term is at least about 4 weeks to about 48 weeks.

[0109] In some embodiments, the Disclosure provides a method for reducing the expression of a target gene in a subject, comprising administering to the subject a certain dose of an expression repressor described herein or a nucleic acid encoding an expression repressor, wherein the expression repressor comprises (i) a DNA target-directed moiety that binds to a target sequence in a region, and (ii) a DNA methyltransferase.

[0110] In some embodiments, the Disclosure provides a method for reducing the expression of a target gene in a subject, comprising administering to the subject a certain dose of an expression repressor system described herein comprising at least one expression repressor (e.g., 1, 2, 3, 4, 5 or more expression repressors described herein), or a nucleic acid encoding at least one expression repressor, wherein the at least one expression repressor comprises (i) a DNA target-directed moiety that binds to a target sequence in a region, and (ii) a DNA methyltransferase.

[0111] In some embodiments, the expression of the target gene is reduced compared to before administration or compared to a control subject that has not received the dose. In some embodiments, the expression of the target gene is reduced over a long period after administration. In some embodiments, the expression of the target gene is reduced over a long period after administration until the subject receives the next dose of the repressor or repressor system.

[0112] In some embodiments, the expression of the target gene is reduced for a long period after administration until the next dose of the gene repressor or gene repressor system is administered to the subject, where the long period is at least about 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, or 28 days. In some embodiments, the long period is at least about 21 days. In some embodiments, the long period is at least about 28 days.

[0113] In some embodiments, the expression of the target gene is reduced for a long period after administration until the subject receives the next dose of the gene repressor or gene repressor system, where the long period is approximately 10 to 100 days, 20 to 90 days, 20 to 80 days, 20 to 70 days, 20 to 60 days, 25 to 75 days, 25 to 65 days, 30 to 100 days, 30 to 90 days, 30 to 80 days, or 30 to 70 days. In some embodiments, the long period is approximately 21 to 100 days. In some embodiments, the long period is approximately 21 to 200 days. In some embodiments, the long period is approximately 28 to 100 days. In some embodiments, the long period is approximately 28 to 200 days.

[0114] In some embodiments, the expression of the target gene is reduced for a long period after administration until the subject receives the next dose of the repressor or repressor system, where a long period is at least about 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 7 weeks, 8 weeks, 9 weeks, 10 weeks, 11 weeks, or 12 weeks. In some embodiments, a long period is at least about 3 weeks. In some embodiments, a long period is at least about 4 weeks.

[0115] In some embodiments, the expression of the target gene is reduced for a long period after administration until the next dose of the repressor or repressor system is administered to the subject, where long period is approximately 1 to 48 weeks, approximately 1 to 36 weeks, approximately 1 to 24 weeks, approximately 1 to 12 weeks, approximately 2 to 48 weeks, approximately 2 to 36 weeks, approximately 2 to 24 weeks, approximately 2 to 12 weeks, approximately 3 to 48 weeks, approximately 3 to 36 weeks, approximately 3 to 24 weeks, approximately 3 to 12 weeks, approximately 4 to 48 weeks, approximately 4 to 36 weeks, approximately 4 to 24 weeks, or approximately 4 to 12 weeks. In some embodiments, long period is at least approximately 3 to 12 weeks. In some embodiments, long period is at least approximately 3 to 24 weeks. In some embodiments, long period is at least approximately 3 to 48 weeks. In some embodiments, long period is at least approximately 4 to 12 weeks. In some embodiments, the long term is at least about 4 weeks to about 24 weeks. In some embodiments, the long term is at least about 4 weeks to about 48 weeks.

[0116] Treatment method In some embodiments, the Disclosure provides a method for treating a condition related to the expression of a target gene, comprising administering to a subject a certain dose of an expression repressor described herein, or a nucleic acid encoding an expression repressor, wherein the expression repressor comprises (i) a DNA target-directed moiety that binds to a target sequence in a region of the genome containing the target gene, and (ii) a DNA methyltransferase, thereby increasing DNA methylation at a site in the region of the genome containing the target gene.

[0117] In some embodiments, the Disclosure provides a method for treating a pathological condition related to the expression of a target gene, comprising administering to a subject a certain dose of a repressor system described herein comprising at least one repressor (e.g., 1, 2, 3, 4, 5 or more repressors described herein) or a nucleic acid encoding at least one repressor, wherein the at least one repressor comprises (i) a DNA target-directed portion that binds to a target sequence in a region of the genome containing the target gene, and (ii) a DNA methyltransferase, thereby increasing DNA methylation at a site in the region of the genome containing the target gene.

[0118] In some embodiments, the Disclosure provides a method for treating a condition associated with the overexpression of a target gene, comprising administering to a subject a certain dose of an expression repressor described herein, or a nucleic acid encoding an expression repressor, wherein the expression repressor comprises (i) a DNA target-directed moiety that binds to a target sequence in a region of the genome containing the target gene, and (ii) a DNA methyltransferase, thereby increasing DNA methylation at a site in the region of the genome containing the target gene.

[0119] In some embodiments, the Disclosure provides a method for treating a condition associated with the overexpression of a target gene, comprising administering to a subject a certain dose of a repressor system described herein comprising at least one repressor (e.g., 1, 2, 3, 4, 5 or more repressors described herein) or a nucleic acid encoding at least one repressor, wherein the at least one repressor comprises (i) a DNA target-directed portion that binds to a target sequence in a region of the genome containing the target gene, and (ii) a DNA methyltransferase, thereby increasing DNA methylation at a site in the region of the genome containing the target gene.

[0120] In some embodiments, this method reduces the expression of the target gene (e.g., in the target tissue or target cell population). In some embodiments, the level of expression of the target gene is reduced compared to the level before administration. In some embodiments, the level of expression of the target gene is equivalent to that of a control subject that has not received the dose.

[0121] In certain embodiments, the Disclosure provides a method for treating a condition related to the expression of a target gene, comprising administering to a subject a certain dose of an expression repressor described herein, or a nucleic acid encoding an expression repressor, wherein the expression repressor comprises (i) a DNA target-directed moiety that binds to a target sequence in a region of the genome containing the target gene, and (ii) a DNA methyltransferase, the target gene containing one or more mutations, and having increased DNA methylation at a site in the region of the genome containing the target gene.

[0122] In some embodiments, the Disclosure provides a method for treating a pathological condition related to the expression of a target gene, comprising administering to a subject a certain dose of a repressor system described herein comprising at least one repressor (e.g., 1, 2, 3, 4, 5 or more repressors described herein) or a nucleic acid encoding at least one repressor, wherein the at least one repressor comprises (i) a DNA target-directed portion that binds to a target sequence in a region of the genome containing the target gene, and (ii) a DNA methyltransferase, wherein the target gene contains one or more mutations and has increased DNA methylation at a site in the region of the genome containing the target gene.

[0123] In some embodiments, DNA methylation at the site increases over a long period after administration. In some embodiments, DNA methylation at the site increases over a long period after administration, until the subject receives the next dose of the inhibitor or inhibitor system.

[0124] In some embodiments, this method reduces the expression of a target gene (e.g., in a target tissue or target cell population). In some embodiments, the level of expression of the target gene is reduced compared to the level before administration or compared to the level of a control. In some embodiments, the expression of the target gene remains reduced for a long period after administration until the subject receives the next dose of the repressor or repressor system.

[0125] In some embodiments, the longest period is at least about 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, or 28 days. In some embodiments, the longest period is at least about 21 days. In some embodiments, the longest period is at least about 28 days. In some embodiments, the longest period is about 10 to about 100 days, about 20 to about 90 days, about 20 to about 80 days, about 20 to about 70 days, about 20 to about 60 days, about 25 to about 75 days, about 25 to about 65 days, about 30 to about 100 days, about 30 to about 90 days, about 30 to about 80 days, or about 30 to about 70 days. In some embodiments, the longest period is about 21 to about 100 days. In some embodiments, the longest period is approximately 21 to 200 days. In some embodiments, the longest period is approximately 28 to 100 days. In some embodiments, the longest period is approximately 28 to 200 days. In some embodiments, the longest period is at least approximately 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 7 weeks, 8 weeks, 9 weeks, 10 weeks, 11 weeks, or 12 weeks. In some embodiments, the longest period is at least approximately 3 weeks. In some embodiments, the longest period is at least approximately 4 weeks. In some embodiments, the long term is approximately 1 to 48 weeks, approximately 1 to 36 weeks, approximately 1 to 24 weeks, approximately 1 to 12 weeks, approximately 2 to 48 weeks, approximately 2 to 36 weeks, approximately 2 to 24 weeks, approximately 2 to 12 weeks, approximately 3 to 48 weeks, approximately 3 to 36 weeks, approximately 3 to 24 weeks, approximately 3 to 12 weeks, approximately 4 to 48 weeks, approximately 4 to 36 weeks, approximately 4 to 24 weeks, or approximately 4 to 12 weeks. In some embodiments, the long term is at least approximately 3 to 12 weeks. In some embodiments, the long term is at least approximately 3 to 24 weeks. In some embodiments, the long term is at least approximately 3 to 48 weeks. In some embodiments, the long term is at least approximately 4 to 12 weeks. In some embodiments, the long term is at least approximately 4 to 24 weeks. In some embodiments, a long period is at least about 4 weeks to about 48 weeks.

[0126] In some embodiments, the pathological condition associated with the expression of the target gene is a neoplasm. In some embodiments, the pathological condition is tumorigenesis. In some embodiments, the pathological condition is cancer. In some embodiments, cancer is associated with a poor prognosis. In some embodiments, the pathological condition is cancer and the target gene is an oncogene. In some embodiments, the method reduces the expression of the oncogene, thereby treating the cancer.

[0127] In some embodiments, the pathological condition associated with the expression of the target gene is a metabolic disorder. In some embodiments, the metabolic disorder is weight gain, diabetes, cardiovascular disease, liver disease, stroke, or a combination thereof. In some embodiments, the metabolic disorder is hereditary. In some embodiments, the metabolic disorder is related to lifestyle. In some embodiments, the pathological condition is a metabolic disorder in which the transcription or translation product of the target gene is located in a metabolic pathway, and dysregulation of the metabolic pathway due to, for example, abnormal levels of metabolite production, disruption of metabolite secretion, disruption of metabolite degradation, or a combination thereof contributes to the disorder. In some embodiments, the method reduces the expression of the target gene, thereby treating the metabolic disorder.

[0128] In some embodiments, the pathological condition associated with the expression of the target gene is an infectious disease. In some embodiments, the pathological condition associated with the expression of the target gene is an autoimmune disease. In some embodiments, the pathological condition associated with the expression of the target gene is an inflammatory disease. In some embodiments, the pathological condition associated with the expression of the target gene is a lung disease.

[0129] Expression suppressor In some embodiments, the disclosure provides an expression repressor for reducing the expression of a target gene. In some embodiments, the expression repressor comprises a DNA target-directed moiety and an effector domain. In some embodiments, the DNA target-directed moiety binds to a target sequence in a region of the genome containing the target gene. In some embodiments, the region of the genome is or contains an IGD containing the target gene. In some embodiments, the target sequence is located in or near a transcriptional regulatory element (e.g., a promoter) operably linked to the target gene. In some embodiments, the effector domain increases DNA methylation at a site in the region of the genome containing the target gene. In some embodiments, the site is a span of bases containing CpG islands (e.g., 300 to about 2,000 bases) (e.g., a span of bases with a CpG sequence frequency higher than the CpG sequence frequency across the entire genome). In some embodiments, when the expression repressor is introduced into cells, the DNA target-directed moiety localizes the effector domain to a region of the genome, and the effector domain increases DNA methylation at the site, thereby reducing the expression of the target gene. In some embodiments, the site of increased DNA methylation is located at or near a promoter operably linked to the target gene. In some embodiments, the site of increased DNA methylation is located at or near the transcription start site of the target gene. In some embodiments, the target sequence is located at or near a site of increased DNA methylation.

[0130] target sequence In some embodiments, the DNA target-directing moiety binds to a target sequence of the target gene. In some embodiments, the target gene is a human gene. In some embodiments, the target gene is a gene associated with a human disease or disorder.

[0131] In some embodiments, the DNA target-directing moiety binds to a target sequence in a genomic region containing the target gene. In some embodiments, the DNA target-directing moiety binds to a target sequence in a transcriptional regulatory element operably linked to the target gene. In some embodiments, the DNA target-directing moiety binds to a target sequence in a promoter operably linked to the target gene. In some embodiments, the DNA target-directing moiety binds to a target sequence in an enhancer operably linked to the target gene. In some embodiments, the target sequence is located in or near a site that targets the effector function of an expression repressor (e.g., in or near a site containing a CpG islet).

[0132] In some embodiments, the DNA target-directed moiety includes a ZF that binds to a target sequence in a genomic region containing the target gene. In some embodiments, the DNA target-directed moiety includes a ZF that binds to a target sequence on or near the target gene or a transcriptional regulatory element operably linked to the target gene. In some embodiments, the DNA target-directed moiety includes a TALE that binds to a target sequence in a genomic region containing the target gene. In some embodiments, the DNA target-directed moiety includes a TALE that binds to a target sequence on or near the target gene or a transcriptional regulatory element operably linked to the target gene. In some embodiments, the DNA target-directed moiety includes a site-induced nuclease (e.g., a catalytically inactive site-induced nuclease) that binds to a target sequence in a genomic region containing the target gene. In some embodiments, the DNA target-directed moiety includes a site-induced nuclease (e.g., a catalytically inactive site-induced nuclease) that binds to or near the target gene or a transcriptional regulatory element operably linked to the target gene.

[0133] In some embodiments, the DNA target-directing moiety binds to a target sequence located in a genomic region containing an IGD (Intragingive Genetic Diagram) containing the target gene. In some embodiments, the DNA target-directing moiety binds to a target sequence located in an IGD containing the target gene. In some embodiments, the target sequence is located in or near an enhancer of the IGD. In some embodiments, the target sequence is located in or near the promoter of the IGD. In some embodiments, the target sequence is located in or near a CpG islet of the IGD. In some embodiments, the target sequence is in close proximity to a CpG islet.

[0134] In some embodiments, the DNA target-directed moiety includes a ZF that binds to a target sequence in a genomic region including an IGD containing the target gene. In some embodiments, the DNA target-directed moiety includes a ZF that binds to a target sequence in an IGD containing the target gene. In some embodiments, the DNA target-directed moiety includes a TALE that binds to a target sequence in a genomic region including an IGD containing the target gene. In some embodiments, the DNA target-directed moiety includes a TALE that binds to a target sequence in an IGD containing the target gene. In some embodiments, the DNA target-directed moiety includes a site-inducible nuclease (e.g., a catalytically inactive site-inducible nuclease) that binds to a target sequence in a genomic region including an IGD containing the target gene. In some embodiments, the DNA target-directed moiety includes a site-inducible nuclease (e.g., a catalytically inactive site-inducible nuclease) that binds to a target sequence in an IGD containing the target gene.

[0135] In some embodiments, the site-directed nuclease comprises a Cas nuclease described herein (e.g., a catalytically inactive Cas nuclease) and a gRNA containing a spacer sequence corresponding to a target sequence. The spacer sequence is a sequence that defines the target sequence. The target sequence is located in double-stranded genomic DNA having a single strand containing the target sequence, which includes a protospacer sequence adjacent to a PAM sequence, referred to as the "PAM strand," and a second strand complementary to the PAM strand, referred to as the "non-PAM strand." Both the gRNA spacer sequence and the target sequence are complementary to the non-PAM strand of the genomic DNA molecule. As used herein, a spacer sequence "complementary to" the target sequence refers to a guide sequence that binds to the non-PAM strand of the target sequence by Watson-Crick base pairing, wherein the spacer sequence has sufficient complementarity to the PAM strand to enable the non-Cas nuclease to target the target sequence of the genomic DNA molecule. In some embodiments, the spacer sequence has up to one, two, or three mismatches with respect to the target sequence of the genomic DNA molecule, where the spacer sequence has sufficient complementarity with non-PAM strands to enable the Cas nuclease to target the target sequence of the genomic DNA molecule.

[0136] In some embodiments, the DNA target-directing moiety binds to a target sequence in a genomic region containing an IGD containing the target gene, where the target sequence is either upstream of or at the 5' boundary of the IGD. In some embodiments, the target sequence is between the 5' and 3' boundaries of the IGD. In some embodiments, the target sequence is either downstream of or at the 3' boundary of the IGD. In some embodiments, the DNA target-directing moiety binds to a target sequence in the IGD, where the target sequence is in a region containing a transcriptional regulatory element (e.g., a promoter or enhancer) (e.g., a 0.5-2kb region). In some embodiments, the region includes a promoter. In some embodiments, the target sequence is in the promoter. In some embodiments, the region includes an enhancer. In some embodiments, the target sequence is in the enhancer. In some embodiments, the DNA target-directing moiety binds to a target sequence in the IGD, where the target sequence is in a region containing a CpG island (e.g., a 0.5-2kb region). In some embodiments, the target sequence is in a CpG island.

[0137] The length of the target sequence depends on the DNA target-directing moiety used. In some embodiments, the DNA target-directing moiety contains a ZF, and the target sequence is approximately 10 to approximately 50 nucleotides, approximately 10 to approximately 40 nucleotides, approximately 10 to approximately 30 nucleotides, approximately 10 to approximately 20 nucleotides, or approximately 15 to approximately 20 nucleotides. In some embodiments, the DNA target-directing moiety contains a ZF, and the target sequence is approximately 10, approximately 11, approximately 12, approximately 13, approximately 14, approximately 15, approximately 16, approximately 17, approximately 18, approximately 19, or approximately 20 nucleotides. In some embodiments, the DNA target-directing moiety contains a ZF, and the target sequence is 15 nucleotides. In some embodiments, the DNA target-directing moiety contains a ZF, and the target sequence is 16 nucleotides. In some embodiments, the DNA target-directing moiety contains a ZF, and the target sequence is 17 nucleotides. In some embodiments, the DNA target-directing moiety contains a ZF, and the target sequence is 18 nucleotides. In some embodiments, the DNA target-directing moiety contains a ZF, and the target sequence is 19 nucleotides. In some embodiments, the DNA targeting portion includes a ZF, and the target sequence is 20 nucleotides.

[0138] In some embodiments, the DNA target-directed portion includes a TALE, and the target sequence is approximately 10 to approximately 50 nucleotides, approximately 10 to approximately 40 nucleotides, approximately 10 to approximately 30 nucleotides, approximately 10 to approximately 20 nucleotides, or approximately 15 to approximately 20 nucleotides. In some embodiments, the DNA target-directed portion includes a TALE, and the target sequence is approximately 10, approximately 11, approximately 12, approximately 13, approximately 14, approximately 15, approximately 16, approximately 17, approximately 18, approximately 19, or approximately 20 nucleotides. In some embodiments, the DNA target-directed portion includes a TALE, and the target sequence is 15 nucleotides. In some embodiments, the DNA target-directed portion includes a TALE, and the target sequence is 16 nucleotides. In some embodiments, the DNA target-directed portion includes a TALE, and the target sequence is 17 nucleotides. In some embodiments, the DNA target-directed portion includes a TALE, and the target sequence is 18 nucleotides. In some embodiments, the DNA target-directed portion includes a TALE, and the target sequence is 19 nucleotides. In some embodiments, the DNA targeting portion includes a TALE, and the target sequence is 20 nucleotides.

[0139] In some embodiments, the DNA targeting moiety comprises a site-induced nuclease (e.g., a catalytically inactive site-induced nuclease), and the target sequence is approximately 10 to approximately 50 nucleotides, approximately 10 to approximately 40 nucleotides, approximately 10 to approximately 30 nucleotides, approximately 10 to approximately 20 nucleotides, or approximately 15 to approximately 20 nucleotides. In some embodiments, the DNA targeting moiety comprises a site-induced nuclease (e.g., a catalytically inactive site-induced nuclease), and the target sequence is approximately 10, approximately 11, approximately 12, approximately 13, approximately 14, approximately 15, approximately 16, approximately 17, approximately 18, approximately 19, or approximately 20 nucleotides. In some embodiments, the DNA targeting moiety comprises a site-induced nuclease (e.g., a catalytically inactive site-induced nuclease), and the target sequence is 15 nucleotides. In some embodiments, the DNA targeting moiety includes a site-induced nuclease (e.g., a catalytically inactive site-induced nuclease), and the target sequence is 16 nucleotides. In some embodiments, the DNA targeting moiety includes a site-induced nuclease (e.g., a catalytically inactive site-induced nuclease), and the target sequence is 17 nucleotides. In some embodiments, the DNA targeting moiety includes a site-induced nuclease (e.g., a catalytically inactive site-induced nuclease), and the target sequence is 18 nucleotides. In some embodiments, the DNA targeting moiety includes a site-induced nuclease (e.g., a catalytically inactive site-induced nuclease), and the target sequence is 19 nucleotides. In some embodiments, the DNA targeting moiety includes a site-induced nuclease (e.g., a catalytically inactive site-induced nuclease), and the target sequence is 20 nucleotides.

[0140] In some embodiments, the target sequence is 10 to 50 nucleotides (e.g., 10 to 40, 10 to 30, 15 to 30, 15 to 25, or 15 to 20 nucleotides) in the genomic region containing the target gene. In some embodiments, the target sequence is about 10 to about 50 nucleotides, about 10 to about 40 nucleotides, about 10 to about 30 nucleotides, about 10 to about 20 nucleotides, or about 15 to about 20 nucleotides in the genomic region containing the target gene. In some embodiments, the target sequence is about 10, about 11, about 12, about 13, about 14, about 15, about 16, about 17, about 18, about 19, or about 20 nucleotides in the genomic region containing the target gene.

[0141] In some embodiments, the target sequence is 10 to 50 nucleotides (e.g., 10 to 40, 10 to 30, 15 to 30, 15 to 25, or 15 to 20 nucleotides) in the genomic region containing the IGD that includes the target gene. In some embodiments, the target sequence is about 10 to about 50 nucleotides, about 10 to about 40 nucleotides, about 10 to about 30 nucleotides, about 10 to about 20 nucleotides, or about 15 to about 20 nucleotides in the genomic region containing the IGD that includes the target gene. In some embodiments, the target sequence is about 10, about 11, about 12, about 13, about 14, about 15, about 16, about 17, about 18, about 19, or about 20 nucleotides in the genomic region containing the IGD that includes the target gene.

[0142] In some embodiments, the target sequence is 10 to 50 nucleotides (e.g., 10 to 40, 10 to 30, 15 to 30, 15 to 25, or 15 to 20 nucleotides) of a region of the IGD (e.g., a 0.1 to 2 kb region), and the region includes a transcriptional regulatory element (e.g., a promoter or enhancer). In some embodiments, the target sequence is about 10 to about 50 nucleotides, about 10 to about 40 nucleotides, about 10 to about 30 nucleotides, about 10 to about 20 nucleotides, or about 15 to about 20 nucleotides) of a region of the IGD (e.g., a 0.1 to 2 kb region), and the region includes a transcriptional regulatory element (e.g., a promoter or enhancer). In some embodiments, the target sequence is approximately 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 nucleotides of the IGD region (e.g., a 0.1–2 kb region), and the region includes a transcriptional regulatory element (e.g., a promoter or enhancer).

