Compositions and methods for epigenetic regulation of HBV gene expression

Abstract

The present invention relates to compositions, methods, strategies, and treatment approaches related to epigenetic modification of Hepatitis B virus (HBV) genes.

Description

[Technical Field]

[0001] cross reference

[0001] This application claims the benefit of U.S. Provisional Patent Application No. 63 / 409,607, filed September 23, 2022, U.S. Provisional Patent Application No. 63 / 502,328, filed May 15, 2023, U.S. Provisional Patent Application No. 63 / 516,063, filed July 27, 2023, and U.S. Provisional Patent Application No. 63 / 581,229, filed September 7, 2023, each of which is incorporated by reference in its entirety into this specification. [Background technology]

[0002] Despite the availability of treatments, chronic hepatitis B (CHB) remains a major unmet medical need, with 250 million hepatitis B virus (HBV) carriers worldwide and approximately 800,000 deaths annually due to HBV-related liver disease. Currently approved CHB therapies result in functional cure rates (defined as persistent HBsAg loss and undetectable serum HBV after completing a course of treatment) of less than 20%. Therefore, improved clinical approaches targeting HBV are needed. Summary of the Invention

[0003]

[0003] Some aspects of the present disclosure provide systems, compositions, strategies and methods for epigenetic modification of HBV, including HBV, in host cells and organisms.