[0143] In some embodiments, the target sequence is 10 to 50 nucleotides (e.g., 10 to 40, 10 to 30, 15 to 30, 15 to 25, or 15 to 20 nucleotides) of a transcriptional regulatory element (e.g., promoter or enhancer) in the IGD. In some embodiments, the target sequence is about 10 to about 50 nucleotides, about 10 to about 40 nucleotides, about 10 to about 30 nucleotides, about 10 to about 20 nucleotides, or about 15 to about 20 nucleotides of a transcriptional regulatory element (e.g., promoter or enhancer) in the IGD. In some embodiments, the target sequence is about 10, about 11, about 12, about 13, about 14, about 15, about 16, about 17, about 18, about 19, or about 20 nucleotides of a transcriptional regulatory element (e.g., promoter or enhancer) in the IGD.

[0144] In some embodiments, the target sequence is 10 to 50 nucleotides (e.g., 10 to 40, 10 to 30, 15 to 30, 15 to 25, or 15 to 20 nucleotides) in a region of the IGD (e.g., a 0.1 to 2 kb region), and the region includes the promoter. In some embodiments, the target sequence is within the range of the promoter or overlaps with it. In some embodiments, the target sequence is about 10 to about 50 nucleotides, about 10 to about 40 nucleotides, about 10 to about 30 nucleotides, about 10 to about 20 nucleotides, or about 15 to about 20 nucleotides in a region of the IGD (e.g., a 0.1 to 2 kb region), and the region includes the promoter. In some embodiments, the target sequence is about 10, about 11, about 12, about 13, about 14, about 15, about 16, about 17, about 18, about 19, or about 20 nucleotides in a region of the IGD (e.g., a 0.1 to 2 kb region), and the region includes the promoter.

[0145] In some embodiments, the target sequence is 10–50 nucleotides of the promoter in IGD (e.g., 10–40, 10–30, 15–30, 15–25, or 15–20 nucleotides). In some embodiments, the target sequence is about 10–50 nucleotides, about 10–40 nucleotides, about 10–30 nucleotides, about 10–20 nucleotides, or about 15–20 nucleotides of the promoter in IGD. In some embodiments, the target sequence is about 10, about 11, about 12, about 13, about 14, about 15, about 16, about 17, about 18, about 19, or about 20 nucleotides of the promoter in IGD.

[0146] In some embodiments, the target sequence is 10 to 50 nucleotides (e.g., 10 to 40, 10 to 30, 15 to 30, 15 to 25, or 15 to 20 nucleotides) of the IGD region (e.g., the 0.1 to 2 kb region), and the region includes an enhancer. In some embodiments, the target sequence is within the range of the enhancer or overlaps with it. In some embodiments, the target sequence is about 10 to about 50 nucleotides, about 10 to about 40 nucleotides, about 10 to about 30 nucleotides, about 10 to about 20 nucleotides, or about 15 to about 20 nucleotides) of the IGD region (e.g., the 0.1 to 2 kb region), and the region includes an enhancer. In some embodiments, the target sequence is approximately 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 nucleotides in a region of the IGD (e.g., a 0.1–2 kb region), and the region includes an enhancer.

[0147] In some embodiments, the target sequence is 10–50 nucleotides of the enhancer in the IGD (e.g., 10–40, 10–30, 15–30, 15–25, or 15–20 nucleotides). In some embodiments, the target sequence is about 10–50 nucleotides, about 10–40 nucleotides, about 10–30 nucleotides, about 10–20 nucleotides, or about 15–20 nucleotides of the enhancer in the IGD. In some embodiments, the target sequence is about 10, about 11, about 12, about 13, about 14, about 15, about 16, about 17, about 18, about 19, or about 20 nucleotides of the enhancer in the IGD.

[0148] In some embodiments, the target sequence is 10 to 50 nucleotides (e.g., 10 to 40, 10 to 30, 15 to 30, 15 to 25, or 15 to 20 nucleotides) in a region of the IGD (e.g., a 0.1 to 2 kb region), and the region includes a CTCF binding site (e.g., a CTCF binding site on the boundary of the IGD or a CTCF binding site on the IGD). In some embodiments, the target sequence is within the range of a CTCF binding site or overlaps with it. In some embodiments, the target sequence is about 10 to about 50 nucleotides, about 10 to about 40 nucleotides, about 10 to about 30 nucleotides, about 10 to about 20 nucleotides, or about 15 to about 20 nucleotides in a region of the IGD (e.g., a 0.1 to 2 kb region), and the region includes a CTCF binding site (e.g., a CTCF binding site on the boundary of the IGD or a CTCF binding site on the IGD). In some embodiments, the target sequence is approximately 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 nucleotides in a region of the IGD (e.g., a 0.1–2 kb region), and the region includes a CTCF binding site (e.g., a CTCF binding site at the boundary of the IGD or a CTCF binding site in the IGD).

[0149] In some embodiments, the target sequence is 10 to 50 nucleotides (e.g., 10 to 40, 10 to 30, 15 to 30, 15 to 25, or 15 to 20 nucleotides) in a region of the IGD (e.g., a 0.1 to 2 kb region), and the region includes CpG islands. In some embodiments, the target sequence is within the range of CpG islands or overlaps with them. In some embodiments, the target sequence is about 10 to about 50 nucleotides, about 10 to about 40 nucleotides, about 10 to about 30 nucleotides, about 10 to about 20 nucleotides, or about 15 to about 20 nucleotides in a region of the IGD (e.g., a 0.1 to 2 kb region), and the region includes CpG islands. In some embodiments, the target sequence is approximately 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 nucleotides in a region of IGD (e.g., a 0.1–2kb region), and the region contains CpG islands.

[0150] DNA targeting moiety This disclosure provides an expression repressor comprising a DNA target-directed moiety that specifically targets, for example, a genomic sequence element (e.g., a promoter, TSS, or anchor sequence) located in, adjacent to, and / or operably linked to a target gene. In some embodiments, the DNA target-directed moiety specifically binds to a DNA sequence, for example, a DNA sequence associated with the target gene. Any molecule or compound that specifically binds to a DNA sequence can be used as the DNA target-directed moiety.

[0151] In some embodiments, the DNA target-directed moiety targets, for example, a component of the genome complex by binding. In some embodiments, the DNA target-directed moiety targets, for example, a transcription regulatory sequence (e.g., a promoter or enhancer) operably linked to a target gene by binding. In some embodiments, the DNA target-directed moiety targets, for example, a target gene or a portion of a target gene by binding. The target of the DNA target-directed moiety may be referred to as its targeted component. The targeted component may include, but is not limited to, a promoter, enhancer, anchor sequence, exon, intron, UTR coding sequence, splice site, or transcription start site, the target gene, or any genomic sequence element operably linked to the target gene itself. In some embodiments, the DNA target-directed moiety specifically binds to one or more target anchor sequences (e.g., intracellular) and does not bind to non-targeted anchor sequences (e.g., intracellular).

[0152] In some embodiments, the DNA targeting moiety includes a CRISPR / Cas domain (e.g., a catalytically inactive CRISPR / Cas domain), a TAL effector domain, a Zn finger domain, a peptide nucleic acid (PNA), or a nucleic acid molecule.

[0153] In some embodiments, the expression repressor of the Disclosure comprises one DNA targeting moiety. In some embodiments, the expression repressor comprises multiple DNA targeting moieties, each DNA targeting moiety not binding to another DNA targeting moiety in a detectable manner, for example, not binding at all.

[0154] In some embodiments, the DNA target-directing moiety has the following sequences: 500, 450, 400, 350, 300, 250, 200, 150, 100, 50, 40, 30, 20, 10, 9, 8, 7, 6, 5, 4, 3, 2, 1, 0.9, 0.8, 0.7, 0.6, 0.5, 0.4, 0.3, 0.2, 0.1, 0.09, 0.08, 0.07, 0.06, 0.05, 0.04, 0.03, 0.02, 0.01, 0 The KD is 0.005, 0.002, or 0.001 nM or less (and optionally, at least 50, 40, 30, 20, 10, 9, 8, 7, 6, 5, 4, 3, 2, 1, 0.9, 0.8, 0.7, 0.6, 0.5, 0.4, 0.3, 0.2, 0.1, 0.09, 0.08, 0.07, 0.06, 0.05, 0.04, 0.03, 0.02, 0.01, 0.005, 0.002, or 0.001 nM). In some embodiments, the DNA target-directing moiety is bound to its target sequence with a KD of 0.001 nM to 500 nM, for example, 0.1 nM to 5 nM, for example, about 0.5 nM. In some embodiments, the DNA target-directing moiety binds to the non-target sequence with a KD of at least 500, 600, 700, 800, 900, 1000, 2000, 5000, 10,000, or 100,000 nM (and optionally, shows no noticeable binding to the non-target sequence). In some embodiments, the DNA target-directing moiety does not substantially bind to the non-target sequence.

[0155] CRISPR / Cas domain In some embodiments, the DNA targeting moiety includes a CRISPR / Cas domain. A CRISPR / Cas protein may comprise a CRISPR / Cas effector and, optionally, one or more other domains. The CRISPR / Cas domain typically has structural and / or functional similarities to a clustered and regularly arranged short palindromic sequence repeat (CRISPR) system, e.g., a protein involved in the Cas protein. The CRISPR / Cas domain optionally includes a guide RNA, e.g., a single guide RNA (sgRNA). In some embodiments, the CRISPR / Cas domain non-covalently binds to the gRNA contained within the CRISPR / Cas domain.

[0156] The CRISPR system is an adaptive defense system initially discovered in bacteria and archaea. The CRISPR system uses RNA-guided nucleases, referred to as CRISPR-associated or "Cas" endonucleases (e.g., Cas9 or Cpfl), to cleave foreign DNA. For example, in a typical CRISPR / Cas system, the endonuclease is guided to a target nucleotide sequence (e.g., a site in the genome to be sequence-edited) by a sequence-specific, non-coding "guide RNA" that targets a single-stranded or double-stranded DNA sequence. Three classes (I-III) of CRISPR systems have been identified. Class II CRISPR systems use a single Cas endonuclease (rather than multiple Cas proteins). One class II CRISPR system includes a type II Cas endonuclease such as Cas9, a CRISPR RNA ("crRNA"), and a transactivating crRNA ("tracrRNA"). The crRNA typically contains a "guide RNA," which is an RNA sequence of approximately 20 nucleotides corresponding to the target DNA sequence. crRNA also contains a region that binds to tracrRNA, and this binding creates a partially double-stranded structure, which, when cleaved by RNase III, results in a crRNA / tracrRNA hybrid. The crRNA / tracrRNA hybrid then leads to Cas9 endonuclease recognizing and cleaving a target DNA sequence. The target DNA sequence must generally be adjacent to a “protospacer fringe motif” ("PAM") specific to a given Cas endonuclease; however, PAM sequences appear throughout a given genome.CRISPR endonucleases identified from various prokaryotic species have unique PAM sequence requirements; examples of PAM sequences include 5'-NGG (Streptococcus pyogenes), 5'-NNAGAA (Streptococcus thermophilus CRISPR1), 5'-NGGNG (Streptococcus thermophilus CRISPR3), and 5'-NNNGATT (Neisseria meningiditis). Some endonucleases, such as Cas9 endonucleases, are associated with G-rich PAM sites, e.g., 5'-NGG, and perform blunt-end cleavage of target DNA at a position 3 nucleotides upstream (towards its 5' side) from the PAM site. Other Class II CRISPR systems include the smaller V-type endonuclease Cpfl, which is smaller than Cas9; examples include AsCpfl (derived from Acidaminococcus sp.) and LbCpfl (derived from Lachnospiraceae sp.). Cpfl-associated CRISPR arrays process to mature crRNA without requiring tracrRNA; in other words, the Cpfl system requires only the Cpfl nuclease and crRNA to cleave the target DNA sequence. The Cpfl endonuclease is associated with T-rich PAM sites, e.g., 5'-TTN. Cpfl can also recognize the 5'-CTA PAM motif. Cpfl cleaves target DNA by introducing shifted or adherent-end double-strand breaks with a 4 or 5 nucleotide 5' overhang, for example, by cutting the target DNA with a 5-nucleotide shifted or adherent-end cut located 18 nucleotides downstream (3' side) from the PAM site on the coding strand and 23 nucleotides downstream from the PAM site on the complementary strand; this 5-nucleotide overhang resulting from such a shifted break allows for more precise genome editing by homologous recombination DNA insertion compared to insertion in blunt-end cuts.For example, see Zetsche et al. (2015) Cell, 163:759-771.

[0157] The techniques provided herein may utilize a variety of CRISPR-related (Cas) genes or proteins, and the selection of the Cas protein will depend on the detailed conditions of the method. Specific examples of Cas proteins include the Class II system, which includes Casl, Cas2, Cas3, Cas4, Cas5, Cas6, Casl, Cas8, Cas9, Cas10, Cpfl, C2C1, or C2C3. In some embodiments, the Cas protein, e.g., the Cas9 protein, may be derived from any of the various prokaryotic species. In some embodiments, a detailed Cas protein, e.g., a detailed Cas9 protein, is selected to recognize a detailed protospacer-adjacent motif (PAM) sequence. In some embodiments, the DNA targeting moiety includes a sequence-targeting polypeptide, such as a Cas protein, e.g., Cas9. In certain embodiments, the Cas protein, e.g., the Cas9 protein, may be obtained from bacteria or archaea, or may be synthesized using known methods. In certain embodiments, the Cas protein may be derived from Gram-positive or Gram-negative bacteria.In certain embodiments, the Cas protein is found in the genera Streptococcus (e.g., Streptococcus pyogenes or S. thermophilus), Francisella (e.g., F. novicida), Staphylococcus (e.g., Staphylococcus aureus), and Acidaminococcus (e.g., Acidaminococcus species). It may be derived from the genera sp.)BV3L6), Neisseria (e.g., Neisseria meningitidis), Cryptococcus, Corynebacterium, Haemophilus, Eubacterium, Pasteurella, Prevotella, Veillonella, or Marinobacter.

[0158] In some embodiments, for the Cas protein to bind and / or function, the Cas protein must have a protospacer adjacency motif (PAM) present or adjacent to the target DNA sequence. In some embodiments, the PAM is NGG, YG, NNGRRT, NNNRRT, NGA, TYCV, TATV, NTTN, or NNNGATT (wherein N represents any nucleotide, Y represents C or T, R represents A or G, and V represents A or C or G) from 5' to 3', or comprises the same. In some embodiments, the Cas protein is a protein listed in Table 1. In some embodiments, the Cas protein comprises one or more mutations that alter its PAM. In some embodiments, the Cas protein comprises E1369R, E1449H, and R1556A mutations or similar substitutions in the amino acids corresponding to the aforementioned positions. In some embodiments, the Cas protein includes E782K, N968K, and R1015H mutations or similar substitutions in the amino acids corresponding to the aforementioned positions. In some embodiments, the Cas protein includes DI 135V, R1335Q, and T1337R mutations or similar substitutions in the amino acids corresponding to the aforementioned positions. In some embodiments, the Cas protein includes S542R and K607R mutations or similar substitutions in the amino acids corresponding to the aforementioned positions. In some embodiments, the Cas protein includes S542R, K548V, and N552R mutations or similar substitutions in the amino acids corresponding to the aforementioned positions.

[0159] [Table 1]

[0160] In some embodiments, the Cas protein is modified to inactivate the nuclease, for example, to become a nuclease-deficient Cas. In some embodiments, the Cas protein is the Cas9 protein. While wild-type Cas9 causes double-strand breaks (DSBs) at specific DNA sequences targeted by the gRNA, several functionally modified CRISPR endonucleases are available; for example, the “nickase” version of Cas9 causes only single-strand breaks; and catalytically inactive Cas9 ("dCas9") does not cleave target DNA. In some embodiments, when dCas binds to a DNA sequence, transcription at that site may be interfered with by steric hindrance. In some embodiments, the DNA targeting moiety is or includes catalytically inactive Cas, for example, dCas. Many catalytically inactive Cas proteins are known in the art. In some embodiments, dCas9 includes mutations in each endonuclease domain of the Cas protein, for example, D10A and H840A mutations.

[0161] In some embodiments, the catalytically inactive Cas9 protein, e.g., dCas9, contains a D11A mutation or similar substitution in the amino acid corresponding to the said position. In some embodiments, the catalytically inactive Cas9 protein, e.g., dCas9, contains an H969A mutation or similar substitution in the amino acid corresponding to the said position. In some embodiments, the catalytically inactive Cas9 protein, e.g., dCas9, contains an N995A mutation or similar substitution in the amino acid corresponding to the said position. In some embodiments, the catalytically inactive Cas9 protein, e.g., dCas9, contains D11A, H969A, and N995A mutations or similar substitutions in the amino acids corresponding to the said position.

[0162] In some embodiments, the catalytically inactive Cas9 protein, e.g., dCas9, contains a D10A mutation or similar substitution in the amino acid corresponding to the said position. In some embodiments, the catalytically inactive Cas9 protein, e.g., dCas9, contains an H557A mutation or similar substitution in the amino acid corresponding to the said position. In some embodiments, the catalytically inactive Cas9 protein, e.g., dCas9, contains D10A and H557A mutations or similar substitutions in the amino acids corresponding to the said position.

[0163] In some embodiments, the catalytically inactive Cas9 protein, e.g., dCas9, contains a D839A mutation or similar substitution in the amino acid corresponding to the aforementioned position. In some embodiments, the catalytically inactive Cas9 protein, e.g., dCas9, contains an H840A mutation or similar substitution in the amino acid corresponding to the aforementioned position. In some embodiments, the catalytically inactive Cas9 protein, e.g., dCas9, contains an N863A mutation or similar substitution in the amino acid corresponding to the aforementioned position. In some embodiments, the catalytically inactive Cas9 protein, e.g., dCas9, contains D10A, D839A, H840A, and N863A mutations or similar substitutions in the amino acids corresponding to the aforementioned positions.

[0164] In some embodiments, the catalytically inactive Cas9 protein, e.g., dCas9, contains an E993A mutation or similar substitution at the amino acid corresponding to the aforementioned position.

[0165] In some embodiments, the catalytically inactive Cas9 protein, e.g., dCas9, contains a D917A mutation or similar substitution in the amino acid corresponding to the said position. In some embodiments, the catalytically inactive Cas9 protein, e.g., dCas9, contains an E1006A mutation or similar substitution in the amino acid corresponding to the said position. In some embodiments, the catalytically inactive Cas9 protein, e.g., dCas9, contains a D1255A mutation or similar substitution in the amino acid corresponding to the said position. In some embodiments, the catalytically inactive Cas9 protein, e.g., dCas9, contains D917A, E1006A, and D1255A mutations or similar substitutions in the amino acids corresponding to the said position.

[0166] In some embodiments, the catalytically inactive Cas9 protein, e.g., dCas9, contains a D16A mutation or similar substitution in the amino acid corresponding to the said position. In some embodiments, the catalytically inactive Cas9 protein, e.g., dCas9, contains a D587A mutation or similar substitution in the amino acid corresponding to the said position. In some embodiments, the catalytically inactive Cas9 protein, e.g., dCas9, contains an H588A mutation or similar substitution in the amino acid corresponding to the said position. In some embodiments, the catalytically inactive Cas9 protein, e.g., dCas9, contains an N611A mutation or similar substitution in the amino acid corresponding to the said position. In some embodiments, the catalytically inactive Cas9 protein, e.g., dCas9, contains D16A, D587A, H588A, and N611A mutations or similar substitutions in the amino acids corresponding to the said position.

[0167] In another embodiment, the disclosure relates to an expression repressor or polypeptide comprising one or more (e.g., one) DNA targeting moieties and one or more effector domains, wherein the one or more DNA targeting moieties are or comprise a CRISPR / Cas domain comprising a Cas protein, e.g., a catalytically inactive Cas9 protein, e.g., dCas9, or a functional variant or fragment thereof. In some embodiments, dCas9 comprises the amino acid sequence of SEQ ID NO: 26.

[0168] In some embodiments, dCas9 is encoded by the nucleic acid sequence of sequence number 27.

[0169] In some embodiments, the DNA targeting moiety includes a gRNA or a Cas domain linked to it (e.g., covalently linked). The gRNA is a short synthetic RNA consisting of a “scaffold” sequence required for Cas-protein binding and a user-defined targeting sequence of approximately 20 nucleotides for a genomic target. In practice, guide RNA sequences are generally 17–24 nucleotides long (e.g., 19, 20, or 21 nucleotides) and designed to be complementary to the target nucleic acid sequence. Custom gRNA generators and algorithms for designing effective guide RNAs are commercially available. Gene editing has also been achieved using chimeric “single guide RNA” (“sgRNA”), which is an engineered (synthetic) single RNA molecule that mimics the naturally occurring crRNA-tracrRNA complex and contains both tracrRNA (for nuclease binding) and at least one crRNA (for guiding the nuclease to the sequence targeted for editing). Chemically modified sgRNAs have also been shown to be effective for use with Cas proteins; see, for example, Hendel et al. (2015) Nature Biotechnol, 985-991.

[0170] In some embodiments, the gRNA comprises a nucleic acid sequence complementary to the target sequence described herein. In some embodiments, the gRNA comprises a nucleic acid sequence that is at least 90, 95, 99, or 100% complementary to the target sequence described herein. In some embodiments, the gRNA used with a DNA target-directing moiety containing a Cas molecule is an sgRNA.

[0171] TAL domain In some embodiments, the DNA targeting moiety is or includes a TAL effector (sometimes also referred to herein as "TALE") domain. A TAL effector domain, for example, a TAL effector domain that specifically binds to a given 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., located at the N-terminal and / or C-terminal ends of the plurality of TAL effector domains), where each TAL effector repeat recognizes a nucleotide. In some embodiments, a TAL effector protein may comprise a TAL effector domain and, optionally, one or more other domains. Many TAL effector domains are known to those skilled in the art and are commercially available, for example, from Thermo Fisher Scientific.

[0172] TAL effector proteins are naturally occurring effector proteins secreted by numerous bacterial pathogen species, including the plant pathogen Xanthomonas genus. They regulate gene expression in host plants and promote bacterial colonization and survival. Specific binding of TAL effectors is based on a central repeat domain (a repeating variable duovalent domain, RVD domain), which is typically a tandem arrangement of nearly identical repeats of 33 or 34 amino acids.

[0173] The members of the TAL effector family differ primarily in the number and order of repeats. The number of repeats ranges from 1.5 to 33.5, with C-terminal repeats typically being shorter (e.g., about 20 amino acids) and generally referred to as "half-replies." Each repeat in a TAL effector is characterized by a one-base-pair correlation where different types of repeats exhibit different base-pair specificity (one repeat recognizes one base pair on the target gene sequence). Generally, fewer repeats result in weaker protein-DNA interactions. It has been shown that 6.5 repeats are sufficient to achieve reporter gene transcription (Scholze et al., 2010).

[0174] The variability between repeats predominantly occurs at amino acid positions 12 and 13, and is therefore referred to as "hypervariable." As shown in Table 2, which lists exemplary repeat variable duo (RVD) and their correspondence with nucleic acid base targets, this is involved in the specificity of interaction with target DNA promoter sequences.

[0175] [Table 2]

[0176] Therefore, TAL effector repeats can be modified to target specific DNA sequences. Further studies have shown that RVD NK can target G. The target sites of TAL effectors also tend to contain T adjacent to the 5' base targeted by the initial repeat, although the exact mechanism of this recognition is unknown. To date, more than 113 TAL effector sequences are known. Non-exclusive examples of TAL effectors from the genus Xanthomonas include Hax2, Hax3, Hax4, AvrXa7, AvrXa10, and AvrBs3.

[0177] Therefore, in some embodiments, the TAL effector repeat of the TAL effector domain of the present disclosure is any bacterial species (e.g., African strain of Xanthomonas oryzae pv. Oryzae (Yu et al. 2011), Xanthomonas campestris pv. raphani strain 756C, and Xanthomonas oryzae pv. Oryzicola strain BLS256 (Bogdanove et al.) TAL effectors may be derived from Xanthomonas species, such as al. 2011. When used herein, the TAL effector domains relating to this disclosure include an RVD domain and one or more flanking sequences (sequences at the N-terminal and / or C-terminal ends of the RVD domain) from naturally occurring TAL effectors. In some embodiments, this may include more or fewer repeats than the RVD of the naturally occurring TAL effector domain. The TAL effector domains of this disclosure target a given DNA sequence based on the above codes and others known in the art. The TAL effector domain is designed to achieve the following: The number of TAL effector repeats (e.g., monomers or modules) and one or more specific sequences thereof are selected based on the desired DNA target sequence. For example, TAL effector repeats may be removed or added to fit the specific target sequence. In one embodiment, the TAL effector domain of the Disclosure comprises 6.5 to 33.5 TAL effector repeats. In another embodiment, the TAL effector domain of the Disclosure comprises 8 to 33.5 TAL effector repeats, e.g., 10 to 25 TAL effector repeats, e.g., 10 to 14 TAL effector repeats.

[0178] In some embodiments, the TAL effector domain includes TAL effector repeats corresponding to a perfect match with the DNA target sequence. In some embodiments, mismatches between the repeats and target base pairs on the DNA target sequence are tolerated as long as the repressor system, e.g., the repressor containing the TAL effector domain, can still function. Generally, TAL binding affinity is inversely correlated with the number of mismatches. In some embodiments, the TAL effector domain of the repressor of this disclosure contains no more than seven, six, five, four, three, two, or one mismatch with the target DNA sequence, or optionally, no mismatches. While we do not wish to be constrained by theory, generally, the fewer the number of TAL effector repeats in the TAL effector domain, the fewer the number of mismatches that can be tolerated while the repressor or repressor system, e.g., the repressor containing the TAL effector domain, can still function. Binding affinity is thought to depend on the sum of the matching repeat DNA combinations. For example, a TAL effector domain with 25 or more TAL effector repeats may be able to tolerate up to 7 mismatches.

[0179] In addition to the TAL effector repeat, in some embodiments, the TAL effector domain of the present disclosure may include additional sequences derived from naturally occurring TAL effectors. The lengths of one or more C-terminal and / or N-terminal sequences included on either side of the TAL effector repeat portion of the TAL effector domain may vary and can be selected by those skilled in the art, for example, based on the study of Zhang et al. (2011). Zhang et al. characterized several C-terminal and N-terminal truncation mutants of TAL effector-based proteins derived from Hax3 and identified key elements that contribute to optimal binding to target sequences and, consequently, to transcriptional activation. Generally, transcriptional activity was found to be inversely correlated with the length of the N-terminal. With respect to the C-terminal, important elements were identified in DNA-binding residues within the first 68 amino acids of the Hax3 sequence. Thus, in some embodiments, the TAL effector domain of the repressor of the present disclosure includes the first 68 amino acids of the C-terminal TAL effector repeat of a naturally occurring TAL effector. Therefore, in one embodiment, the TAL effector domain of the present disclosure comprises: 1) one or more TAL effector repeats derived from naturally occurring TAL effectors; 2) at least 70, 80, 90, 100, 110, 120, 130, 140, 150, 170, 180, 190, 200, 220, 230, 240, 250, 260, 270, 280 amino acids or more from naturally occurring TAL effectors on the N-end side of the TAL effector repeat; and / or 3) at least 68, 80, 90, 100, 110, 120, 130, 140, 150, 170, 180, 190, 200, 220, 230, 240, 250, 260 amino acids or more from naturally occurring TAL effectors on the C-end side of the TAL effector repeat.

[0180] In some embodiments, the regulatory agent includes a DNA target-directing moiety containing an engineered DNA-binding domain (DBD), for example, a TAL effector containing a TAL effector repeat that binds to a target sequence, for example, a promoter or transcription start site (TSS) sequence operably linked to a target gene, for example, a sequence adjacent to a transcription regulatory element, for example, an anchor sequence of an anchor sequence-mediated conjugate (ASMC) containing the target gene, for example, a sequence adjacent to an anchor sequence. In some embodiments, the TAL effector domain can be engineered to deliver an epigenetic effector domain to a target site.

[0181] Zn finger domain In some embodiments, the DNA targeting moiety is or includes a Zn finger domain. The Zn finger domain includes a Zn finger, e.g., a naturally occurring Zn finger or an engineered Zn finger, or a fragment thereof. Many Zn fingers are known to those skilled in the art and are commercially available, for example, from Sigma-Aldrich. Generally, a Zn finger domain contains multiple Zn fingers, each Zn finger recognizing 3 nucleotides. A Zn finger protein may include a Zn finger domain and, optionally, one or more other domains.

[0182] In some embodiments, the Zn finger molecule comprises a naturally occurring Zn finger protein engineered to bind to a target DNA sequence of choice. For example, Beerli, et al. (2002) Nature Biotechnol. 20:135-141; Pabo, et al. (2001) Ann. Rev. Biochem. 70:313-340; Isalan, et al. (2001) Nature Biotechnol. 19:656-660; Segal, et al. (2001) Curr. Opin. Biotechnol. 12:632-637; Choo, et al. al. (2000) Curr.Opin.Struct.Biol.10:411-416; US Patent No. 6,453,242; US Patent No. 6,534,261; US ​​Patent No. 6,59 Specification No. 9,692; Specification No. 6,503,717; Specification No. 6,689,558; Specification No. 7,030,215; Specification No. 6,794,136; Specification No. 7,067,3 See Specification No. 17; Specification No. 7,262,054; Specification No. 7,070,934; Specification No. 7,361,635; Specification No. 7,253,273; and U.S. Patent Application Publication No. 2005 / 0064474; Specification No. 2007 / 0218528; Specification No. 2005 / 0267061 (all of which are incorporated herein by reference as a whole).

[0183] Manipulated Zn fingers may possess novel binding specificity compared to naturally occurring Zn fingers. Manipulation methods include, but are not limited to, rational design and various selections. Rational design includes, for example, using a database containing triplet (or quadruplet) nucleotide sequences and individual Zn finger amino acid sequences, where each triplet or quadruplet nucleotide sequence is associated with one or more amino acid sequences of zinc fingers that bind to that particular triplet or quadruplet sequence. See, for example, U.S. Patent Nos. 6,453,242 and 6,534,261 (in whole, incorporated herein by reference).

[0184] Exemplary selection methods, including phage displays and two-hybrid systems, are disclosed in U.S. Patent Nos. 5,789,538; 5,925,523; 6,007,988; 6,013,453; 6,410,248; 6,140,466; 6,200,759; and 6,242,568; as well as in International Publications 98 / 37186; 98 / 53057; 00 / 27878; and 01 / 88197 and UK Patent No. 2,338,237. In addition, for example, International Publication 02 / 077227 describes enhanced binding specificity for zinc finger proteins.

[0185] In addition, as disclosed in these and other references, zinc finger and / or multi-finger zinc finger domains may be linked together using any suitable linker sequence, including, for example, linkers of 5 amino acid length or longer. See also U.S. Patent No. 6,479,626; No. 6,903,185; and No. 7,153,949 for exemplary linker sequences of 6 amino acid length or longer. The proteins described herein may include any combination of suitable linkers between the individual zinc fingers of the protein. In addition, enhancement of binding specificity for zinc finger binding domains is also described, for example, in International Publication No. 02 / 077227.

[0186] The design and construction methods for Zn fingers and expression repressors (and the polynucleotides encoding them) are known to those skilled in the art, as seen in U.S. Patent No. 6,140,081, No. 5; No. 789,538; No. 6,453,242; No. 6,534,261; No. 5,925,523; No. 6,007,988; No. 6,013,453; and No. 6,200,759; International Publication No. 95 / 19431; International Publication No. 96 / 0616 Details are provided in Pamphlet No. 6; Pamphlet No. 98 / 53057; Pamphlet No. 98 / 54311; Pamphlet No. 00 / 27878; Pamphlet No. 01 / 60970; Pamphlet No. 01 / 88197; Pamphlet No. 02 / 099084; Pamphlet No. 98 / 53058; Pamphlet No. 98 / 53059; Pamphlet No. 98 / 53060; Pamphlet No. 02 / 016536; and Pamphlet No. 03 / 016496.

[0187] In certain embodiments, the DNA target-directing moiety includes a Zn finger domain containing an engineered zinc finger that binds to a target DNA sequence (in a sequence-specific manner). In some embodiments, the Zn finger domain includes one Zn finger or a fragment thereof. In some embodiments, the Zn finger domain includes multiple Zn fingers (or fragments thereof), e.g., 2, 3, 4, 5, 6 or more Zn fingers (and optionally 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, or 2 or fewer Zn fingers). In some embodiments, the Zn finger domain includes at least 3 Zn fingers. In some embodiments, the Zn finger domain includes 4, 5, or 6 Zn fingers. In some embodiments, the Zn finger domain includes 8, 9, 10, 11, or 12 Zn fingers. In some embodiments, a Zn finger domain containing 3 Zn fingers recognizes a target DNA sequence containing 9 or 10 nucleotides. In some embodiments, a Zn finger domain containing 4 Zn fingers recognizes a target DNA sequence containing 12 to 14 nucleotides. In some embodiments, a Zn finger domain containing six Zn fingers recognizes a target DNA sequence containing 18 to 21 nucleotides.

[0188] In some embodiments, the DNA targeting domain includes a two-handed zinc finger protein. A two-handed zinc finger protein is a protein in which two clusters of zinc fingers are separated by intervening amino acids, so that two zinc finger domains bind to two discontinuous target DNA sequences. An example of a two-handed zinc finger binding protein is SIP1, in which a cluster of four zinc fingers is located at the amino terminus of the protein and a cluster of three zinc fingers is located at the carboxyl terminus (see Remade, et al. (1999) EMBO Journal 18(18):5073-5084). Each cluster of zinc fingers in these domains can bind to its own target sequence, and the space between the two target sequences may contain a large number of nucleotides.

[0189] In some embodiments, the expression repressor includes a DNA target-directing moiety including a Zn finger domain, which contains a Zn finger (ZFN) that binds to an engineered DNA-binding domain (DBD), for example, a sequence adjacent to a target gene, for example, a sequence adjacent to a transcriptional regulatory element, for example, an anchor sequence of an anchor sequence sex-mediated conjugate (ASMC) containing the target gene, for example, a target sequence operably linked to a sequence adjacent to an anchor sequence, for example, a promoter or transcription start site (TSS) sequence. In some embodiments, the ZFN can be engineered to deliver an epigenetic effector molecule to the target site.

[0190] Effector Domain In some embodiments, the expression repressors of the Disclosure comprise one or more effector domains. In some embodiments, the effector domains, when used as part of an expression repressor or expression repression system described herein, reduce the expression of a target gene in a cell.

[0191] In some embodiments, the effector domain has a function independent of the binding of the DNA targeting moiety. For example, the effector domain may target a genomic sequence element or genomic complex component that is adjacent to the genomic sequence element targeted by the DNA targeting moiety, for example, by binding to it or by recruiting a transcription factor. As a further example, the effector domain may include enzymatic activity, such as gene modification function.

[0192] In some embodiments, the effector domain includes a transcriptional repressor moiety. In some embodiments, the effector domain includes a DNA modification function, such as a DNA methyltransferase. In some embodiments, the effector domain includes a polypeptide that increases DNA methylation. In some embodiments, the effector domain includes a polypeptide that induces DNA methylation of CpG islands (i.e., regions in the genome containing high concentrations of CpG residues). In some embodiments, the effector domain includes a DNA methyltransferase enzyme (DNMT). In some embodiments, the effector domain includes a polypeptide that forms a complex for epigenetic modification. In some embodiments, the polypeptide forms a complex that increases DNA methylation. In some embodiments, the effector domain promotes DNA methylation, for example, directly or indirectly. For example, the effector domain can indirectly promote DNA methylation by recruiting endogenous proteins that methylate DNA. In some embodiments, the effector domain directly promotes DNA methylation by catalyzing the transfer of a methyl group from cytosine to C5.

[0193] In some embodiments, the effector domain is or comprises a protein selected from MQ1, DNMT1, DNMT3A1, DNMT3A2, DNMT3B1, DNMT3B2, DNMT3B3, DNMT3B4, DNMT3B5, DNMT3B6, DNMT3L, or any functional variant or fragment thereof.

[0194] In some embodiments, the effector domain is a protein selected from or comprising a polypeptide of a sequence having at least 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% identity with any of the above referenced sequences, including DNMT3A (e.g., human DNMT3A) (e.g., the protein encoded by NP_072046.2 or NM_022552.4); DNMT3B (e.g., the protein encoded by NP_008823.1 or NM_006892.4); DNMT3L (e.g., the protein encoded by NP_787063.1 or NM_175867.3); DNMT3A / 3L complex; bacterial MQ1 (e.g., the protein encoded by CAA35058.1 or P15840.3); any of these functional fragments; or a polypeptide of a sequence having at least 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% identity with any of the above referenced sequences.

[0195] In another embodiment, the disclosure relates to an expression repressor or polypeptide comprising one or more (e.g., one) DNA targeting moieties and one or more effector domains, wherein the one or more effector domains are or comprise MQ1, e.g., bacterial MQ1, or a functional variant or fragment thereof. In some embodiments, MQ1 is Spiroplasma MQ1 of the class Mollicutes. In some embodiments, MQ1 is Spiroplasma monobiae MQ1. In some embodiments, MQ1 is MQ1 derived from strain ATCC 33825 and / or MQ1 corresponding to Uniprot ID P15840. In some embodiments, MQ1 comprises the amino acid sequence of SEQ ID NO: 2. In some embodiments, MQ1 comprises the amino acid sequence of SEQ ID NO: 30. In some embodiments, the effector domains described herein include sequence number 29 or 31, or sequences that are at least 80, 85, 90, 95, 99, or 100% identical thereto, or sequences that have no more than 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 different positions.

[0196] In some embodiments, MQ1 is encoded by the nucleotide sequence of SEQ ID NO: 29 or 31. In some embodiments, the nucleic acids described herein include the sequence of SEQ ID NO: 98, 100 or a sequence of at least 80, 85, 90, 95, 99, or 100% identity therewith, or a sequence in which the number of positions different therefrom does not exceed 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1.

[0197] In some embodiments, the MQ1 used in the polypeptide or expression repressor described herein is a mutant containing, for example, one or more mutations compared to wild-type MQ1 (e.g., SEQ ID NO: 2). In some embodiments, the MQ1 mutant contains one or more amino acid substitutions, deletions, or insertions compared to wild-type MQ1, for example, MQ1 of SEQ ID NO: 28. In some embodiments, the MQ1 mutant contains a K297P substitution. In some embodiments, the MQ1 mutant contains an N299C substitution. In some embodiments, the MQ1 mutant contains an E301Y substitution. In some embodiments, the MQ1 mutant contains a Q147L substitution (e.g., and has reduced DNA methyltransferase activity compared to wild-type MQ1). In some embodiments, the MQ1 mutant contains K297P, N299C, and E301Y substitutions (e.g., and has reduced DNA binding affinity compared to wild-type MQ1). In some embodiments, MQ1 mutants include Q147L, K297P, N299C, and E301Y substitutions (for example, with reduced DNA methyltransferase activity and DNA binding affinity compared to wild-type MQ1).

[0198] In some embodiments, the polypeptide or repressor is a fusion protein comprising MQ1 or an effector domain containing it, and a zinc finger domain, TAL domain, or CRISPR / Cas domain, dCas9 domain, or a DNA target-directing moiety containing it. In some embodiments, the polypeptide or repressor includes additional moieties as described herein. In some embodiments, the polypeptide or repressor reduces the expression of a target gene. In some embodiments, the polypeptide or repressor may be used in methods of regulating gene expression, e.g., reducing it, treating a disease, or epigenetically modifying a target gene or transcriptional regulatory element as described herein, for example, instead of a repressor system. In some embodiments, the repressor system comprises two or more (e.g., two, three, or four) repressors, the first repressor comprising an effector domain containing MQ1, e.g., bacterial MQ1, or a functional variant or fragment thereof.

[0199] In another embodiment, the disclosure relates to an expression repressor or polypeptide comprising one or more (e.g., one) DNA targeting moieties and one or more effector domains, wherein the one or more effector domains are or comprise DNMT1, e.g., human DNMT1, or a functional variant or fragment thereof. In some embodiments, DNMT1 is human DNMT1, e.g., human DNMT1 corresponding to gene ID 1786, e.g., human DNMT1 corresponding to UniProt ID P26358.2. In some embodiments, DNMT1 comprises the amino acid sequence of SEQ ID NO: 103. In some embodiments, the effector domains described herein comprise the sequence relating to SEQ ID NO: 103 or a sequence of at least 80, 85, 90, 95, 99, or 100% identity thereto, or a sequence 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 different positions therefrom.

[0200] In some embodiments, DNMT1 is encoded by the nucleotide sequence of SEQ ID NO: 104. In some embodiments, the nucleic acids described herein include the sequence of SEQ ID NO: 104 or a sequence that is at least 80, 85, 90, 95, 99, or 100% identical thereto, or a sequence in which the number of positions different therefrom does not exceed 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1.

[0201] In some embodiments, the DNMT1 used in the polypeptide or repressor described herein is a mutant containing, for example, one or more mutations compared to the DNMT sequence of SEQ ID NO: 32. In some embodiments, the effector domain contains one or more amino acid substitutions, deletions, or insertions compared to wild-type DNMT1. In some embodiments, the polypeptide is a fusion protein comprising a repressor domain containing DNMT1 or the same, and a DNA targeting moiety. In some embodiments, the DNA targeting moiety is or contains a zinc finger domain, a TAL domain, or a CRISPR / Cas domain, for example, a dCas9 domain. In some embodiments, the repressor system comprises two or more (e.g., two, three, or four) repressors, the first repressor comprising an effector domain containing DNMT1 or a functional mutant or fragment thereof.

[0202] In another embodiment, the disclosure relates to an expression repressor or polypeptide comprising one or more (e.g., one) DNA targeting moieties and one or more effector domains, wherein the one or more effector domains are or comprise a DNMT3a / 3L complex, or a functional variant or fragment thereof. In some embodiments, the one or more effector domains are or comprise a DNMT3a / 3L complex fusion construct. In some embodiments, the DNMT3a / 3L complex comprises DNMT3A (e.g., human DNMT3A) (e.g., the protein encoded by NP_072046.2 or NM_022552.4). In some embodiments, the DNMT3a / 3L complex comprises DNMT3L (e.g., the protein encoded by NP_787063.1 or NM_175867.3). In some embodiments, DNMT3a / 3L comprises the amino acid sequence of SEQ ID NO: 34 or SEQ ID NO: 35. In some embodiments, the effector domains described herein include sequence number 34 or sequence number 35, or sequences that are at least 80, 85, 90, 95, 99, or 100% identical thereto, or sequences that have no more than 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 different positions.

[0203] In some embodiments, DNMT3a / 3L is encoded by the nucleotide sequence of SEQ ID NO: 28. In some embodiments, the nucleic acids described herein include the sequence of SEQ ID NO: 28 or a sequence of at least 80, 85, 90, 95, 99, or 100% identity therewith, or a sequence in which the number of positions different therefrom does not exceed 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1.

[0204] In some embodiments, the DNMT3a / 3L used in the polypeptide or repressor described herein is a mutant containing, for example, one or more mutations compared to DNMT3a / 3L of SEQ ID NO: 34 or SEQ ID NO: 35. In some embodiments, the DNMT3a / 3L mutant contains one or more amino acid substitutions, deletions, or insertions compared to SEQ ID NO: 34 or SEQ ID NO: 35. In some embodiments, the polypeptide or repressor is a fusion protein comprising an effector domain containing DNMT3a / 3L or therein, and a DNA targeting moiety. In some embodiments, the DNA targeting moiety is or contains a zinc finger domain, a TAL domain, or a CRISPR / Cas domain, for example, a dCas9 domain. In some embodiments, the repressor system comprises two or more (e.g., 2, 3, or 4) repressors, the first repressor comprising an effector domain containing DNMT3a / 3L or a functional mutant or fragment thereof.

[0205] In some embodiments, a candidate effector domain may be determined to be suitable for use as an effector domain by methods known to those skilled in the art. For example, a candidate effector domain may be tested by assay to determine whether it reduces the expression of a target gene in a cell, for example, by reducing the level of RNA transcript encoded by the target gene (e.g., measured by RNASeq or Northern blotting) or by reducing the level of protein encoded by the target gene (e.g., measured by ELISA), when the candidate effector domain is located in the cell nucleus and is appropriately localized (e.g., by a DNA target-directing moiety, for example, to a target gene or a transcriptional regulatory element operably linked to the target gene).

[0206] In some embodiments, the repressor comprises multiple effector domains, where each effector domain does not bind to another effector domain in a detectable manner, for example, not binding at all. In some embodiments, the repressor system comprises a first repressor comprising a first effector domain and a second repressor comprising a second effector domain, where the first effector domain does not bind to the second effector domain in a detectable manner, for example, not binding at all.

[0207] In some embodiments, the repressor system comprises a plurality of repressors, each member of which comprises an effector domain, where each effector domain does not bind to another effector domain in a detectable manner, for example, it does not bind. In some embodiments, the repressor system comprises a first repressor comprising a first effector domain and a second repressor comprising a second effector domain, where the first effector domain does not bind to the second effector domain in a detectable manner, for example, it does not bind. In some embodiments, the repressor system comprises a first repressor comprising a first effector domain and a second repressor comprising a second effector domain, where the first effector domain does not bind to another first effector domain in a detectable manner, for example, it does not bind, and the second effector domain does not bind to another second effector domain in a detectable manner, for example, it does not bind. In some embodiments, the effector domains used in the compositions and methods described herein function in monomeric form, for example, in non-dimeric form.

[0208] In some embodiments, the effector domain comprises a biologically active fragment of the effector domain. As used herein, “biologically active fragment of the effector domain” refers to a portion (e.g., “minimal” or “core” domain) that maintains the function of the effector domain (e.g., completely, partially, or minimally). In some embodiments, fusion of all or part of one or more effector domains of a DNA methylase or enzyme that plays a role in DNA demethylation (e.g., DNMT3a, DNMT3b, DNMT3L, DNMT inhibitors, or combinations thereof) produces a chimeric protein linked to a polypeptide, which is useful in the methods described herein.

[0209] additional part The expression repressor may further comprise one or more additional segments (e.g., one or more target-directing segments and one or more effector domains). In some embodiments, the additional segments are selected from tagging or monitoring segments, cleavable segments (e.g., cleavable segments located between the DNA target-directing segment and the effector domain or at the N-terminus or C-terminus of a polypeptide), small molecules, membrane transport polypeptides, or drug segments.

[0210] Expression suppressor system In some embodiments, this disclosure provides an expression repression system comprising two or more expression repressors described herein. In some embodiments, the expression repression system comprises two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, or more (and optionally 15, fourteen, thirteen, twelve, eleven, ten, nine, eight, seven, six, five, four, three, or two or fewer) expression repressors.

[0211] In some embodiments, the expression repressor system includes a first expression repressor comprising (i) a first DNA target-directed moiety that binds to a first target sequence as described herein, and (ii) a first effector domain (e.g., DNA methyltransferase); and at least one additional expression repressor comprising (i) a second DNA target-directed moiety that binds to a second target sequence as described herein, and (ii) a second effector domain (e.g., DNA methyltransferase). In some embodiments, the first target sequence is different from the second target sequence.

[0212] In some embodiments, the expression repressor system comprises a first expression repressor comprising (i) a first DNA target-directed moiety that binds to a first target sequence as described herein, and (ii) a first effector domain (e.g., DNA methyltransferase); and at least one additional expression repressor comprising (i) a second DNA target-directed moiety that binds to a second target sequence as described herein, and (ii) a second effector domain (e.g., DNA methyltransferase), wherein the first target sequence is different from the second target sequence. In some embodiments, the first effector domain is the same as the second effector domain. In some embodiments, the first effector domain is different from the second effector domain.

[0213] In some embodiments, the expression repressor system includes at least 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 additional expression repressors.

[0214] In some embodiments, the repressor system comprises a first repressor comprising (i) a first DNA target-directed moiety that binds to a first target sequence as described herein, and (ii) a first effector domain (e.g., DNA methyltransferase); and at least 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 additional repressors, each of which comprises (i) a DNA target-directed moiety that binds to a target sequence as described herein, and (ii) an effector domain (e.g., DNA methyltransferase), and the target sequences of each additional repressor are different from and different from the first target sequence. In some embodiments, the effector domain of each additional repressor is the same as or different from the first effector domain.

[0215] In some embodiments, each of the repressors in the repressor system binds to another target sequence described herein.

[0216] In some embodiments, each of the repressors in the repressor system is formulated in the same composition. In some embodiments, each of the repressors in the repressor system is formulated in different compositions.

[0217] In some embodiments, the repressors in the repressor system each contain a different DNA target-directed moiety (e.g., the first, second, third, or further repressors each contain a different DNA target-directed moiety). For example, the repressor system may include a first repressor and a second repressor, where the first repressor contains a first target-directed moiety (e.g., a Cas9 domain, a TAL effector domain, or a Zn finger domain), and the second repressor contains a second target-directed moiety different from the first target-directed moiety (e.g., a Cas9 domain, a TAL effector domain, or a Zn finger domain). In some embodiments, "different" means containing distinct types of target-directed moieties, for example, the first target-directed moiety contains a Cas9 domain and the second DNA target-directed moiety contains a Zn finger domain. In some embodiments, "different" means including distinct variants of the same type of target-directing moiety, for example, the first target-directing moiety includes a first Cas9 domain (e.g., of the first species) and the second target-directing moiety includes a second Cas9 domain (e.g., of the second species). In some embodiments, when the repressor system includes two or more target-directing moieties of the same type, for example, two or more Cas9 or ZF domains, the target-directing moieties specifically bind to two or more different target sequences. For example, in a repressor system including two or more Cas9 domains, the two or more Cas9 domains may be selected or modified so that they bind only to the gRNA corresponding to their target sequence (e.g., and do not show sufficient binding to the gRNA corresponding to the target of another Cas9 domain). In a further example, in an expression repressor system comprising two or more effector moieties, the two or more effector moieties may be selected or modified so that they bind only to their own target sequences (for example, and do not show sufficient binding to the target sequences of other effector moieties).

[0218] In some embodiments, the expression repressor system comprises three or more expression repressors, where two or more of the expression repressors include the same DNA target-directed moiety. For example, the expression repressor system may include three expression repressors, where the first and second expression repressors both include a first DNA target-directed moiety, and the third expression repressor includes a second different DNA target-directed moiety. As a further example, the expression repressor system may include four expression repressors, where the first and second expression repressors both include a first DNA target-directed moiety, and the third and fourth expression repressors include a second different DNA target-directed moiety. As a further example, the expression repressor system may include five expression repressors, where the first and second expression repressors both include a first DNA target-directed moiety, the third and fourth expression repressors both include a second different DNA target-directed moiety, and the fifth expression repressor includes a third different DNA target-directed moiety. As stated above, "different" may mean containing different types of DNA target-directed regions, or containing distinct variants of the same type of target-directed region.

[0219] In some embodiments, the repressors in an expression repressor system each bind to a different target sequence as described herein (for example, the first, second, third, or further repressors each bind to a different DNA sequence). For example, the expression repressor system may include a first repressor and a second repressor, where the first repressor binds to a first target sequence as described herein, and the second repressor binds to a second target sequence as described herein. In some embodiments, "different" may mean that there is at least one non-identical position between the target sequence to which one repressor binds and the target sequence to which another repressor binds, or that at least one position present in the target sequence to which one repressor binds is not present in the target sequence to which another repressor binds.

[0220] In some embodiments, each repressor in the repressor system contains a different effector domain (for example, the first, second, third, or further repressors each contain different effector portions). In some embodiments, the first effector domain is a DNA methyltransferase as described herein, and the effector domain of each additional repressor in the repressor system is the same as or different from the first effector domain. In some embodiments, the first effector domain includes a DNA methyltransferase as described herein, or a functional fragment or variant thereof, and the effector domain of each additional repressor in the repressor system includes a DNA methyltransferase, or a functional fragment or variant thereof, or a histone modifying enzyme, or a functional fragment or variant thereof. In some embodiments, the histone modifying enzyme is selected from histone methyltransferase, histone deacetylase, and histone demethylase.

[0221] In some embodiments, the first effector domain comprises a DNA methyltransferase described herein, or a functional fragment or variant thereof, and each additional effector domain of the repressor system 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, Selected from SIRT3, SIRT4, SIRT5, SIRT6, SIRT7, SIRT8, SIRT9, KDM1A (i.e., LSD1), KDM1B (i.e., LSD2), KDM2A, KDM2B, KDM5A, KDM5B, KDM5C, KDM5D, KDM4B, NO66, SETDB1, SETDB2, EHMT2 (i.e., G9A), EHMT1 (i.e., GLP), SUV39H1, EZH2, EZH1, SUV39H2, SETD8, SUV420H1, SUV420H2, KRAB, MeCP2, HP1, RBBP4, REST, FOG1, SUZ12 and their functional variants or fragments.

[0222] In some embodiments, the first effector domain includes DNA methyltransferase activity (e.g., MQ1, DNMT1, DNMT3A1, DNMT3A2, DNMT3B1, DNMT3B2, DNMT3B3, DNMT3B4, DNMT3B5, DNMT3B6, DNMT3L, or any functional variant or fragment thereof), and the effector domain of each additional repressor in the repressor system includes DNA methyltransferase activity (e.g., MQ1, DNMT1, DNMT3A1, DNMT3A2, DNMT3B1, DNMT3B2, DNMT3B3, DNMT3B4, DNMT3B5, DNMT3B6, DNMT3L, or any functional variant or fragment thereof), and transcription repressor activity (e.g., KRAB, MeCP2, HP1, R The effector domain of each additional repressor is one of those described in International Publication No. 2022 / 132195; International Publication No. 2022 / 067033; or U.S. Patent No. 11,312,955 (as incorporated herein by reference).

[0223] In some embodiments, two or more (e.g., all) of the repressors in the repressor system are not covalently associated with each other, for example, each repressor is not covalently associated with any other repressor. In other embodiments, two or more of the repressors in the repressor system are covalently associated with each other. In some embodiments, the repressor system includes a first and second repressor located on the same polypeptide, for example, as a fusion molecule, linked by a peptide bond and optionally by a linker. In some embodiments, the peptide is a self-cleaving peptide, for example, a T2A self-cleaving peptide. In some embodiments, the repressor system includes a first and second repressor linked by a non-peptide bond, for example, conjugated to each other.

[0224] Method for creating expression suppressors In some embodiments, the proteins or polypeptides of the compositions of this disclosure may be biochemically synthesized using standard solid-phase methods. Such methods include exclusionary solid-phase synthesis, partial solid-phase synthesis, fragment condensation, and classical solution synthesis. These methods can be used when the peptide is relatively short (e.g., 10 kDa) and / or cannot be produced by recombinant techniques (i.e., not encoded by nucleic acid sequence), and therefore different chemistry is involved.

[0225] Solid-phase synthesis procedures are well known in the art and are described in detail by John Morrow Stewart and Janis Dillaha Young, Solid Phase Peptide Syntheses, 2nd Ed., Pierce Chemical Company, 1984; and Coin, I., et al., Nature Protocols, 2:3247-3256, 2007.

[0226] Recombination methods can be used for long peptides. Methods for creating recombinant therapeutic polypeptides are common in the art. See, for example, Smales & James (Eds.), Therapeutic Proteins: Methods and Protocols (Methods in Molecular Biology), Humana Press (2005); and Crommelin, Sindelar & Meibohm (Eds.), Pharmaceutical Biotechnology: Fundamentals and Applications, Springer (2013).

[0227] The exemplary methods for producing expression repressors or polypeptides described herein involve expression in mammalian cells; however, recombinant proteins can also be produced using insect cells, yeast, bacteria, or other cells under the control of a suitable promoter. Mammalian expression vectors may include non-transcription elements such as a replication origin, a suitable promoter, and other 5' or 3' flanking non-transcription sequences, as well as, optionally, 5' or 3' untranslated sequences such as ribosome binding sites, polyadenylation sites, splice donor / receptor sites, and termination sequences. DNA sequences derived from the SV40 viral genome, e.g., the SV40 origin, initial promoter, splice, and polyadenylation sites, may be used to provide other genetic elements necessary for the expression of heterologous DNA sequences. Cloning and expression vectors suitable for use in bacterial, fungal, yeast, and mammalian cell hosts are described in Green & Sambrook, Molecular Cloning: A Laboratory Manual (Fourth Edition), Cold Spring Harbor Laboratory Press (2012).

[0228] In some embodiments, large quantities of expression repressors or polypeptides are desired, which can be generated using techniques such as those described in Brian Bray, Nature Reviews Drug Discovery, 2:587-593, 2003; and Weissbach & Weissbach, 1988, Methods for Plant Molecular Biology, Academic Press, NY, Section VIII, pp 421-463.

[0229] Various mammalian cell culture systems can be used for the expression and production of recombinant proteins. Examples of mammalian expression systems include, without limitation, CHO cells, COS cells, HeLA and BHK cell lines. For host cell culture methods for producing protein therapeutics, see, for example, Zhou and Kantardjieff (Eds.), Mammalian Cell Cultures for Biologies Manufacturing (Advances in Biochemical Engineering / Biotechnology), Springer (2014). The compositions described herein may include vectors encoding recombinant proteins, such as viral vectors, e.g., lentiviral vectors. In some embodiments, the vector, e.g., viral vector, may contain nucleic acids encoding recombinant proteins. The compositions described herein may include lipid nanoparticles encapsulating vectors encoding recombinant proteins, such as viral vectors, e.g., lentiviral vectors. In some embodiments, the lipid nanoparticles encapsulating the vector, e.g., viral vector, may contain nucleic acids encoding recombinant proteins.

[0230] The purification of protein-based therapeutics is described, for example, in Franks, Protein Biotechnology: Isolation, Characterization, and Stabilization, Humana Press (2013); and Cutler, Protein Purification Protocols (Methods in Molecular Biology), Humana Press (2010). The formulation of protein-based therapeutics is described in Meyer (Ed.), Therapeutic Protein Drug Products: Practical Approaches to Formulation in the Laboratory, Manufacturing, and the Clinic, Woodhead Publishing Series (2012).

[0231] Proteins contain one or more amino acids. Amino acids include any compounds and / or substances that can be incorporated into a polypeptide chain, for example, through the formation of one or more peptide bonds. In some embodiments, amino acids have a general structure H2N-C(H)I-COOH. In some embodiments, amino acids are naturally occurring amino acids. In some embodiments, amino acids are non-natural amino acids; in some embodiments, amino acids are D-amino acids; in some embodiments, amino acids are L-amino acids. "Standard amino acid" refers to any of the 20 standard L-amino acids commonly found in naturally occurring peptides. "Non-standard amino acid" refers to any amino acid other than a standard amino acid, whether it is synthetically prepared or obtained from a natural source. In some embodiments, amino acids may include carboxyl-end and / or amino-end amino acids in a polypeptide, and may include structural modifications compared to the general structure described above. For example, in some embodiments, amino acids may be modified and / or substituted (e.g., amino groups, carboxylic acid groups, one or more protons, and / or hydroxyl groups) by methylation, amidation, acetylation, pegylation, glycosylation, phosphorylation, and / or substitution compared to the general structure. In some embodiments, such modifications may alter the cyclic half-life of the polypeptide containing the modified amino acid, for example, compared to one containing an otherwise identical unmodified amino acid. In some embodiments, such modifications do not significantly alter the relevant activity of the polypeptide containing the modified amino acid, compared to one containing an otherwise identical modified amino acid. As will be apparent from the context, in some embodiments, the term “amino acid” may refer to a free amino acid; in some embodiments, it may refer to an amino acid residue of a polypeptide.

[0232] Nucleic acids of this disclosure In another embodiment, this specification provides nucleic acids encoding repressors or repressor systems of the Disclosure. In some embodiments, the repressors may be provided by a composition comprising nucleic acids encoding the repressors, wherein the nucleic acids associate with other sequences sufficient to achieve expression in the system of interest (e.g., in specific cells, tissues, organisms, etc.).

[0233] In some embodiments, the Disclosure provides compositions of nucleic acids encoding repressors or fragments thereof. In some embodiments, the nucleic acid may be DNA, RNA, or any other nucleic acid moiety or entity as described herein, or may include such, and may be prepared by any technique as described herein or otherwise available in the art (e.g., synthesis, cloning, amplification, in vitro or in vivo transcription, etc.). In some embodiments, the nucleic acids encoding the repressors or fragments thereof provided are operably associated with one or more replication, replication, and / or expression signals appropriate and / or sufficient to achieve the integration, replication, and / or expression of the provided nucleic acid in a system of interest (e.g., in a particular cell, tissue, organism, etc.).

[0234] In some embodiments, the composition for delivering the repressor or repressor system described herein is a vector, for example, a viral vector, comprising one or more nucleic acids encoding one or more components of the repressor or repressor as described herein.

[0235] In some embodiments, the Disclosure provides compositions of nucleic acids encoding an expression repressor, one or more expression repressors, or fragments thereof. In some embodiments, the nucleic acids provided may be, or include, DNA, RNA, or any other nucleic acid moieties or entities as described herein, and may be prepared by any technique as described herein or otherwise available in the art (e.g., synthesis, cloning, amplification, in vitro or in vivo transcription). Examples of nucleic acid sequences include, without limitation, DNA, RNA, modified oligonucleotides (e.g., chemically modified, such as modifications altering the backbone, sugar molecules, and / or nucleic acid bases), and artificial nucleic acids. In some embodiments, examples of nucleic acid sequences include, without limitation, genomic DNA, cDNA, peptide nucleic acid (PNA) or peptide oligonucleotide conjugates, locked nucleic acid (LNA), cross-linked nucleic acid (BNA), polyamide, triple-stranded oligonucleotide, modified DNA, antisense DNA oligonucleotide, tRNA, mRNA, rRNA, modified RNA, miRNA, gRNA, and siRNA or other RNA or DNA molecules. In some embodiments, a nucleic acid provided encoding an expression repressor, one or more expression repressors, or a polypeptide fragment thereof is operably associated with one or more replication, integration, and / or expression signals appropriate and / or sufficient to achieve integration, replication, and / or expression of the provided nucleic acid in a system of interest (e.g., in a particular cell, tissue, organism, etc.).

[0236] In some embodiments, nucleic acid sequences are approximately 2 to 5000 nt in length, approximately 10 to 100 nt, approximately 50 to 150 nt, approximately 100 to 200 nt, approximately 150 to 250 nt, approximately 200 to 300 nt, approximately 250 to 350 nt, approximately 300 to 500 nt, approximately 10 to 1000 nt, approximately 50 to 1000 nt, approximately 100 to 1000 nt, approximately 1000 to 2000 nt, approximately 2000 to 3000 nt, approximately 3000 to 4000 nt, approximately 4000 to 5000 nt, or any range in between.

[0237] In some embodiments, the composition for delivering the repressor or repressor system described herein is an RNA, for example mRNA, containing one or more nucleic acids encoding the repressor as described herein.

[0238] In some embodiments, the nucleic acids of this disclosure include nucleosides, e.g., purines or pyrimidines, e.g., adenine, cytosine, guanine, thymine, and uracil. In some embodiments, the nucleic acid sequence includes one or more nucleoside analogs. Nucleoside analogs include, but are not limited to, 5-fluorouracil; 5-bromouracil, 5-chlorouracil, 5-iodouracil, hypoxanthine, xanthine, 4-acetylcytosine, 4-methylbenzimidazole, 5-(carboxyhydroxylmethyl)uracil, 5-carboxymethylaminomethyl-2-thiouridine, 5-carboxymethylaminomethyluracil, dihydrouracil, dihydrouridine, β-D-ga Lactosylqueosin (β-D-galactosylqueosin), inosine, N6-isopentenyladenine, 1-methylguanine, 1-methylinosine, 2,2-dimethylguanine, 2-methyladenine, 2-methylguanine, 3-methylcytosine, 5-methylcytosine, N6-adenine, 7-methylguanine, 5-methylaminomethyluracil, 5-methoxyaminomethyl-2-thiouracil, β-D-mannosylqueosin ((β-D-man (nosylqueosine), 5'-methoxycarboxymethyluracil, 5-methoxyuracil, 2-methylthio-N6-isopentenyladenine, uracil-5-oxyacetic acid (v), weibtoxosin, pseudouracil, queosine, 2-thiocytosine, 5-methyl-2-thiouracil, 2-thiouracil, 4-thiouracil, 5-methyluracil, uracil-5-oxyacetic acid methyl ester, uracil-5-oxyacetic acid (v), 5 This includes methyl-2-thiouracil, 3-(3-amino-3-N-2-carboxypropyl)uracil, (acp3)w, 2,6-diaminopurine, 3-nitropyrrole, inosine, thiouridine, quosin, waiosin, diaminopurine, isoguanine, isocytosine, diaminopyrimidine, 2,4-difluorotoluene, isoquinoline, pyrrolo[2,3-β]pyridine, and any other substances that can base pair with a purine or pyrimidine side chain.Additional qualifications are publicly known and are described, for example, in International Publication No. 2012 / 019168; International Publication No. 2015 / 038892; International Publication No. 2015 / 038892; International Publication No. 2015 / 089511; International Publication No. 2015 / 196130; International Publication No. 2015 / 196118; and International Publication No. 2015 / 196128.

[0239] mRNA In one embodiment, the Specified Public Service provides RNA, e.g., mRNA, that encodes an expression repressor or expression repressor system as described herein. In some embodiments, the mRNA includes an open reading frame (ORF), e.g., a codon sequence that can be translated into a peptide or protein, e.g., an expression repressor or expression repressor system.

[0240] Open Reading Frame (ORF) An open reading frame (ORF) includes a start codon at its 5' end and a subsequent nucleotide region, typically in multiples of three nucleotides in length. In some embodiments, the ORF ends with a stop codon (e.g., TAA, TAG, or TGA). In certain embodiments, the ORF may be isolated or incorporated into a longer nucleic acid sequence, such as a vector or mRNA. ORFs are also known in the art as protein-coding regions.

[0241] In some embodiments, the mRNA of this disclosure includes, for example, an ORF encoding a DNA targeting portion and / or effector domain of an expression repressor or expression repressor system described herein. In certain embodiments, the ORF includes a sequence-optimized sequence. The sequence-optimized nucleotide sequences disclosed herein are distinctly different from the corresponding wild-type nucleotide acid sequences and other known optimized nucleotide sequences, for example, these sequence-optimized nucleic acids have unique compositional characteristics.

[0242] In some embodiments, mRNA comprises bicistronic RNA. As used herein, bicistronic RNA is typically RNA containing two ORFs, preferably mRNA. In some embodiments, mRNA comprises polycistronic RNA. As used herein, polycistronic RNA is typically RNA containing more than two ORFs, preferably mRNA.

[0243] In some embodiments, the nucleic acid encoding the repressor system is a polycistronic sequence. In some embodiments, the polycistronic sequence is a bicistronic sequence. In some embodiments, the polycistronic sequence includes a sequence encoding a first repressor and a sequence encoding a second repressor. In some embodiments, the polycistronic sequence encodes a self-cleavable peptide sequence, such as a 2A peptide sequence, such as a T2A peptide sequence or a P2A sequence. In some embodiments, the polycistronic sequence encodes a T2A peptide sequence and a P2A peptide sequence.

[0244] In some embodiments, the bisistronic construct further includes a poly-A tail. In some embodiments, during transcription of the bisistronic gene construct, a single mRNA transcript encoding a first repressor and a second repressor is produced, which, during translation, is cleaved, for example, after a glycine residue within the range of the 2A peptide, producing the first and second repressors as two separate proteins. In some embodiments, the first and second repressors are separated by "ribosome skipping". In some embodiments, the first and / or second repressors retain fragments of the 2A peptide even after ribosome skipping. In some embodiments, the expression levels of the first and second repressors are equal. In some embodiments, the expression levels of the first and second repressors are different. In some embodiments, the protein level of the first repressor is within approximately 1%, 2%, 5%, or 10% (greater or less) of the protein level of the second repressor.

[0245] In some embodiments, a system encoded by a bicistronic nucleic acid reduces the expression of target genes in cells by at least 1%, at least 2%, at least 3%, at least 4%, at least 5%, at least 10%, at least 20%, at least 30%, at least 40%, or at least 50% compared to a otherwise similar system in which the first and second expression repressors are encoded by monocistronic nucleic acids.

[0246] Untranslated area (UTR) In certain embodiments, a polynucleotide (e.g., mRNA) encoding a repressor or repressor system of the present disclosure further includes a 5'UTR and / or a translation initiation sequence. The natural 5'UTR has features that function in the initiation of protein translation. It contains a signature, e.g., a Kozak sequence commonly involved in ribosome-mediated translation initiation of many genes. The 5'UTR may also form secondary structures that function to further facilitate translation by binding to elongation factors. Those skilled in the art will recognize that manipulating these features can enhance the stability and protein production of the polynucleotides of the present disclosure. Untranslated regions useful in the design and manufacture of polynucleotides include, for example, those disclosed without limitation in International Publication No. 2014 / 164253 (see also U.S. Patent Application Publication No. 2016 / 0022840).

[0247] Other non-UTR sequences may be used as regions or subregions within a polynucleotide. For example, introns or fragments of intron sequences may be incorporated into a polynucleotide region without limitation. In some embodiments, the incorporation of one or more intron sequences may increase protein production and / or polynucleotide levels.

[0248] A combination of features may be included in adjacent regions, and the combination may be contained within other features. For example, an ORF may be adjacent to a 5'UTR which may contain a potent Kozak translation initiation signal, and / or a 3'UTR which may contain an oligo(dT) sequence for template addition of a polyA tail. The 5'UTR may contain a first polynucleotide fragment and a second polynucleotide fragment from the same and / or different genes, such as the 5'UTR described in U.S. Patent Application Publication No. 2010 / 0293625.

[0249] The UTR, or fragment thereof, may be oriented in the same orientation as in the original transcript from which it was selected, or its orientation and / or position may be altered. For example, a 5' or 3' UTR may be inverted, shortened, lengthened, or made up with one or more other 5' UTRs or 3' UTRs. In some embodiments, the UTR sequence may be modified in any way compared to a reference sequence, e.g., an endogenous UTR. For example, a 3' or 5' UTR may be altered in terms of orientation or position compared to a wild-type or native UTR by including additional nucleotides, nucleotide deletions, or nucleotide swapping or transposition.

[0250] In some embodiments, two copies of the same UTR are coded in either series or substantially series. In some embodiments, more than two copies of the same UTR are coded in either series or substantially series.

[0251] In some embodiments, adjacent regions, for example, adjacent ORFs, may be heterogeneous. In some embodiments, the 5' untranslated region may originate from a different species than the 3' untranslated region. The untranslated region may also include translation enhancer elements (TEEs). For example, TEEs are described, without limitation, in U.S. Patent Application Publication No. 2009 / 0226470.

[0252] In certain embodiments, the polynucleotide (e.g., mRNA) encoding a repressor or repressor system further comprises a 3'UTR. The 3'-UTR is the segment of mRNA immediately following the translation termination codon. In some embodiments, the 3'-UTR contains a regulatory region that affects gene expression post-transcriptionally. Such regulatory regions within the 3'-UTR may affect the polyadenylation, translation efficiency, localization, and / or stability of the mRNA. In some embodiments, the 3'-UTR contains a binding site for a regulatory protein and / or microRNA. In some embodiments, the 3'-UTR has a silencer region that binds to a repressor protein to inhibit mRNA expression. In other embodiments, the 3'-UTR comprises an AU-rich element (ARE). The protein may bind to the ARE to affect the stability and / or decay rate of the mRNA. In some embodiments, the 3'-UTR contains the sequence of SEQ ID NO: 54, which leads to the addition of an adenine residue in the poly(A) tail to the end of the mRNA transcript.

[0253] terminal modification In some embodiments, the mRNA described herein includes one or more terminal modifications, such as a 5' cap structure and / or a poly-A tail (e.g., 100-200 nucleotides long). The 5' cap structure may be selected from the group consisting of CapO, Capl, ARCA, inosine, Nl-methyl-guanosine, 2'-fluoro-guanosine, 7-deaza-guanosine, 8-oxo-guanosine, 2-amino-guanosine, LNA-guanosine, and 2-azido-guanosine. In some cases, the modified RNA also includes a 5'UTR and a 3'UTR containing at least one Kozak sequence. Such modifications are known and are described, for example, in International Publication No. 2012 / 135805 and International Publication No. 2013 / 052523. Additional terminal modifications are described, for example, in International Publication No. 2014 / 164253, International Publication No. 2016 / 011306, International Publication No. 2012 / 045075, and International Publication No. 2014 / 093924.

[0254] A polynucleotide comprising mRNA encoding an expression repressor or expression repressor system according to the present disclosure may further comprise a 5' cap. The 5' cap can bind to mRNA cap-binding proteins (CBPs), thereby increasing mRNA stability. This cap may further aid in the removal of 5' adjacent introns during mRNA splicing.

[0255] In some embodiments, a polynucleotide comprising mRNA encoding an expression repressor or expression repressor system of the Disclosure includes a non-hydrolyzable cap structure that prevents detachment. In some embodiments, the non-hydrolyzable cap structure increases the half-life of the mRNA. Hydrolysis of the cap structure requires cleavage of the 5'-ppp-5' phosphorodiester bond; thus, modified nucleotides can be used during the capping reaction. Modified guanosine nucleotides, such as α-thio-guanosine, α-methylphosphonic acid, and selenol-phosphate nucleotides, may also be suitable for use in the Disclosure.

[0256] In certain embodiments, the 5' cap comprises 2'-O-methylation of the ribose sugar of the 5'-terminus and / or pre-5'-terminus nucleotide at the 2'-hydroxyl group of the sugar ring. In some embodiments, the cap may include cap analogues that differ chemically from naturally occurring (i.e., endogenous, wild-type, or physiological) 5'-caps, i.e., synthetic cap analogues, chemical caps, chemical cap analogues, or structural / functional cap analogues. The cap analogues may be synthesized chemically (i.e., non-enzymatically) or enzymatically and / or linked to the polynucleotides of the Disclosure.

[0257] In certain embodiments, mRNA encoding an expression repressor or expression repressor system of the present disclosure can be configured to produce a 5'-cap structure by enzymatic capping after production (e.g., IVT or chemical synthesis).

[0258] In certain embodiments, the 5'-terminated cap may include an endogenous cap or a cap analogue. In certain embodiments, the 5'-terminated cap may include a guanine analogue. Suitable guanine analogues include, without limitation, inosine, N1-methyl-guanosine, 2'-fluoro-guanosine, 7-deaza-guanosine, 8-oxo-guanosine, 2-amino-guanosine, LNA-guanosine, and 2-azido-guanosine.

[0259] In some embodiments, the mRNA encoding the repressor or repressor system of the Disclosure further comprises a poly(A) tail. In some embodiments, one or more terminal groups can be incorporated into the poly(A) tail for stabilization. Such poly(A) tail may also contain a structural moiety or a 2'-O-methyl modification, as taught, for example, by Li et al. (2005) Current Biology 15:1501-1507.

[0260] In some embodiments, the poly-A tail, when present, is longer than 30 nucleotides. In some embodiments, the poly-A tail is longer than 35 nucleotides (e.g., at least about 35, 40, 45, 50, 55, 60, 70, 80, 90, 100, 120, 140, 160, 180, 200, 250, 300, 350, 400, 450, 500, 600, 700, 800, 900, 1,000, 1,100, 1,200, 1,300, 1,400, 1,500, 1,600, 1,700, 1,800, 1,900, 2,000, 2,500, or 3,000 nucleotides).

[0261] In some embodiments, the polyA tail is designed to be the length of the entire polynucleotide or the length of a specific region of the polynucleotide. For example, this may be based on the length of the coding region, the length of a specific feature or region, or the length of the product expressed from the polynucleotide. Thus, in some embodiments, the polyA tail may be 10, 20, 30, 40, 50, 60, 70, 80, 90, or 100% longer than the polynucleotide or a fragment thereof.

[0262] In some embodiments, a modified nucleotide at the 3' end of a polyA tail may be used to link one or more polynucleotides together by a polyA-binding protein (PABP) via the 3' end of the PABP.

[0263] In some embodiments, the repressor or mRNA encoding the repressor comprises, is essentially, or consists of a 5' terminal cap, a 5' UTR, an open reading frame (ORF), a 3' UTR, and a poly(A) tail.

[0264] In some embodiments, modified mRNA may be cyclized or concatemerized to generate a translationally competent molecule that facilitates the interaction between the poly(A)-binding protein and the 5'-end binding protein. The mechanism of cyclization or concatemerization may occur through at least three different pathways: 1) a chemical pathway, 2) an enzymatic pathway, and 3) a ribozyme-catalyzed pathway. The newly formed 5'- / 3'- bond may be an intramolecular or intermolecular bond. Such modifications are described, for example, in International Publication No. 2013 / 151736.

[0265] Recombinant expression vector Nucleic acids, or nucleic acids encoding expression repressors or expression repressor systems, as described herein may be incorporated into vectors. Vectors, including those derived from retroviruses such as lentiviruses, are suitable tools for achieving long-term gene transfer, enabling the long-term stable incorporation of transgenes and their transmission to daughter cells. Examples of suitable vectors include expression vectors, replication vectors, probe-generating vectors, and sequencing vectors. In some embodiments, expression vectors may be supplied to cells in the form of viral vectors. Viral vector technology is well known in the art and is described in various virology and molecular biology manuals. Suitable viruses as vectors include, but are not limited to, retroviruses, adenoviruses, adeno-associated viruses, herpesviruses, and lentiviruses. Generally, suitable vectors have a replication origin, promoter sequence, convenient restriction endonuclease site, and one or more selectable markers that are functional in at least one organism.

[0266] The expression of natural or synthetic nucleic acids is typically achieved by operably ligating the nucleic acid encoding the gene of interest to a promoter and incorporating the construct into an expression vector. The vector may be suitable for replication and integration in eukaryotes. A typical cloning vector contains transcription and translation terminators, initiation sequences, and promoters useful for expressing the desired nucleic acid sequence. Additional promoter elements, such as enhancer sequences, can modulate the transcription initiation frequency. Typically, these sequences are located 30–110 bp upstream of the transcription initiation site, although in recent years, several promoters have been shown to also contain functional elements downstream of the transcription initiation site.

[0267] In some embodiments, the expression repressors or expression repressor systems described herein act on enhancer sequences. In some embodiments, the enhancer sequence is an enhancer, stretch enhancer, shadow enhancer, locus regulatory region (LCR), or superenhancer. In some embodiments, the superenhancer comprises a cluster of enhancers and other regulatory elements. In some embodiments, these sequences are located 0.2–2 Mb upstream or downstream of the transcription start site. In some embodiments, this region is a non-coding region. In some embodiments, this region is related to long-range regulation of the target gene. In some embodiments, this region is cell type specific. In some embodiments, the superenhancer modifies (e.g., increases or decreases) target gene expression by recruiting the target gene promoter. In some embodiments, the superenhancer interacts with the target gene promoter through an enhancer docking site. In some embodiments, the enhancer docking site is an anchor sequence. In some embodiments, the enhancer docking site is located at least 100 bp, 200 bp, 500 bp, 1000 bp, 1500 bp, 2000 bp, or 3000 bp away from the target gene promoter. In some embodiments, the super-enhancer region is at least 100 bp, at least 200 bp, at least 300 bp, at least 500 bp, at least 1 kb, at least 2 kb, at least 3 kb, at least 5 kb, at least 10 kb, at least 15 kb, at least 20 kb, or at least 25 kb in length.

[0268] The spacing between promoter elements is often flexible, so that promoter function is maintained even if elements are inverted or move relative to each other. For example, in the thymidine kinase (TK) promoter, activity only begins to decrease when the spacing between promoter elements is increased to about 50 bp. Although we do not wish to be constrained by theory, it is hypothesized that in some promoters, individual elements may function cooperatively or independently in activating transcription.

[0269] One example of a promoter suitable for use in this disclosure is the pre-early cytomegalovirus (CMV) promoter sequence. This promoter sequence is a potent constitutive promoter sequence capable of driving high levels of expression of any polynucleotide sequence operably ligated to it. In some embodiments, a suitable promoter is elongation growth factor-1a (EF-1a). Alternatively, other constitutive promoter sequences may also be used, but are not limited to, the Simian virus 40 (SV40) early promoter, mouse mammary cancer virus (MMTV), human immunodeficiency virus (HIV) long-terminal repeat (LTR) promoter, MoMuLV promoter, avian leukemia virus promoter, Epstein-Barr virus pre-early promoter, Roussarcoma virus promoter, and, but are not limited to, human gene promoters including actin promoters, myosin promoters, hemoglobin promoters, and creatine kinase promoters.

[0270] This disclosure should not be construed as being limited to the use of any particular promoter or category of promoter (e.g., constitutive promoter). For example, in some embodiments, inductive promoters are intended as part of this disclosure. In some embodiments, the use of an inductive promoter provides a molecular switch capable of turning on the expression of a polynucleotide sequence to which it is operably ligated when such expression is desired. In some embodiments, the use of an inductive promoter provides a molecular switch capable of turning off expression when expression is not desired. Examples of inductive promoters include, but are not limited to, metallothionine promoters, glucocorticoid promoters, progesterone promoters, and tetracycline promoters.

[0271] In some embodiments, the expression vector to be introduced may also include either or both a selectable marker gene or a reporter gene to facilitate the identification and selection of expressing cells from a cell population to be transfected or infected via the viral vector. In some embodiments, the selectable marker may be supported on a separate DNA fragment and used in the cotransfection procedure. Both the selectable marker and the reporter gene may be flanked by appropriate transcriptional regulatory sequences to enable expression in host cells. Useful selectable markers include, for example, antibiotic resistance genes such as neomycin.

[0272] In some embodiments, a reporter gene may be used to identify potentially transfected cells and / or to determine the functionality of transcriptional regulatory sequences. Generally, a reporter gene is a gene that is not present in or expressed in the recipient source, and whose expression is manifested by some readily detectable characteristic, such as enzymatic activity or visible fluorescence, which encodes a polypeptide. Reporter gene expression is assayed at a suitable time after the DNA has been introduced into recipient cells. Suitable reporter genes may include genes encoding luciferase, β-galactosidase, chloramphenicol acetyltransferase, secreted alkaline phosphatase, or green fluorescent protein genes (e.g., Ui-Tei et al., 2000 FEBS Letters 479:79-82). Suitable expression systems are well known and can be prepared using known techniques or are commercially available. Generally, a construct with the minimum 5' adjacent region exhibiting the highest level of reporter gene expression is identified as the promoter. Such a promoter region may be linked to a reporter gene and used to determine the regulatory capacity of a drug related to the transcription driven by the promoter.

[0273] cell This disclosure further relates, in part, to cells containing the repressors or repressor systems described herein. Any cells known to those skilled in the art, for example, cell lines, such as cell lines suitable for the expression of recombinant polypeptides, are suitable for containing the repressors or repressor systems described herein. In some embodiments, cells, for example, cell lines, may be used to express the repressors or repressor systems described herein, for example, one or more repressors. In some embodiments, cells, for example, cell lines, may be used to express or amplify the repressors or repressor systems described herein, for example, nucleic acids encoding one or more repressors, for example, vectors. In some embodiments, cells contain the repressors or repressor systems described herein, for example, nucleic acids encoding one or more repressors.

[0274] In some embodiments, the cell comprises a first component of the expression repression system, e.g., a first nucleic acid encoding a first expression repressor, and a second component of the expression repression system, e.g., a second nucleic acid encoding a second expression repressor. In some embodiments, when the cell contains a nucleic acid encoding an expression repression system comprising two or more expression repressors, the sequences encoding each expression repressor are placed in separate nucleic acid molecules, e.g., different vectors, e.g., a first vector encoding a first expression repressor and a second vector encoding a second expression repressor. In some embodiments, the sequences encoding each expression repressor are placed in the same nucleic acid molecule, e.g., the same vector. In some embodiments, part or all of the nucleic acid encoding the expression repression system is integrated into the genomic DNA of the cell. In some embodiments, the nucleic acid encoding the first expression repressor of the expression repression system is integrated into the genomic DNA of the cell, and the nucleic acid encoding the second expression repressor of the expression repression system is not integrated into the genomic DNA of the cell (e.g., is located on a vector). In some embodiments, one or more nucleic acids encoding the first and second expression repressors of the expression repression system are integrated into the genomic DNA of the cell, e.g., at the same (e.g., adjacent or co-localized) or different sites of the genomic DNA.

[0275] Examples of cells that may contain and / or express the expression repression systems or expression repressors described herein include, but are not limited to, hepatocytes, neurons, endothelial cells, muscle cells, and lymphocytes.

[0276] Method for preparing RNA Methods for the production and purification of modified RNAs are known and disclosed in the art. For example, without limitation, modified RNAs are produced using in vitro transcription (IVT) enzyme synthesis. Methods for the production of IVT polynucleotides are known in the art and are described in WO 2013 / 151666, WO 2013 / 151668, WO 2013 / 151663, WO 2013 / 151669, WO 2013 / 151670, WO 2013 / 151664, WO 2013 / 151665, WO 2013 / 151671, WO 2013 / 151672, WO 2013 / 151667, and WO 2013 / 151736. Purification methods include contacting a sample under conditions such that an RNA transcript binds to a surface linked to multiple thymidines or derivatives thereof and / or multiple uracils or derivatives thereof (poly T / U), and eluting the purified RNA transcript from the surface (WO 2014 / 152031); using ion (e.g., anion) exchange chromatography that enables the separation of long RNAs up to 10,000 nucleotides in length by a scalable method (WO 2014 / 144767); and subjecting the modified RMNA sample to DNase treatment (WO 2014 / 152030), which includes purifying RNA transcripts containing poly A tails.

[0277] In the field of human diseases, antibodies, viruses, and modified RNAs encoding proteins in various in vivo settings are publicly known, for example, in International Publication Nos. 2013 / 151666, 2013 / 151668, 2013 / 151663, 2013 / 151669, 2013 / 151670, and 2013 / This is disclosed in Table 6 of Brochure No. 151664, Brochure No. 2013 / 151665, and Brochure No. 2013 / 151736; Tables 6 and 7 of International Publication Brochure No. 2013 / 151672; Tables 6, 178, and 179 of International Publication Brochure No. 2013 / 151671; and Tables 6, 185, and 186 of International Publication Brochure No. 2013 / 151667. Any of the above can be synthesized as IVT polynucleotides, chimeric polynucleotides, or cyclic polynucleotides and linked to the polypeptides described herein, each of which may contain one or more modified nucleotides or terminal modifications.

[0278] In some embodiments, the repressors include or consist of proteins and can therefore be prepared by methods for preparing proteins as known in the art, for example, as provided in this disclosure. In some embodiments, one or more repressors in a repressor system, for example, in a repressor system, include one or more proteins and can therefore be prepared by methods for preparing proteins. As those skilled in the art will understand, methods for preparing proteins or polypeptides (which may include preparing drugs as described herein) are common in the art. See, for example, Smales & James (Eds.), Therapeutic Proteins: Methods and Protocols (Methods in Molecular Biology), Humana Press (2005); and also Crommelin, Sindelar & Meibohm (Eds.), Pharmaceutical Biotechnology: Fundamentals and Applications, Springer (2013).

[0279] delivery lipid particles The expression repressors or expression repressor systems described herein can be delivered using any biological delivery system / formulation, including particles, e.g., nanoparticle delivery systems. Nanoparticles include particles with dimensions (e.g., diameter) of about 1 to about 1000 nanometers, about 1 to about 500 nanometers, about 1 to about 100 nm, about 30 nm to about 200 nm, about 50 nm to about 300 nm, about 75 nm to about 200 nm, about 100 nm to about 200 nm, and any range in between. Nanoparticles have a composite structure of nanoscale dimensions. In some embodiments, nanoparticles are typically spherical, but different forms are possible depending on the nanoparticle composition. The portion of the nanoparticle that is in contact with the environment which is external to the nanoparticle is generally identified as the surface of the nanoparticle. In some embodiments, the nanoparticles have a maximum dimension in the range of 25 nm to 200 nm. Nanoparticles described herein include, but are not limited to, solid, semi-solid, emulsion, or colloidal nanoparticles, and delivery systems which may be provided in any of these forms. Nanoparticle delivery systems may include, but are not limited to, lipid-based systems, liposomes, micelles, microvesicles, exosomes, or gene guns. In one embodiment, the nanoparticles are lipid nanoparticles (LNPs). In some embodiments, LNPs are particles comprising multiple lipid molecules physically associated with each other by intermolecular forces.

[0280] In some embodiments, the LNP may contain multiple components, for example, three to four components. In one embodiment, an expression repressor or a pharmaceutical composition containing the expression repressor (or a nucleic acid encoding it, or a pharmaceutical composition containing the expression repressor nucleic acid) is encapsulated in the LNP. In one embodiment, an expression repressor system or a pharmaceutical composition containing the expression repressor system (or a nucleic acid encoding it, or a pharmaceutical composition containing the expression repressor system nucleic acid) is encapsulated in the LNP. In some embodiments, the nucleic acid encoding the first expression repressor and the nucleic acid encoding the second expression repressor reside on the same LNP. In some embodiments, the nucleic acids encoding the first expression repressor and the nucleic acid encoding the second expression repressor reside on different LNPs. Preparation of the LNP and encapsulation of modifiers may be used, and / or adapted from Rosin et al, Molecular Therapy, vol. 19, no. 12, pages 1286-2200, December 2011. In some embodiments, the lipid nanoparticle compositions disclosed herein are useful for the expression of mRNA-encoded proteins. In some embodiments, when nucleic acids are present in lipid nanoparticles, they exhibit resistance to degradation by nucleases in aqueous solutions.

[0281] In some embodiments, the LNP formulation may include CCD lipids, neutral lipids, and / or helper lipids. In some embodiments, the LNP formulation includes ionizable lipids. In some embodiments, the ionizable lipid may be a cationic lipid that can be readily protonated, an ionized cationic lipid, or an amine-containing lipid. In some embodiments, the lipid is a cationic lipid that can exist in a positively charged or neutral form depending on the pH. In some embodiments, the cationic lipid is a lipid that has the ability to be positively charged, for example, under physiological conditions. In some embodiments, the lipid particles include cationic lipids in the formulation together with one or more of the following: neutral lipids, ionizable amine-containing lipids, biodegradable alkyl lipids, steroids, phospholipids including polyunsaturated lipids, structural lipids (e.g., sterols), PEG, cholesterol, and polymer-conjugated lipids.

[0282] In some embodiments, the LNP formulation (e.g., MC3 and / or SSOP) includes cholesterol and / or helper lipids. The LNP may be, for example, microspheres (in some embodiments, substantially spherical, including monolayer and multilayer vesicles and lamellar lipid bilayers).

[0283] In some embodiments, the LNP may include an aqueous core containing, for example, a nucleic acid encoding an expression repressor or system as disclosed herein, which is referred to herein as the “cargo.” In some embodiments of this disclosure, the cargo of the LNP formulation includes at least one guide RNA. In some embodiments, the cargo, for example, the nucleic acid encoding an expression repressor or system as disclosed herein, may be adsorbed onto the surface of the LNP, for example, an LNP containing a cationic lipid. In some embodiments, the cargo, for example, the nucleic acid encoding an expression repressor or system as disclosed herein, may be associated with the LNP. In some embodiments, the cargo, for example, the nucleic acid encoding an expression repressor or system as disclosed herein, may be encapsulated in the LNP, for example, fully encapsulated and / or partially encapsulated.

[0284] In some embodiments, the LNP containing the cargo may be administered for systemic delivery, for example, to deliver a therapeutically effective dose of the cargo that can result in widespread exposure to the active agent within the organism. Systemic delivery of lipid nanoparticles can be by any means known in the art, for example, intravenous, intra-arterial, subcutaneous, and intraperitoneal delivery, without limitation. In some embodiments, systemic delivery of lipid nanoparticles is by intravenous delivery. In some embodiments, the LNP containing the cargo may be administered for local delivery, for example, to deliver the active agent directly to a target site within the organism.

[0285] In some embodiments, LNP may be delivered locally to a disease site, e.g., a tumor, or other target site, e.g., an inflammatory site, or a target organ, e.g., the liver, lungs, stomach, colon, pancreas, uterus, breast, lymph nodes, etc. In some embodiments, LNP as disclosed herein may be delivered locally to specific cells, e.g., hepatocytes, astrocytes, Kupffer cells, endothelial cells, alveolar cells, and / or epithelial cells. In some embodiments, LNP as disclosed herein may be delivered locally to a specific site, e.g., a tumor site, e.g., by subcutaneous or orthotopic administration. LNP may be formulated as an emulsion, a dispersed phase in micelles, or as an internal phase in a suspension. In some embodiments, LNP is biodegradable. In some embodiments, LNP does not accumulate or cause in vivo toxicity to cytotoxic levels at therapeutically effective doses. In some embodiments, LNP does not accumulate or cause in vivo toxicity to cytotoxic levels after repeated administration at therapeutically effective doses. In some embodiments, LNPs do not evoke an innate immune response that would lead to substantially adverse effects at therapeutically effective doses.

[0286] In some embodiments, the LNP used includes formula 4-(dimethylamino)butanoic acid (6Z,9Z,28Z,31Z)-heptatriaconta-6,9,28,31-tetraen-19-yl or ssPalmO-phenyl-P4C2 (ssPalmO-Phe, SS-OP). In some embodiments, the LNP formulation comprises the formula, 4-(dimethylamino)butanoic acid (6Z,9Z,28Z,31Z)-heptatriaconta-6,9,28,31-tetraen-19-ylbutanoate (MC3), 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), cholesterol, 1,2-dimyristoyl-rac-glycero-3-methoxypolyethylene glycol-2000 (PEG2k-DMG), for example, MC3 LNP or ssPalmO-phenyl-P4C2 (ssPalmO-Phe, SS-OP), 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), cholesterol, 1,2-dimyristoyl-rac-glycero-3-methoxypolyethylene glycol-2000 (PEG2k-DMG), for example, SSOP-LNP.

[0287] Liposomes are spherical vesicular structures composed of a monolayer or multilayer lipid bilayer surrounding an internal aqueous compartment and a relatively impermeable outer lipophilic phospholipid bilayer. Liposomes can be anionic, neutral, or cationic. They are biocompatible, non-toxic, capable of delivering both hydrophilic and lipophilic drug molecules, protecting their cargo from degradation by plasma enzymes, and transporting their load across biological membranes and the blood-brain barrier (BBB) ​​(see, for example, Spuch and Navarro, Journal of Drug Delivery, vol. 2011, Article ID 469679, 12 pages, 2011).

[0288] Vesicles can be composed of several different types of lipids; however, phospholipids are most commonly used for the formation of liposomes as drug carriers. Vesicles may, for example, contain DOTMA, DOTAP, DOTIM, and DDAB individually, or together with cholesterol to form DOTMA and cholesterol, DOTAP and cholesterol, DOTIM and cholesterol, and DDAB and cholesterol. Methods for preparing multilayer vesicle lipids are known in the art (see, for example, U.S. Patent No. 6,693,086, the teaching relating to the preparation of multilayer vesicle lipids is incorporated herein by reference). Vesicle formation can occur spontaneously when a lipid film is mixed with an aqueous solution, but it can also be accelerated by applying force in the form of shaking using a homogenizer, sonicator, or extruder (see, for example, Spuch and Navarro, Journal of Drug Delivery, vol. 2011, Article ID 469679, 12 pages, 2011). Extruded lipids can be prepared by extruding them through a reducing filter, as described in Templeton et al, Nature Biotech, 15:647-652, 1997 (this instruction regarding the preparation of extruded lipids is incorporated herein by reference).

[0289] Viral vector In some embodiments, viral vector systems may be used in the methods and compositions described herein. Suitable viral vector systems for use include, for example, (a) adenovirus vectors (e.g., Ad5 / F35 vector); (b) retrovirus vectors, including, but not limited to, lentivirus vectors (including embedded competent or embedded-deficient lentivirus vectors), Moloney's mouse leukemia virus, etc.; (c) adeno-associated virus vectors; (d) herpes simplex virus vectors; (e) SV40 vector; (f) polyomavirus vectors; (g) papillomavirus vectors; (h) picornavirus vectors; (i) poxvirus vectors such as orthopox, e.g., vaccinia virus vector or avipox, e.g., canarypox or fowlpox; and (j) helper-dependent or gutless adenoviruses. Replication-deficient viruses may also be advantageous. Different vectors will be incorporated into or not incorporated into the cell genome. The construct may include a viral sequence for transfection, if necessary. Alternatively, the construct may be incorporated into an episomal replication vector, such as EPV and EBV vectors. See, for example, U.S. Patent Nos. 6,534,261; 6,607,882; 6,824,978; 6,933,113; 6,979,539; 7,013,219; and 7,163,824 (each of which is incorporated herein by reference in its entirety). Vectors, including those derived from retroviruses such as lentiviruses, are suitable tools for achieving long-term gene transfer, enabling the long-term stable incorporation of transgenes and their transmission to daughter cells. Examples of vectors include expression vectors, replication vectors, probe-generating vectors, and sequencing vectors. In certain embodiments, expression vectors may be supplied to cells in the form of viral vectors. Viral vector technology is well known in the art and is described in various virology and molecular biology manuals.

[0290] In some embodiments, suitable viral vectors for use in the present invention are adeno-associated virus vectors, such as recombinant adeno-associated virus vectors. Recombinant adeno-associated virus vectors (rAAV) are gene delivery systems based on deficient and non-pathogenic parvovirus adeno-associated virus type 2. In some embodiments, the vector is derived from a plasmid containing only the AAV 145 bp reverse terminal repeat adjacent to the transgene expression cassette. Efficient gene transfer and stable transgene delivery by integration into the genome of transduced cells are key features of this vector system. (Wagner et al., Lancet 351:91171702-3 (1998), Kearns et al., Gene Ther. 9:748-55 (1996)). AAV serotypes, including AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, and AAV9, can be used in the present invention. High titers of replication-deficient recombinant adenovirus vectors (Ad) can be produced, which can easily infect several different cell types. Most adenovirus vectors are engineered so that the trans gene replaces the Ad E1a, E1b, and / or E3 genes; the replication-deficient vector is then propagated in a suitable cell system that supplies the deleted gene function in trans, such as HEK293 and its variants.

[0291] Ad vectors can transduce multiple types of tissues in vivo, including non-dividing differentiated cells found in the liver, kidneys, and muscles. Conventional Ad vectors have high transport capacity. In clinical trials, Ad vectors have been used in polynucleotide therapy for antitumor immunization via intramuscular injection (Sterman et al., Hum. Gene Ther. 7:1083-9 (1998)). Further examples of the use of adenovirus vectors for gene transfer in clinical trials include Rosenecker et al., Infection 24:15-10 (1996); Sterman et al., Hum.Gene Ther. 9:71083-1089 (1998); Welsh et al., Hum.Gene Ther. 2:205-18 (1995); Alvarez et al., Hum.Gene Ther. 5:597-613 (1997); Topf et al., Gene Ther. 5:507-513 (1998); and Sterman et al., Hum.Gene Ther. 7:1083-1089 (1998).

[0292] Packaging cells are used to form viral particles capable of infecting host cells. Examples of such cells include, without limitation, HEK293 cells and their variants, Ψ2 cells, and PA317 cells. Viral vectors used in gene therapy are typically created by a producing cell line that packages the nucleic acid vector into viral particles. The vector typically contains the minimum viral sequence necessary for packaging and subsequent integration into the host (if applicable), with other viral sequences replaced by an expression cassette encoding the protein to be expressed. Missing viral functions are supplied trans by the packaging cell line. For example, AAV vectors used in gene therapy typically contain only the reverse-terminal repeat (ITR) sequence from the AAV genome necessary for packaging and integration into the host genome. In some embodiments, viral DNA is packaged into a cell line, which also contains helper plasmids encoding other AAV genes, i.e., rep and cap, but lacks the ITR sequence. In certain embodiments, the cell line is also infected with adenovirus as a helper. Helper viruses facilitate the replication of AAV vectors and the expression of AAV genes from helper plasmids. In certain embodiments, the helper plasmids are not packaged in meaningful quantities because they lack an ITR sequence. In certain embodiments, adenovirus contamination can be reduced, for example, by heat treatment that is more susceptible to adenoviruses than AAV.

[0293] Pharmaceutical composition This disclosure further relates, in part, to expression repressors or expression repressor systems described herein, for example, pharmaceutical compositions comprising one or more expression repressors, to pharmaceutical compositions comprising nucleic acids encoding one or more expression repressors, and / or expression repressors or expression repressor systems described herein, for example, compositions for delivering one or more expression repressors to cells, tissues, organs, and / or subjects.

[0294] As used herein, the term “pharmaceutical composition” means an active agent formulated with one or more pharmaceutically acceptable carriers (e.g., pharmaceutically acceptable carriers known to those skilled in the art) (e.g., an expression repressor or nucleic acid of an expression repressor, e.g., an expression repressor system, e.g., one or more expression repressors of an expression repressor system, or nucleic acids encoding them). In some embodiments, the active agent is present in a unit dose appropriate for administration in a therapeutic regime that exhibits a statistically significant probability of achieving a predetermined therapeutic effect when administered to a relevant population. In some embodiments, a pharmaceutical composition comprising an expression repressor of the Disclosure comprises an expression repressor or one or more nucleic acids encoding it. In some embodiments, a pharmaceutical composition comprising an expression repressor system of the Disclosure comprises each of the expression repressors of the expression repressor system or one or more nucleic acids encoding them (e.g., if the expression repressor system comprises a first expression repressor and a second expression repressor, the pharmaceutical composition comprises the first and second expression repressors). In some embodiments, the pharmaceutical composition comprises expression repressors that do not comprise all of the expression repressor system comprising multiple expression repressors. For example, the expression repressor system may include a first expression repressor and a second expression repressor, the first pharmaceutical composition may include the first expression repressor or a nucleic acid encoding it, and the second pharmaceutical composition may include the second expression repressor or a nucleic acid encoding it. In some embodiments, the pharmaceutical composition may include a co-formulation of one or more expression repressors or one or more nucleic acids encoding them.

[0295] In some embodiments, the pharmaceutical composition is suitable for the following, namely, oral administration, for example, aqueous medicine (aqueous or non-aqueous solution or suspension), tablets, for example, those targeted for buccal, sublingual, and systemic absorption, bolus, powders, granules, pastes applied to the tongue; for example, sterile solutions or suspensions, or parenteral administration by, for example, subcutaneous, intramuscular, intravenous or epidural injection as sustained-release formulations; for example, topical application as creams, ointments, or controlled-release patches, or sprays applied to the skin, lung, or mouth; for example, intravaginal or intrarectal as pessaries, creams, or foams; sublingual; intraocular; transdermal; or may be specially formulated for administration in solid or liquid form, including those suitable for nasal, lung, and / or other mucosal surfaces.

[0296] As used herein, the term "pharmaceutically acceptable" refers to compounds, materials, compositions, and / or dosage forms that are suitable for use in contact with human and animal tissue without undue toxicity, irritation, allergic reaction, or other problems or complications, within the scope of sound medical judgment and commensurate with a reasonable risk-benefit ratio.

[0297] As used herein, the term “pharmaceutically acceptable carrier” means a pharmaceutically acceptable material, composition, or medium, such as a liquid or solid filler, diluent, excipient, or solvent encapsulant, that is involved in transporting or delivering the compound of the subject from one organ or part of the body to another organ or part of the body. Each carrier must be “acceptable” in the sense that it is compatible with the other components of the formulation and is not harmful to the patient. In some embodiments, for example, materials that can act as pharmaceutically acceptable carriers include sugars such as lactose, glucose, and sucrose; starches such as corn starch and potato starch; celluloses such as sodium carboxymethylcellulose, ethylcellulose, and cellulose acetate, and their derivatives; tragacanth powder; malt; gelatin; talc; excipients such as cocoa butter and suppository wax; oils such as peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, corn oil, and soybean oil; glycols such as propylene glycol; polyols such as glycerin, sorbitol, mannitol, and polyethylene glycol; esters such as ethyl oleate and ethyl laurate; agar; buffers such as magnesium hydroxide and aluminum hydroxide; alginic acid; pyrogen-free water; isotonic saline; Ringer's solution; ethyl alcohol; pH buffer solution; polyesters, polycarbonates, and / or polyanhydrous materials; and other non-toxic and suitable substances used in pharmaceutical formulations.

[0298] As used herein, the term “pharmaceutically acceptable salt” means a salt of a compound that is suitable for use in a pharmaceutical context, i.e., a salt that is suitable for use in contact with human and lower animal tissues without excessive toxicity, irritation, allergic reactions, etc., within reasonable limits of medical judgment, and that has a reasonable risk-benefit ratio. Pharmacochemically acceptable salts are well known in the art. For example, SMBerge, et al. describes pharmaceutically acceptable salts in detail in J. Pharmaceutical Sciences, 66:1-19 (1977).

[0299] In some embodiments, pharmaceutically acceptable salts include, but are not limited to, non-toxic acid addition salts, which are amino group salts formed with inorganic acids such as hydrochloric acid, hydrobromic acid, phosphoric acid, sulfuric acid, and perchloric acid, or with organic acids such as acetic acid, maleic acid, tartaric acid, citric acid, succinic acid, or malonic acid, or by other methods used in the art, such as ion exchange. In some embodiments, pharmaceutically acceptable salts include, but are not limited to, adipine salts, alginates, ascorbic acid, aspartates, benzenesulfonates, benzoates, bisulfates, borates, butyrates, camphorates, camphor sulfons, citrates, cyclopentanepropionates, digluconates, dodecyl sulfates, ethanesulfonates, formates, fumarates, glucoheptonates, glycerophosphates, gluconates, hemisulfates, heptanoates, hexanoates, hydroiodides, and 2-hydroxysulfates. - Examples include ethanesulfonates, lactobionates, lactates, laurates, lauryl sulfates, malates, maleates, malons, methanesulfonates, 2-naphthalenesulfonates, nicotinates, nitrates, oleates, oxalates, palmitates, pamoates, pectates, persulfates, 3-phenylpropionates, phosphates, picrates, pivalates, propions, stearates, succinates, sulfates, tartrates, thiocyanates, toluenesulfonates, undecanoates, and valersates. Typical alkali metal salts or alkaline earth metal salts include sodium, lithium, potassium, calcium, and magnesium. In some embodiments, pharmaceutically acceptable salts may include, where appropriate, non-toxic ammonium, quaternary ammonium, and amine cations formed using counterions such as halides, hydroxides, carboxylates, sulfates, phosphates, nitrates, alkyls having 1 to 6 carbon atoms, sulfonates, and arylsulfonates. In various embodiments, this disclosure provides pharmaceutical compositions described herein, comprising pharmaceutically acceptable excipients.Pharmaceutically acceptable excipients include generally safe, non-toxic, and desirable excipients useful in the preparation of pharmaceutical compositions, and include excipients acceptable for veterinary use and human medicinal use. Such excipients may be solid, liquid, semi-solid, or, in the case of aerosol compositions, gaseous.

[0300] Pharmaceutical preparations may, in the case of tablets, be prepared by grinding, mixing, granulating, and compressing as necessary; or, in the case of hard gelatin capsules, by conventional pharmaceutical techniques involving grinding, mixing, and filling. When a liquid carrier is used, the preparation may be in the form of a syrup, elixir, emulsion, or aqueous or non-aqueous solution or suspension. Such liquid formulations may be administered directly orally.

[0301] In some embodiments, the pharmaceutical composition may be formulated for delivery to cells and / or to a subject via any route of administration. Modes of administration to a subject may include injection, infusion, inhalation, intranasal, intraocular, topical delivery, intercannular delivery, or oral ingestion. Injections include, without limitation, intravenous, intramuscular, intraarterial, subarachnoid, intraventricular, intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal, transtracheal, subcutaneous, subepidermal, intra-articular, subcapsular, subarachnoid, intraspinal, intracerebrospinal, and intrasternal injections and infusions. In some embodiments, administration includes, for example, aerosol inhalation by spray therapy. In some embodiments, administration may be systemic (e.g., oral, rectal, nasal, sublingual, buccal, or parenteral), enteral (e.g., systemic effect, but delivered through the gastrointestinal tract), or topical (e.g., topical application to the skin, or intravitreal injection). In some embodiments, one or more compositions are administered systemically. In some embodiments, the administration is other than parenteral administration, and the therapeutic agent is a parenteral therapeutic agent. In some embodiments, administration may be via the bronchi (e.g., by bronchial infusion), buccal, skin (this may be one or more of the following, e.g., topical, intradermal, interdermal, transdermal, etc., into the dermis), enteral, intra-arterial, intradermal, intragastric, intramedullary, intramuscular, intranasal, intraperitoneal, subarachnoid, intravenous, intraventricular, intra-organ (e.g., intrahepatic), mucosa, nasal cavity, oral, rectal, subcutaneous, sublingual, topical, trachea (e.g., by intratracheal infusion), vagina, vitreous humor, etc. In some embodiments, the administration may be a single dose. In some embodiments, the administration may involve dose administration, which is intermittent (e.g., multiple doses spaced apart in time) and / or cyclical (e.g., individual doses spaced apart by a common time interval). In some embodiments, administration may involve continuous administration (e.g., perfusion) over a selected period of time. In some embodiments, during a single treatment or over a treatment regimen, the subject may be given 6, 8, 10, 12, 15, 20 or more doses.

[0302] In some embodiments, administration may be given as needed, for example, as long as symptoms associated with the disease, disorder, or condition persist. In some embodiments, repeated administration may be indicated throughout the subject's lifetime. The duration of treatment may vary, for example, 1 day, 2 days, 3 days, 1 week, 2 weeks, 1 month, 2 months, 3 months, 6 months, 1 year, or longer.

[0303] Dosage The dosage of the drug or composition administered may vary based on factors such as the condition under treatment, the severity of the disease, personal parameters of the subject including age, physiological condition, size, and weight, the duration of treatment, the type of treatment to be performed (if any), the detailed route of administration, and similar factors. Thus, the dosage of the drugs described herein may depend on these various parameters. The dosage of the composition administered may also vary depending on other factors such as the sex of the subject, the overall medical condition, and the severity of the disorder to be treated. While it may be desirable to provide subjects with a single intravenous infusion of a dosage of approximately 1 mg / kg to 6 mg / kg of the modulated drug or combination of modulated drugs disclosed herein, lower or higher dosages may also be administered depending on the circumstances. The dosage may be repeated as needed, for example, once a day (for example, over 1 to 30 days), once every 3 days (for example, over 1 to 30 days), once every 5 days (for example, over 1 to 30 days), or once a week (for example, over 1 to 6 weeks or 2 to 5 weeks). In some embodiments, the dosage may be, but is not limited to, 1.0 mg / kg to 6 mg / kg, 1.0 mg / kg to 5 mg / kg, 1.0 mg / kg to 4 mg / kg, 1.0 mg / kg to 3.0 mg / kg, 1.5 mg / kg to 3.0 mg / kg, 1.0 mg / kg to 1.5 mg / kg, 1.5 mg / kg to 3 mg / kg, 3 mg / kg to 4 mg / kg, 4 mg / kg to 5 mg / kg, or 5 mg / kg to 6 mg / kg. The dosage may be administered multiple times, for example, once a week, or twice a week, once every week, or once every two weeks. In some embodiments, subjects are provided with multiple intravenous infusions of dosages of the modulo agent or combination of modulo agents disclosed herein, ranging from approximately 1 mg / kg to 6 mg / kg; however, depending on the circumstances, lower or higher dosages may also be administered.

[0304] The modulated agent or combination of modulated agents as disclosed herein may be administered as a single dose every 3 to 5 days, and repeated for a total of at least 3 doses. Alternatively, the modulated agent or combination of modulated agents as disclosed herein may be administered at 3 mg / kg every 5 days for 25 days. Alternatively, the modulated agent or combination of modulated agents as disclosed herein may be administered at 1.0 to 5.0 mg / kg every 3 to 5 days for 1 to 10 doses. Alternatively, the modulated agent or combination of modulated agents as disclosed herein may be administered at 3.0 mg / kg every 5 days for 3 doses, followed by 3 doses every 3 days. Alternatively, the modulated agent or combination of modulated agents as disclosed herein may be administered at 1.0 to 3.0 mg / kg every 5 days for 4 doses, followed by 3 doses every 3 days. Alternatively, the modulated agent or combination of modulated agents as disclosed herein may be administered at 6 mg / kg every 5 days for 1 to 10 doses. Alternatively, the modulated agent or combination of modulated agents as disclosed herein may be administered at 3 mg / kg every 5 days for 1 to 10 doses. Alternatively, the modulated agent or combination of modulated agents as disclosed herein may be administered at 1.5 mg / kg every 5 days for 2 doses, at 3 mg / kg every 5 days for 3 doses, and at 3 mg / kg every 3 days for 1 dose. Alternatively, the modulated agent or combination of modulated agents as disclosed herein may be administered at 6 mg / kg every 5 days or at 1.5 mg / kg once daily for 5 days, followed by a 2-day rest period. The dose administration schedule can be repeated at any other interval, and the dosage may be administered via various parenteral routes, with appropriate adjustments to the dosage and schedule. In some embodiments, the dose administration of a modulo agent or combination of modulo agents may include doses of 1.0 mg / kg to 6.0 mg / kg, which are optionally administered weekly, twice weekly, or every other week.Those skilled in the art will recognize that various factors, including age, sex, weight, and the severity of the disorder to be treated, may be considered when selecting the dosage of modulo agents or combinations of modulo agents as disclosed herein, and that the dosage and / or frequency of administration may be increased or decreased during the course of treatment. Dosage may be repeated as needed, with evidence that a reduction in tumor volume was observed after only 2 to 8 doses. The effect of the dosages and administration schedules disclosed herein on the overall body weight of the subject is minimal compared to cisplatin, sorafenib, or small molecule control agents. Methods of the subject may include the measurement of tumor response at regular intervals using CT and / or PET / CT, or MRI. Blood levels of tumor markers may also be monitored. Dosage and / or administration schedule may be adjusted as needed based on imaging and / or blood marker level results.

[0305] In some embodiments, the expression inhibitor or expression inhibitor system is administered to the patient in combination with non-statin lipid modification therapy. In some embodiments, the non-statin lipid modification therapy includes therapeutic agents selected from the group consisting of ezetimibe, fibrates, niacin, ω-3 fatty acids, and bile acid resins.

[0306] In some embodiments, in a method for treating a subject with cancer, the compositions disclosed herein may be administered in combination with one or more therapeutic agents or methods selected from surgical resection, tyrosine kinase inhibitors (TKIs), e.g., sorafenib, bromodomain inhibitors, e.g., BET inhibitors, e.g., JQ1, e.g., BET672, e.g., virabrecib, MEK inhibitors (e.g., trametinib), orthotopic liver transplantation, radiofrequency ablation, immunotherapy, immune checkpoint + anti-vascular endothelial growth factor combination therapy, photodynamic therapy (PDT), laser therapy, brachytherapy, radiotherapy, transcatheter arterial chemistry or radioembolization, stereotactic radiotherapy, chemotherapy, and / or systemic chemotherapy to treat the disease or disorder.

[0307] The pharmaceutical compositions relating to this disclosure can be delivered in therapeutically effective doses. The exact therapeutically effective dose is the amount of composition that would produce the most effective result in terms of therapeutic efficacy in a given subject. This amount will vary depending on a variety of factors, including, but not limited to, the properties of the therapeutic compound (including activity, pharmacokinetics, pharmacodynamics, and bioavailability), the physiological conditions of the subject (including age, sex, type and stage of disease, overall health status, responsiveness to a given dose, and type of pharmacotherapy), the properties of one or more pharmaceutically acceptable carriers in the formulation, and / or the route of administration.

[0308] Administration In some embodiments, the Disclosure provides a method for delivering a therapeutic agent, comprising administering a composition as described herein to a target, wherein the modulating agent is the therapeutic agent, and / or the delivery of the therapeutic agent causes an alteration of gene expression compared to gene expression in the absence of the therapeutic agent.

[0309] The methods provided in the various embodiments herein may be used in any of the embodiments further outlined herein. In some embodiments, for example, one or more compositions comprising an expression repressor or expression repressor system described herein are targeted to specific cells or one or more specific tissues.

[0310] For example, in some embodiments, one or more compositions comprising, for example, an expression repressor or expression repressor system described herein are targeted to liver, epithelial, binding, muscle, reproductive, and / or nerve tissue or cells. In some embodiments, the composition targets cells or tissues of specific organ systems, such as the cardiovascular system (heart, vascular system); the digestive system (esophagus, stomach, liver, gallbladder, pancreas, intestine, colon, rectum, and anus); the endocrine system (hypothalamus, pituitary gland, pineal gland or pineal gland, thyroid gland, parathyroid gland, adrenal gland); the excretory system (kidney, ureter, bladder); the lymphatic system (lymph, lymph nodes, lymphatic vessels, tonsils, pharyngeal tonsils, thymus, spleen); the cutaneous system (skin, hair, nails); the muscular system (e.g., skeletal muscle); the nervous system (brain, spinal cord, nerves); the reproductive system (ovaries, uterus, mammary glands, testes, vas deferens, seminal vesicles, prostate); the respiratory system (pharynx, larynx, trachea, bronchi, lungs, diaphragm); the skeletal system (bones, cartilage); and / or combinations thereof. In certain embodiments, the expression repressors or expression repressor systems described herein target liver or hepatocytes.

[0311] In some embodiments, the compositions of this disclosure cross the blood-brain barrier, placental membrane, or blood-testis barrier. In some embodiments, the pharmaceutical compositions provided herein are administered systemically. In some embodiments, the administration is other than parenteral administration, and the therapeutic agent is a parenteral therapeutic agent.

[0312] For example, methods and compositions provided herein, including expression inhibitors or expression inhibitor systems described herein, may include pharmaceutical compositions administered in a regimen sufficient to alleviate the symptoms of a disease, disorder, and / or pathological condition. In some embodiments, this disclosure provides a method for delivering a therapeutic agent by administering a composition as described herein.

[0313] The pharmaceutical uses of this disclosure may include compositions as described herein (e.g., modulating agents, e.g., destructive agents).

[0314] In some embodiments, the pharmaceutical compositions of the Disclosure exhibit improved PK / PD, such as increased pharmacokinetics or pharmacodynamics, compared to the active agent alone, including improved targeting, absorption, or transport (e.g., by at least 5%, 10%, 15%, 20%, 30%, 40%, 50%, 60%, 75%, 80%, 90%, or more). In some embodiments, the pharmaceutical compositions exhibit reduced undesirable effects, such as decreased diffusion to non-target sites, off-target activity, or toxic metabolism, compared to the active agent alone (e.g., decreased by at least 5%, 10%, 15%, 20%, 30%, 40%, 50%, 60%, 75%, 80%, 90%, or more), compared to the therapeutic agent alone. In some embodiments, the compositions increase the efficacy of a therapeutic agent and / or reduce its toxicity compared to the active agent alone (e.g., at least 5%, 10%, 15%, 20%, 30%, 40%, 50%, 60%, 75%, 80%, 90%, or more). Furthermore, in certain embodiments, the disclosure provides a method for preventing at least one symptom in a subject who would benefit from the regulation of a target gene, such as a subject having a disease associated with the target gene, by administering the subject a prophylactically effective dose of the agent or composition of the present invention.

[0315] When the target of treatment is a mammal such as a human, the composition may be administered by any means known in the art, including, but not limited to, intracranial (e.g., intravenous, intraparenchymal, and subarachnoid), intravenous, intramuscular, subcutaneous, transdermal, respiratory (aerosol), nasal, rectal, and topical (including buccal and sublingual) administration, including oral, intraperitoneal, or parenteral routes. In certain embodiments, the composition is administered by intravenous infusion or injection. In certain embodiments, the composition is administered by subcutaneous injection.

[0316] In some embodiments, administration of a drug or composition according to the method of the present invention may result in a reduction in the severity, signs, symptoms, or markers of a disease associated with a target gene. In this context, “reduction” means a statistically significant decrease in such level. The reduction (absolute reduction or reduction in the difference between the elevated level and the normal level in the subject) may be, for example, at least about 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95%, or below the detection level of the assay used.

[0317] kit This disclosure further provides a kit comprising one or more repressors or repressor systems as described herein. In some embodiments, the kit comprises a repressor or repressor system (e.g., one or more repressors of a repressor system) and instructions for use of the repressor or repressor system. In some embodiments, the kit comprises a nucleic acid encoding a repressor or a nucleic acid encoding a repressor system or its components (e.g., one or more repressors of a repressor system) and instructions for use of the repressor (and / or the nucleic acid) and / or the repressor system (and / or the nucleic acid). In some embodiments, the kit comprises a cell containing a nucleic acid encoding a repressor or a nucleic acid encoding a repressor system or its components (e.g., one or more repressors of a repressor system) and instructions for use of the cell, the nucleic acid, and / or the repressor or repressor system.

[0318] In some embodiments, the kit includes a container comprising a system comprising two repressors: a first repressor comprising a first DNA target-directing moiety and a first DNA methyltransferase, wherein the first DNA target-directing moiety binds to a first target sequence in a region of the genome containing a target gene (e.g., a transcriptional regulatory element operably linked to the target gene (e.g., a promoter or transcription start site (TSS)) or a sequence adjacent to a transcriptional regulatory element); and a second repressor comprising a second DNA target-directing moiety and a second effector domain, wherein the second DNA target-directing moiety binds to a second target sequence different from the first target sequence.

[0319] In some embodiments, the kit further includes a set of instructions comprising at least one method of treating a disease or regulating, for example, reducing the expression of a target gene in a cell using the composition. In some embodiments, the kit may optionally include a delivery medium for the composition (e.g., lipid nanoparticles). The reagent may be provided suspended in an excipient and / or delivery medium, or as a separate component that can later be combined with the excipient and / or delivery medium. In some embodiments, the kit may optionally include additional therapeutic agents to be co-administered with the composition to affect the regulation of gene expression of a desired target gene. The instructional materials typically include, but are not limited to, written or printed materials. Any medium capable of storing and communicating such instructions to the end user is intended. Such mediums include, but are not limited to, electronic storage media (e.g., magnetic disks, tapes, cartridges, chips), optical media (e.g., CD-ROMs), etc. Such mediums may include the address of an internet site providing such instructional materials.

[0320] In some embodiments, the kit includes a unit dose of an expression repressor or expression repressor system described herein, for example, one or more expression repressors, or a unit dose of a nucleic acid encoding one or more expression repressors, for example, a vector.

[0321] definition As used herein, the singular forms "a," "an," and "the" refer to multiple objects unless otherwise explicitly indicated by the context.

[0322] As used herein, the term “agent” may refer to any chemical class of compound or entity, including, for example, polypeptides, nucleic acids, glycoslipids, small molecules, metals, or combinations or complexes thereof. As will be apparent to those skilled in the art from the context, in some embodiments, the term may refer to an entity that is a cell or organism, or that contains, or is a fraction, extract, or component thereof. Alternatively or in addition, as will be understood in light of the context to those skilled in the art, in some embodiments, the term may be used to refer to a natural product in that it is found in nature and / or obtained from nature. In some embodiments, as will again be understood in light of the context to those skilled in the art, the term may refer to one or more entities that are designed, manipulated and / or produced through human intervention and / or are artificial in that they are not found in nature. In some embodiments, the agent may be available in isolated or pure form; in some embodiments, the agent may be available in crude form. In some embodiments, potential agents may be provided as a collection or library from which active agents can be identified or characterized, for example, by screening. In some embodiments, the term “agent” may refer to a compound or entity that is or contains a polymer; in some embodiments, the term may refer to a compound or entity that contains one or more polymer moieties. In some embodiments, the term “agent” may refer to a compound or entity that is not a polymer and / or substantially does not contain any polymer and / or one or more specific polymer moieties. In some embodiments, the term may refer to a compound or entity that lacks or substantially does not contain any polymer moieties.

[0323] The term “anchor sequence,” as used herein, refers to a nucleic acid sequence recognized by an anchor sequence-mediated conjugate, such as a nucleating agent that binds sufficiently to form a complex. In some embodiments, the anchor sequence comprises one or more CTCF-binding motifs. In some embodiments, the anchor sequence is not located within a gene coding region. In some embodiments, the anchor sequence is located within an intergenetic region. In some embodiments, the anchor sequence is not located within either an enhancer or promoter. In some embodiments, the anchor sequence is located at least 400 bp, at least 450 bp, at least 500 bp, at least 550 bp, at least 600 bp, at least 650 bp, at least 700 bp, at least 750 bp, at least 800 bp, at least 850 bp, at least 900 bp, at least 950 bp, or at least 1 kb away from any transcription start site. In some embodiments, the anchor sequence is located within a region unrelated to genomic imprinting, single-allelic expression, and / or single-allelic epigenetic marking. In some embodiments, the anchor sequence has one or more functions selected from binding to an endogenous nucleating polypeptide (e.g., CTCF), forming an anchor sequence-mediated conjugate through interaction with a second anchor sequence, or sequestering the anchor sequence-mediated conjugate from an enhancer outside the anchor sequence-mediated conjugate. In some embodiments of this disclosure, a technique is provided that can specifically target one or more particular anchor sequences without targeting other anchor sequences (e.g., sequences that may contain a nucleating agent (e.g., CTCF) binding motif in another context); such targeted anchor sequences may be referred to as “target anchor sequences.” In some embodiments, the sequence and / or activity of a target anchor sequence is regulated, while the sequence and / or activity of one or more other anchor sequences that may be present in the same system as the targeted anchor sequence (e.g., in the same cell, and / or in some embodiments on the same nucleic acid molecule—e.g., on the same chromosome) is not regulated. In some embodiments, the anchor sequence contains or is a nucleating polypeptide binding motif. In some embodiments, the anchor sequence is adjacent to the nucleating polypeptide binding motif.

[0324] The term “anchor sequence-mediated conjugate” as used herein refers to a DNA structure, and / or complex, which is present and / or maintained by the physical interaction or binding of at least two anchor sequences in DNA with one or more polypeptides, such as nucleating polypeptides, or one or more proteins and / or nucleic acid entities (such as RNA or DNA), which bind to those anchor sequences to enable spatial proximity and functional binding between the anchor sequences.

[0325] Two events or entities are “related” to each other, as the term is used herein, if the existence, level, form and / or function of one correlates with that of the other. For example, in some embodiments, a particular entity (e.g., polypeptide, gene signature, metabolite, microorganism, etc.) is considered related to a disease, disorder, or condition if its existence, level, form and / or function correlates with the incidence (e.g., in a whole population of the relevant group) and / or susceptibility to that disease, disorder, or condition. In some embodiments, two or more entities are physically “related” to each other if they interact directly or indirectly so that they are in and / or maintain a state of physical proximity to each other. In some embodiments, two or more entities that are physically related to each other are covalently linked; in some embodiments, two or more entities that are physically related to each other are not covalently linked but are non-covalently related, for example, by hydrogen bonds, van der Waals interactions, hydrophobic interactions, magnetism, and combinations thereof. In some embodiments, a DNA sequence is "related to" a target genome or transcription complex when the nucleic acid is at least partially within the target genome or transcription complex, and the expression of the gene in the DNA sequence is affected by the formation or disruption of the target genome or transcription complex.

[0326] As used herein, the term “domain” refers to a section or portion of an entity. In some embodiments, a “domain” relates to a particular structural and / or functional feature of that entity, and thus, even when physically separated from the rest of its parent entity, it substantially or entirely retains that particular structural and / or functional feature. Alternatively or in addition, in some embodiments, a domain may be a section of an entity that substantially retains and / or confers to one or more structural and / or functional features that characterize it in the parent entity, even when separated from its (parent) entity and linked to a different (recipient) entity. In some embodiments, a domain is a section or portion of a molecule (e.g., a small molecule, carbohydrate, lipid, nucleic acid, polypeptide, etc.) or includes it. In some embodiments, a domain is a section of a polypeptide or includes it. In some such embodiments, the domain is characterized by specific structural elements (e.g., specific amino acid sequences or sequence motifs, α-helix features, β-sheet features, coiled-coil features, random-coil features, etc.) and / or by specific functional features (e.g., binding activity, enzymatic activity, folding activity, signal transduction activity, etc.).

[0327] As used herein, the term “CpG sequence” is also called a “CpG site” or “CpG dyad” and is a region of DNA in which a cytosine nucleoside is linked to a guanine nucleoside by a phosphate group from 5' to 3' (i.e., it has a 5'-C-phosphate-linked-G-3'). A CpG sequence is also referred to as a “CpG dinucleotide”.

[0328] A "CpG island," also known as a "CG island," is a region of the genome that contains CpG sequences at a high frequency. Criteria for identifying GpG and CpG islands are publicly known in the art, for example, as described in Bird et al. (1985) Cell 40:91-99). One definition of a CpG island is a region with (1) a length of at least 200 bp, (2) a GC percentage greater than 50%, and (3) an observed-to-predicted CpG ratio greater than 60%. The observed-to-predicted CpG ratio can be calculated in several ways. Two methods for calculating the observed-to-predicted CpG ratio are as follows: (a) (number of Cs × number of Gs) / length of the array (b) ((Number of Cs + Number of Gs) / Length of the array) 2 For example, Gardiner-Garden M, Frommer M (1987). “CpG islands in vertebrate genomes”. Journal of Molecular Biology. DL (2006). “A genome-wide analysis of CpG dinucleotides in the human genome distinguishes two distinct classes of promoters”. Proc Natl Acad Sci USA.103(5):1412-1417.Bibcode:2006PNAS..103.1412S.doi:10.1073 / pnas.0510310103.PMC 1345710.PMID See 16432200. Sources for identifying CpG islands in mammalian genomes (e.g., the hg19 (GRCh37) or hg38 (GRch38) human reference genomes) are publicly known in the art, such as the UCSC Genome Browser (Worldwide Web: genome.ucsc.edu / cgi-bin / hgTrackUi?g=cpgIslandExt). CpG islands are often found near transcription start sites and promoter regions. In fact, many gene promoters are located within or near CpG islands (see, for example, Saxonov et al (2006) PNAS 103:1412-17).

[0329] As used herein, the term “genomic complex” is a complex that brings together two geometrid sequence elements, spaced apart from each other on one or more chromosomes, through the interaction of two or more proteins and / or other components (potentially including geometrid sequence elements). In some embodiments, the geometrid sequence elements are anchor sequences that bind to one or more protein components of the complex. In some embodiments, the geometrid complex may include an anchor sequence-mediated conjugate. In some embodiments, the geometrid sequence elements may be or include a CTCF-binding motif, a promoter, and / or an enhancer. In some embodiments, the geometrid sequence elements include at least one or both of a promoter and / or a regulatory site (e.g., an enhancer). In some embodiments, complex formation occurs by nucleation in one or more geometrid sequence elements and / or by the binding of one or more protein components to one or more geometrid sequence elements. As those skilled in the art will understand, in some embodiments, when the geometrid sites colocalize due to the formation of a complex (e.g., a conjugate), the DNA topology changes in or near one or more geometrid sequence elements, and in some embodiments, between them. In some embodiments, the genome complex comprises an anchor sequence-mediated conjugate, which comprises one or more loops. In some embodiments, the genome complex as described herein is nucleated by a nucleating polypeptide, such as CTCF and / or cohesin. In some embodiments, the genome complex as described herein may include one or more of the following: CTCF, cohesin, non-coding RNA (e.g., eRNA), transcription machinery proteins (e.g., RNA polymerase, one or more transcription factors, selected from the group consisting of TFIIA, TFIIB, TFIID, TFIIE, TFIIF, TFIIH, etc.), transcription regulators (e.g., mediators, P300, enhancer-binding proteins, repressor-binding proteins, histone modifications, etc.).In some embodiments, the genome 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 in some embodiments may interact with each other and / or with one or more genomic sequence elements (e.g., anchor sequences, promoter sequences, regulatory sequences (e.g., enhancer sequences)), thereby constraining a sequence of genomic DNA into a topological configuration (e.g., a loop) that it would not take if the complex were not formed.

[0330] As used herein, the term “part” as it is used herein refers to a defined chemical group or entity having a particular structure and / or activity.

[0331] As used herein, the term “modulatory agent” refers to an agent comprising one or more target-directed moieties and one or more effector moieties that have the ability to alter (e.g., increase or decrease) the expression of a target gene.

[0332] As used herein, in its broadest sense, the term “nucleic acid” refers to an oligonucleotide chain or any compound and / or substance to which it can be incorporated. In some embodiments, a nucleic acid is an oligonucleotide chain or a compound and / or substance to which it can be incorporated by phosphate diester bonds. As will be apparent from the context, in some embodiments, “nucleic acid” refers to individual nucleic acid residues (e.g., nucleotides and / or nucleosides); in some embodiments, “nucleic acid” refers to an oligonucleotide chain comprising individual nucleic acid residues. In some embodiments, “nucleic acid” is RNA or contains it; in some embodiments, “nucleic acid” is DNA or contains it. In some embodiments, a nucleic acid is one or more native nucleic acid residues, contains them, or consists thereof. In some embodiments, a nucleic acid is one or more nucleic acid analogs, contains them, or consists thereof. In some embodiments, a nucleic acid analog differs from a nucleic acid in that it does not utilize a phosphate diester backbone. For example, in some embodiments, the nucleic acid is, contains, or consists of one or more "peptide nucleic acids," which are known in the art, have peptide bonds in their backbone instead of phosphate diester bonds, and are considered to be within the scope of this disclosure. Alternatively or in addition, in some embodiments, the nucleic acid has one or more phosphorothioate bonds and / or 5'-N-phosphoramidite bonds instead of phosphate diester bonds. In some embodiments, the nucleic acid is, contains, or consists of one or more natural nucleosides (e.g., adenosine, thymidine, guanosine, cytidine, uridine, deoxyadenosine, deoxythymidine, deoxyguanosine, and deoxycytidine).In some embodiments, the nucleic acid is, contains, or consists of one or more nucleoside analogs (e.g., 2-aminoadenosine, 2-thiothymidine, inosine, pyrrolo-pyrimidine, 3-methyladenosine, 5-methylcytidine, C-5 propynylcytidine, C-5 propynyluridine, 2-aminoadenosine, C5-bromouridine, C5-fluorouridine, C5-iodouridine, C5-propynyluridine, C5-propynylcytidine, C5-methylcytidine, 2-aminoadenosine, 7-deazaadenosine, 7-deazaguanosine, 8-oxoadenosine, 8-oxoguanosine, 0(6)-methylguanine, 2-thiocytidine, methylated bases, intercalated bases, and combinations thereof). In some embodiments, nucleic acids contain one or more modified sugars (e.g., 2'-fluororibose, ribose, 2'-deoxyribose, arabinose, and hexose) compared to sugars in natural nucleic acids. In some embodiments, nucleic acids have a nucleotide sequence encoding a functional gene product, such as RNA or a protein. In some embodiments, nucleic acids contain one or more introns. In some embodiments, nucleic acids are prepared by one or more of the following: isolation from natural sources, enzymatic synthesis by polymerization based on complementary templates (in vivo or in vitro), replication of recombinant cells or systems, and chemosynthesis. In some embodiments, the nucleic acid is at least 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 20, 225, 250, 275, 300, 325, 350, 375, 400, 425, 450, 475, 500, 600, 700, 800, 900, 1000, 1500, 2000, 2500, 3000, 3500, 4000, 4500, 5000 residues long or longer. In some embodiments, the nucleic acid is partially or entirely single-stranded; in some embodiments, the nucleic acid is partially or entirely double-stranded.In some embodiments, the nucleic acid has a nucleotide sequence comprising at least one element that encodes a polypeptide or is a complement to a sequence encoding one. In some embodiments, the nucleic acid has enzymatic activity.

[0333] As used herein, the terms “nucleating polypeptide” or “conjugated nucleating polypeptide” mean, as used herein, a protein that associates directly or indirectly with an anchor sequence and, by interacting with one or more conjugated nucleating polypeptides (which may interact with an anchor sequence or other nucleic acids), can form a dimer (or a higher-order structure) composed of two or more such conjugated nucleating polypeptides, which may or may not be identical. When conjugated nucleating polypeptides that associate with different anchor sequences associate with each other, and their different anchor sequences are maintained in close physical proximity to one another, the resulting structure is an anchor-mediated conjugate. That is, when a nucleating polypeptide-anchor sequence interacts with another nucleating polypeptide-anchor sequence and approaches close physical proximity, an anchor-mediated conjugate (e.g., a DNA loop, in some cases) is formed, with the anchor sequences as the start and end. As those skilled in the art will immediately understand upon reading this specification, sometimes terms such as “nucleating polypeptide,” “nucleating molecule,” “nucleating protein,” and “conjugated nucleating protein” are used to refer to conjugated nucleating polypeptides. Similarly, as those skilled in the art will immediately understand upon reading this specification, an assembled collection of two or more conjugate nucleating polypeptides (which may, in some embodiments, include multiple copies of the same drug and / or, in some embodiments, include one or more of each of several different drugs) may be referred to as a “complex,” “dimer,” “multimer,” etc.

[0334] As used herein, the phrase “operably linked” refers to a lateral arrangement such that the components described are in a relationship that allows them to function in their intended manner. A “operably linked” transcriptional regulatory element to a functional element, e.g., a gene, is associated with the functional element, e.g., a gene, in such a way that the expression and / or activity of the functional element is realized under conditions compatible with the transcriptional regulatory element. In some embodiments, an “operably linked” transcriptional regulatory element is contiguous with the coding element of interest, e.g., a gene (e.g., covalently linked); in some embodiments, an operably linked transcriptional regulatory element acts in trans with the functional element of interest, e.g., a gene, or otherwise acts detached from it. In some embodiments, “operably linked” means that two nucleic acid sequences are contained on the same nucleic acid molecule. In further embodiments, “operably linked” may further mean that two nucleic acid sequences are in close proximity to each other on the same nucleic acid molecule, e.g., within 1,000, 500, 100, 50, or 10 base pairs of each other, or directly adjacent to each other.

[0335] As used herein, the terms “peptide,” “polypeptide,” and “protein” refer to compounds composed of amino acid residues covalently linked by peptide bonds or by means other than peptide bonds. A protein or peptide may contain at least two amino acids, and there is no limit to the maximum number of amino acids that can constitute a protein sequence or peptide sequence. A polypeptide includes any peptide or protein containing two or more amino acids linked to each other by peptide bonds or by means other than peptide bonds. As used herein, the terms refer to both short chains, often referred to in the art as peptides, oligopeptides, and oligomers, and longer chains, generally referred to in the art as proteins, which contain many different types.

[0336] As used herein, the term “proximity” means that the locations of the first and second sites in the genome are close enough (e.g., within a base span of up to 2,000 bases) that a function directed at the first site results in a desired functional outcome at the second site, and vice versa. For example, in some embodiments, the first site is a target sequence as described herein, and the second site is a site for epigenetic regulation (e.g., a CpG island), where the first and second sites are close enough that when an expression repressor targets the first site by its DNA targeting portion, the desired epigenetic regulation occurs at the second site by its effector domain. In some embodiments, the first site is a site for epigenetic regulation (e.g., a CpG island), and the second site is a transcriptional regulatory element operably linked to the target gene (e.g., a promoter), where the first and second sites are close enough that when an expression repressor introduces epigenetic regulation at the first site via its effector domain, a change in transcriptional regulation occurs at the second site (e.g., transcriptional regulation that leads to a decrease in the expression of the target gene). In some embodiments, the locations of the first and second sites are within a span of approximately 300 to 2,000 bases, or overlap. In some embodiments, the locations of the first and second sites are within a span of approximately 500 to 1,500 bases, or overlap. In some embodiments, the locations of the first and second sites are within a span of approximately 500 to 1,000 bases, or overlap. As used herein, the term “pharmaceutical composition” refers to a formulation in which an active agent (e.g., a modifier, e.g., an expression inhibitor or expression inhibitor system of the present disclosure) is combined with one or more pharmaceutically acceptable carriers. In some embodiments, the active agent is present in a unit dose appropriate for administration in a therapeutic regimen that exhibits a statistically significant probability of achieving a predetermined therapeutic effect when administered to a relevant population.In some embodiments, the pharmaceutical composition may be specially formulated for administration in solid or liquid form, including, for example, oral administration, e.g., liquid drugs (aqueous or non-aqueous solutions or suspensions), tablets, e.g., buccal, sublingual, and systemic absorption, boluses, powders, granules, pastes applied to the tongue; parenteral administration, e.g., sterile solutions or suspensions, or as sustained-release formulations, e.g., by subcutaneous, intramuscular, intravenous, or epidural injection; topical application, e.g., creams, ointments, or controlled-release patches, or sprays applied to the skin, lungs, or oral cavity; and for administration in solid or liquid form, e.g., pessaries, creams, or foams suitable for intravaginal or rectal, sublingual, intraocular, transdermal, or nasal, lung, and / or other mucosal surfaces.

[0337] As used herein, “proximity” means that two sites, for example, nucleic acid sites, are close together so that the binding of an expression repressor to a first site and / or modification of the first site by the expression repressor produces the same or substantially the same effect as the binding and / or modification of the other site. For example, a target-directing moiety may bind to a first site that is proximity to an enhancer (second site), and the effector moiety associated with the target-directing moiety may epigenetically modify the first site so that the expression of the target gene is modified by substantially the same enhancer effect as if the second site (enhancer sequence) had been bound and / or modified. In some embodiments, the regions adjacent to the target gene (e.g., exons, introns, or splice sites within the target gene), regions adjacent to transcriptional regulatory elements operably linked to the target gene, or regions adjacent to anchor sequences are less than 5,000, 4,000, 3,000, 2,000, 1,000, 900, 800, 700, 600, 500, 400, 300, 200, 100, 50, or 25 base pairs from the target gene (e.g., exons, introns, or splice sites within the target gene), transcriptional regulatory elements, or anchor sequences (and optionally, at least 20, 25, 50, 100, 200, or 300 base pairs from the target gene (e.g., exons, introns, or splice sites within the target gene), transcriptional regulatory elements, or anchor sequences).

[0338] As used herein, the term “specific” in reference to an active agent is understood by those skilled in the art to mean that the agent distinguishes between potential target entities or states. For example, in some embodiments, an agent is said to bind “specifically” to a target if it preferentially binds to that target in the presence of one or more competing alternative targets. In some embodiments, specific interaction depends on the presence of specific structural features of the target entity (e.g., epitopes, cracks, binding sites). It should be understood that specificity does not have to be absolute. In some embodiments, specificity may be evaluated in comparison to the specificity of the binding agent to one or more other potential target entities (e.g., competitors). In some embodiments, specificity is evaluated in comparison to a reference specific binding agent. In some embodiments, specificity is evaluated in comparison to a reference nonspecific binding agent. In some embodiments, an agent or entity does not bind to another competing target under conditions that it binds to its target entity in a detectable manner. In some embodiments, the conjugating agent binds to a target entity with a faster on-rate, slower off-rate, increased affinity, decreased dissociation, and / or increased stability compared to one or more competing targets.

[0339] As used herein, the term “specific binding” refers to the ability to distinguish between possible binding partners in the environment in which binding will occur. In some embodiments, a binding agent that interacts with one particular target in the presence of other potential targets is said to “specifically bind” to the target with which it interacts. In some embodiments, specific binding is determined by detecting or determining the degree of competition between the binding agent and its partner; in some embodiments, specific binding is determined by detecting or determining the degree of dissociation of the binding agent-partner complex; in some embodiments, specific binding is determined by detecting or determining the ability of the binding agent to compete with alternative interactions between its partner and another entity; in some embodiments, specific binding is determined by performing such detection or determination over a range of concentrations.

[0340] As used herein, the term “substantially” refers to a qualitative condition that exhibits the full or nearly full range or degree of the desired feature or characteristic. Those skilled in the art will understand that it is rare, if any, for biological and chemical events to go to completion and / or proceed to completeness or to achieve or avoid absolute results. Therefore, in some embodiments herein, the term “substantially” may be used to capture the inherent lack of completeness in many biological and chemical events.

[0341] A drug or entity is considered to “target” another drug or entity if, according to this disclosure, it specifically binds to that other drug or entity under conditions in which they are in contact with each other. In some embodiments, for example, an antibody (or its antigen-binding fragment) targets its cognitive epitope or antigen. In some embodiments, a nucleic acid having a particular sequence targets a nucleic acid with a substantially complementary sequence.

[0342] As used herein, the term “target gene” means, for example, a gene whose expression is targeted for regulation. In some embodiments, the target gene is part of a targeted genomic complex (e.g., a gene located inside, for example, an anchor sequence-mediated conjugate, having at least a portion of its genomic sequence as part of the target genomic complex), and the genomic complex is targeted by one or more regulatory agents as described herein. In some embodiments, the regulation includes inhibition of the expression of the target gene. In some embodiments, the target gene is regulated by the target gene or a transcriptional regulatory element operably linked to the target gene coming into contact with an expression repression system as described herein, for example, one or more repressors. In some embodiments, the target gene is abnormally expressed (e.g., overexpressed) in cells, for example, in the cells of a subject (e.g., a patient).

[0343] As used herein, the term “target-directed portion” means a drug or entity that specifically targets, for example, a regulatory or anchoring sequence of a genome sequence. In some embodiments, the genome sequence element is adjacent to and / or operably linked to a target gene.

[0344] As used herein, the term “therapeutic agent” refers to an agent that, when administered to a subject, exerts a therapeutic effect and / or elicits a desired biological and / or pharmacological effect. In some embodiments, a therapeutic agent is any substance that can be used to reduce, improve, alleviate, inhibit, prevent, delay the onset, reduce the severity, and / or decrease the incidence of one or more symptoms or characteristics of a disease, disorder, and / or pathological condition. In some embodiments, a therapeutic agent includes an expression suppression system as described herein, e.g., an expression suppressor. In some embodiments, a therapeutic agent includes a nucleic acid encoding an expression suppression system as described herein, e.g., an expression suppressor. In some embodiments, a therapeutic agent includes a pharmaceutical composition as described herein.

[0345] As used herein, the term “therapeutic effective dose” means the amount of a substance (e.g., a therapeutic agent, composition, and / or formulation) that, when administered as part of a therapeutic regimen, elicits a desired biological response. In some embodiments, the therapeutic effective dose of a substance is an amount sufficient to treat, diagnose, prevent, and / or delay the onset of a disease, disorder, and / or condition when administered to a subject suffering from or susceptible to such a disease, disorder, and / or condition. As those skilled in the art will understand, the effective dose of a substance may vary depending on factors such as one or more desired biological endpoints, the substance to be delivered, one or more target cells, or one or more target tissues. For example, in some embodiments, the effective dose of a compound in a formulation for the treatment of a disease, disorder, and / or condition is an amount that reduces, improves, alleviates, inhibits, prevents, delays the onset, reduces its severity, and / or decreases its incidence of one or more symptoms or characteristics of the disease, disorder, and / or condition.

[0346] Other Embodiments Embodiment 1. A method for increasing DNA methylation of a target gene, comprising administering to a subject a certain dose of a composition comprising an expression repressor or a nucleic acid molecule encoding an expression repressor, wherein the expression repressor comprises a DNA target-directing moiety and a DNA methyltransferase, and the DNA methylation of the target gene increases in the subject for a period of at least 21 days after administration of the dose, provided that the subject has not received a subsequent dose during that period.

[0347] Embodiment 2. A method for increasing the DNA methylation of a target gene, comprising administering to a subject a first dose of a composition comprising an expression repressor or a nucleic acid molecule encoding an expression repressor, wherein the expression repressor comprises a DNA target-directing moiety and a DNA methyltransferase, and the DNA methylation of the target gene increases in the subject for a period of at least 21 days after administration of the first dose.

[0348] Embodiment 3. A method for reducing the expression of a target gene, comprising administering to a subject a first dose of a composition comprising an expression repressor or a nucleic acid molecule encoding an expression repressor, wherein the expression repressor comprises a DNA target-directing portion and a DNA methyltransferase, and the expression of the target gene is reduced in the subject for a period of at least 21 days after administration of the first dose.

[0349] Embodiment 4. The method according to Embodiment 2 or 3, wherein the subject is not administered the following doses within the specified period.

[0350] Embodiment 5. A method for increasing DNA methylation of a target gene in a cell, comprising contacting the cell with a first dose of a composition comprising an expression repressor or a nucleic acid molecule encoding an expression repressor, wherein the expression repressor comprises a DNA target-directing moiety and a DNA methyltransferase, and the DNA methylation of the target gene increases for a period of at least 21 days after contact with the first dose.

[0351] Embodiment 6. A method for reducing the expression of a target gene in cells, comprising contacting cells with a first dose of a composition comprising an expression repressor or a nucleic acid molecule encoding an expression repressor, wherein the expression repressor comprises a DNA target-directing moiety and a DNA methyltransferase, and the expression of the target gene is reduced for a period of at least 21 days after contact with the first dose.

[0352] Embodiment 7. A method for reducing the expression of a target gene, comprising administering to a subject a certain dose of an expression repressor or a nucleic acid molecule encoding an expression repressor, wherein the expression repressor comprises a DNA target-directing portion and a DNA methyltransferase, and the expression of the target gene is reduced for a period of at least 21 days after the administration of the dose, provided that the subject does not receive a subsequent dose during that period, thereby reducing the expression of the target gene.

[0353] Embodiment 8. The method according to Embodiment 7, wherein the decrease in the expression of the target gene is measured in a tissue sample obtained from the subject compared to a control sample.

[0354] Embodiment 9. The method according to Embodiment 8, wherein the level of one or more biomarkers related to a target gene is reduced in a tissue sample compared to a control sample.

[0355] Embodiment 10. A method for treating a pathological condition related to dysregulation of a target gene, comprising administering to a subject a certain dose of an expression repressor or a nucleic acid molecule encoding an expression repressor, wherein the expression repressor comprises a DNA target-directing portion and a DNA methyltransferase, and the expression of the target gene is reduced and / or DNA methylation of the target gene is increased for a period of at least 21 days after administration of the dose, provided that the subject has not received a subsequent dose during the period, and thereby the pathological condition is treated.

[0356] Embodiment 11. A method for treating a pathological condition related to dysregulation of a target gene, comprising administering to a subject a first dose of an expression repressor or a nucleic acid molecule encoding an expression repressor, wherein the expression repressor comprises a DNA target-directing portion and a DNA methyltransferase, and the expression of the target gene is reduced and / or DNA methylation of the target gene is increased for a period of at least 21 days after administration of the first dose, thereby treating the pathological condition.

[0357] Embodiment 12. The method according to Embodiment 11, wherein the subject is not administered the following dose within the specified period.

[0358] Embodiment 13. The method according to any one of Embodiments 10 to 12, wherein the pathological condition is related to the overexpression of a target gene.

[0359] Embodiment 14. The method according to any one of Embodiments 1 to 13, wherein DNA methylation of a target gene is increased over a period of at least 21 days to 6 months.

[0360] Embodiment 15. The method according to any one of Embodiments 1 to 14, wherein the DNA methylation of the target gene is increased by at least about 1.5 times, 2 times, 3 times, 4 times, 5 times, 10 times, 15 times, 20 times, 30 times, 40 times, or 50 times.

[0361] Embodiment 16. The method according to any one of Embodiments 1 to 14, wherein DNA methylation of the target gene is increased by at least 20 to 50 times.

[0362] Embodiment 17. The method according to any one of Embodiments 1 to 16, wherein the expression of a target gene is reduced.

[0363] Embodiment 18. The method according to any one of Embodiments 1 to 17, wherein the expression of the target gene is reduced by at least 25%.

[0364] Embodiment 19. The method according to any one of Embodiments 1 to 18, wherein a DNA methyltransferase increases the methylation of at least one CpG dinucleotide in a target gene.

[0365] Embodiment 20. The method according to Embodiment 19, wherein at least one CpG dinucleotide is located in the promoter of the target gene.

[0366] Embodiment 21. The method according to any one of Embodiments 1 to 20, wherein the DNA target-directing moiety binds to a region of the target gene.

[0367] Embodiment 22. The method according to any one of Embodiments 1 to 20, wherein the DNA target-directing moiety binds to a region of a promoter, an anchor sequence, or a cis-regulating element.

[0368] Embodiment 23. The method according to Embodiment 22, wherein the anchor array includes a CTCF binding site or a YY1 binding site.

[0369] Embodiment 24. The method according to any one of Embodiments 1 to 20, wherein the DNA targeting portion targets the repressor system to an isolated genomic domain (IGD) containing a target gene.

[0370] Embodiment 25. The method according to any one of Embodiments 1 to 24, wherein the DNA targeting moiety comprises a zinc finger (ZF) domain or a transcription activator-like effector (TALE) domain.

[0371] Embodiment 26. The method according to any one of Embodiments 1 to 25, wherein the DNA methyltransferase is MQ1, DNMT1, DNMT3A1, DNMT3A1, DNMT3B1, DNMT3B2, DNMT3B3, DNMT3B4, DNMT3B5, DNMT3B6, DNMT3L, or a functional variant or fragment thereof.

[0372] Embodiment 27. The method according to Embodiment 26, wherein the DNA methyltransferase is MQ1, or a functional variant or fragment thereof.

[0373] Embodiment 28. The method according to any one of Embodiments 1 to 27, wherein the expression repressor is a fusion protein containing a DNA target-directing moiety operably linked to a DNA methyltransferase.

[0374] Embodiment 29. The method according to Embodiment 28, wherein the DNA targeting portion is linked to a DNA methyltransferase by a linker.

[0375] Embodiment 30. The method according to Embodiment 29, wherein the linker is a peptide linker.

[0376] Embodiment 31. The method according to Embodiment 30, wherein the peptide linker is a Gly-Ser linker.

[0377] Embodiment 32. The method according to any one of Embodiments 1 to 31, wherein the target gene is a gene associated with cancer.

[0378] Embodiment 33. The method according to any one of Embodiments 1 to 31, wherein the target gene is a gene associated with a metabolic disease or disorder.

[0379] Embodiment 34. The method according to any one of Embodiments 1 to 31, wherein the target gene is a pro-inflammatory gene.

[0380] Embodiment 35. The method according to any one of Embodiments 1 to 34, wherein a nucleic acid molecule encoding an expression suppressor is administered to or brought into contact with cells.

[0381] Embodiment 36. The method according to Embodiment 35, wherein the nucleic acid molecule is a messenger RNA (mRNA) encoding an expression repressor.

[0382] Embodiment 37. The method according to Embodiment 36, wherein the mRNA comprises a 3'UTR, a poly(A) tail, a ribosome skipping sequence, or any combination thereof.

[0383] Embodiment 38. The method according to Embodiment 36 or 37, wherein the repressor is a first repressor, and the mRNA comprises a nucleotide sequence encoding a second repressor including a second DNA targeting moiety and an effector domain.

[0384] Embodiment 39. The method according to Embodiment 38, wherein the second expression repressor is a fusion protein comprising a second DNA targeting moiety operably linked to the effector domain.

[0385] Embodiment 40. The method according to Embodiment 38 or 39, wherein the second DNA target-directing moiety binds to a region in the target gene different from the first expression repressor.

[0386] Embodiment 41. The method according to any one of Embodiments 38 to 40, wherein the effector domain is a histone modifying enzyme selected from DNA methyltransferase or histone methyltransferase, histone deacetylase, and histone demethylase.

[0387] Embodiment 42. The method according to Embodiment 41, wherein the DNA methyltransferase is the same DNA methyltransferase as the DNA methyltransferase of the first expression repressor.

[0388] Embodiment 43. The method according to Embodiment 41, wherein the DNA methyltransferase is a different DNA methyltransferase from the DNA methyltransferase of the first expression repressor.

[0389] Embodiment 44. The method according to Embodiment 41, wherein the histone modifying enzyme is histone deacetylase.

[0390] Embodiment 45. The method according to Embodiment 44, wherein the histone deacetylase is 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 thereof.

[0391] Embodiment 46. The method according to Embodiment 41, wherein the histone modifying enzyme is a histone methyltransferase.

[0392] Embodiment 47. The method according to Embodiment 46, wherein the histone methyltransferase is SETDB1, SETDB2, EHMT2, EHMT1, SUV39H1, EZH2, EZH1, SUV39H2, SETD8, SUV420H1, SUV420H2, or a functional variant or fragment thereof.

[0393] Embodiment 48. The method according to Embodiment 41, wherein the effector domain comprises a Kruppel-associated box (KRAB) domain or a functional variant or fragment thereof.

[0394] Embodiment 49. The method according to any one of Embodiments 38 to 48, wherein the mRNA includes a ribosome skipping sequence between the nucleotide sequence encoding the first repressor and the nucleotide sequence encoding the second repressor.

[0395] Embodiment 50. The method according to any one of Embodiments 35 to 49, wherein nucleic acid molecules are encapsulated in lipid nanoparticles.

[0396] Embodiment 51. The method according to Embodiment 50, wherein the lipid nanoparticles include an ionizable cationic lipid.

[0397] Embodiment 52. The method according to Embodiment 50 or 51, wherein the lipid nanoparticles include one or more neutral lipids, ionized cationic amine-containing lipids, essential cationic amine-containing lipids, biodegradable alkyne (alkyn) lipids, steroids, phospholipids, polyunsaturated lipids, structural lipids, PEG lipids, cholesterol, or polymer conjugate lipids.

[0398] Embodiment 53. A method for increasing DNA methylation of a target gene, comprising...

Claims

1. A method for increasing DNA methylation of a target gene, comprising administering to the subject a first dose of a composition comprising lipid nanoparticles comprising mRNA encoding a fusion protein having a DNA target-directing moiety linked to a DNA methyltransferase, with or without a linker, wherein the DNA target-directing moiety comprises a ZF domain or a TALE domain, and the proportion of methylated CpG dinucleotides in the promoter region of the target gene increases by at least 5 times over a period of at least 21 days after administration of the first dose to the subject.

2. The method according to claim 1, wherein the subject is not administered the following dose within the period.

3. The method according to claim 1 or 2, wherein the DNA methylation of the target gene increases over a period of at least 21 days to 6 months.

4. The method according to any one of claims 1 to 3, wherein the proportion of the methylated CpG dinucleotide increases by approximately 5 times, 10 times, 15 times, 20 times, 30 times, 40 times, or 50 times.

5. The method according to any one of claims 1 to 4, wherein the expression of the target gene is reduced.

6. The method according to any one of claims 1 to 5, wherein the expression of the target gene is reduced by at least 25%.

7. The method according to any one of claims 1 to 6, wherein the DNA target-directing portion binds to a region of the target gene.

8. The method according to any one of claims 1 to 6, wherein the DNA target-directing moiety is bound to a region of a promoter, an anchor sequence, or a cis-regulating element.

9. The method according to any one of claims 1 to 8, wherein the anchor array includes a CTCF binding site or a YY1 binding site.

10. The method according to any one of claims 1 to 8, wherein the DNA targeting portion targets the fusion protein to an isolated genomic domain (IGD) containing the target gene.

11. The method according to any one of claims 1 to 12, wherein the DNA methyltransferase is MQ1, DNMT1, DNMT3A1, DNMT3A1, DNMT3B1, DNMT3B2, DNMT3B3, DNMT3B4, DNMT3B5, DNMT3B6, DNMT3L, or a functional variant or fragment thereof.

12. The method according to claim 13, wherein the DNA methyltransferase is MQ1, or a functional variant or fragment thereof.

13. The method according to claim 28, wherein the DNA targeting portion is linked to the DNA methyltransferase by a linker.

14. The method according to claim 15, wherein the linker is a peptide linker.

15. The method according to claim 16, wherein the peptide linker is a Gly-Ser linker.

16. The method according to any one of claims 1 to 17, wherein the target gene is a gene associated with cancer.

17. The method according to any one of claims 1 to 17, wherein the target gene is a gene associated with a metabolic disease or disorder.

18. The method according to any one of claims 1 to 17, wherein the target gene is a pro-inflammatory gene.

19. The method according to any one of claims 1 to 20, wherein the nucleic acid molecule encoding the fusion protein is administered to the target or brought into contact with the cells.

20. The method according to claim 21, wherein the nucleic acid molecule is messenger RNA (mRNA) encoding the fusion protein.

21. The method according to claim 22, wherein the mRNA comprises a 3' UTR, a poly-A tail, a ribosome skipping sequence, or any combination thereof.

22. The method according to any one of claims 21 to 23, wherein the nucleic acid molecule is encapsulated in lipid nanoparticles.

23. The method according to claim 24, wherein the lipid nanoparticles include an ionizable cationic lipid.

24. The method according to claim 24 or 25, wherein the lipid nanoparticles include one or more neutral lipids, ionized cationic amine-containing lipids, essential cationic amine-containing lipids, biodegradable alkyne lipids, steroids, phospholipids, polyunsaturated lipids, structural lipids, PEG lipids, cholesterol, or polymer conjugate lipids.

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