[0004] Some embodiments of the present disclosure provide a method for modifying the epigenetic state of a hepatitis B virus (HBV) gene or genome, comprising contacting the HBV gene or genome with an epigenetic editing system, wherein the epigenetic editing system comprises a first DNA binding domain, a first DNMT domain, and a transcriptional repressor domain, or one or more nucleic acid molecules encoding them, and wherein the first binding domain binds to a first target region of the HBV gene or genome, and wherein the contacting results in a reduction in the number of HBV viral episomes, replication of the HBV gene or genome, and / or expression of a protein product encoded by the HBV gene or genome, wherein the reduction is at least about 20% compared to contacting the HBV gene or genome with a suitable control, and / or the reduction in the number of HBV viral episomes, replication of the HBV gene or genome, or expression of a protein product encoded by the HBV gene or genome is at least about 20% compared to the number, replication, and / or expression in the subject prior to administration. Some embodiments of the present disclosure provide a method of treating HBV infection in a subject, comprising administering an epigenetic editing system to the subject, wherein the epigenetic editing system comprises a first DNA-binding domain, a first DNMT domain, and a transcriptional repressor domain, or one or more nucleic acid molecules encoding them, wherein binding and contacting the first DNA-binding domain to a first target region of an HBV gene or genome results in a reduction in the number of HBV viral episomes, replication of the HBV gene or genome, and / or expression of a protein product encoded by the HBV gene or genome, wherein the reduction is at least about 20% compared to administering a suitable control, and / or the reduction in the number of HBV viral episomes, replication of the HBV gene or genome, or expression of a protein product encoded by the HBV gene or genome is at least about 20% compared to the number, replication, and / or expression in the subject prior to the administering step.Some aspects of the present disclosure provide a method of modulating expression of an HBV gene or genome, comprising contacting the HBV gene or genome with an epigenetic editing system, wherein the epigenetic editing system comprises a first DNA-binding domain, a first DNMT domain, and a transcriptional repressor domain, or one or more nucleic acid molecules encoding them, wherein the first DNA-binding domain binds to a first target region of the HBV gene or genome, and wherein the contacting results in a reduction in expression of a gene product encoded by the HBV gene or genome, optionally wherein the gene product is a nucleic acid or protein, and wherein the reduction is at least about 20% compared to contacting the HBV genome with a suitable control, and / or wherein the reduction of the gene product encoded by the HBV gene or genome is at least about 20% compared to expression in the subject prior to administration. Some embodiments of the present disclosure provide a method for inhibiting viral replication in cells infected with HBV, comprising administering an epigenetic editing system, wherein the epigenetic editing system comprises a first DNA-binding domain, a first DNMT domain, and a transcriptional repressor domain, or one or more nucleic acid molecules encoding them, wherein the first DNA-binding domain binds to a first target region of an HBV gene or genome, and wherein the epigenetic editing system targets the target region of the HBV gene or genome, and wherein the contacting results in a reduction in the number of HBV viral episomes or replication of the HBV gene or genome, wherein the reduction is at least about 20% compared to administering a suitable control, and / or the reduction in the number of HBV viral episomes or replication of the HBV gene or genome is at least about 20% compared to the number and / or replication in the subject prior to the administering step.Some aspects of the present disclosure provide methods comprising administering an epigenetic editing system to a subject in need thereof, wherein the epigenetic editing system comprises a first DNA-binding domain, a first DNMT domain, and a transcriptional repressor domain, or one or more nucleic acid molecules encoding them, wherein binding and contacting the first DNA-binding domain to a first target region of an HBV gene or genome results in a reduction in the number of HBV viral episomes, replication of the HBV gene or genome, or expression of a protein product encoded by the HBV gene or genome, wherein the reduction is at least about 20% compared to administering a suitable control, and / or the reduction in the number of HBV viral episomes, replication of the HBV gene or genome, or expression of a protein product encoded by the HBV gene or genome is at least about 20% compared to the number, replication, and / or expression in the subject prior to the administering step. In some embodiments, the HBV genome is a covalently closed circular DNA (cccDNA) or HBV integrated DNA. In some embodiments, the HBV genome comprises HBV genotype A, HBV genotype B, HBV genotype C, HBV genotype D, HBV genotype E, HBV genotype F, HBV genotype G, or HBV genotype H. In some embodiments, the HBV genome comprises a sequence having at least 80% identity to an HBV genome sequence provided herein. In some embodiments, the first target region is located in a region of the HBV genome within nucleotides 0-303, 1000-2448, or 2802-3182 of the HBV genome provided herein. In some embodiments, the first target region of the HBV genome is located in a CpG island. In some embodiments, the first target region of the HBV genome is located in a promoter. In some embodiments, the first target region of the HBV genome is located in an area of ​​the HBV genome encoding a transcript selected from the group consisting of pgRNA, precure mRNA, preS mRNA, S mRNA, and X mRNA. In some embodiments, the first DNA binding domain comprises a CRISPR-Cas protein.In some embodiments, the epigenetic editing system further comprises a first guide RNA (gRNA) comprising a region complementary to a strand of the first target region. In some embodiments, the gRNA comprises a sequence selected from the gRNAs provided and / or disclosed herein, e.g., in Tables 14 and / or 15. In some embodiments, the first DNA-binding domain comprises a zinc finger protein. In some embodiments, the zinc finger protein comprises a zinc finger motif having a sequence selected from any zinc finger or zinc finger motif provided herein, e.g., in Table 1. In some embodiments, the zinc finger protein comprises the sequence of any of the zinc finger epigenetic repressors provided herein. In some embodiments, the transcriptional repressor domain comprises ZIM3. In some embodiments, the first DNMT domain is a DNMT3A domain or a DNMT3L domain. In some embodiments, the first DNMT domain comprises the sequence of a DNMT domain provided herein. In some embodiments, the epigenetic editing system further comprises a second DNMT domain or a nucleic acid encoding same. In some embodiments, the second DNMT domain is a DNMT3A domain or a DNMT3L domain. In some embodiments, the second DNMT domain comprises a sequence of a DNMT domain provided herein. In some embodiments, the epigenetic editing system comprises a fusion protein or a nucleic acid encoding the same, wherein the fusion protein comprises a first DNA-binding domain, a first DNMT domain, a repressor domain, and a second DNMT domain. In some embodiments, the fusion protein further comprises a nuclear localization sequence (NLS). In some embodiments, the fusion protein comprises a sequence of a fusion protein provided herein. In some embodiments, the epigenetic editing system further comprises a second DNA-binding domain or a nucleic acid encoding the same, wherein the second DNA-binding domain binds to a second target region of the HBV genome. In some embodiments, the second target region is located in a region of the HBV genome within nucleotides 0-303, 1000-2448, or 2802-3182.In some embodiments, the second target region of the HBV genome is located in a CpG island. In some embodiments, the second target region of the HBV genome is located in a promoter. In some embodiments, the second target region of the HBV genome is located in a section of the HBV genome encoding a transcript selected from the group consisting of pgRNA, preCu mRNA, preS mRNA, S mRNA, and X mRNA. In some embodiments, the second DNA-binding domain comprises a CRISPR-Cas protein. In some embodiments, the epigenetic editing system further comprises a second gRNA comprising a region complementary to a strand of the second target region. In some embodiments, the gRNA comprises a sequence selected from the gRNA sequences provided herein, e.g., the sequences provided and / or disclosed in Tables 14 and / or 15. In some embodiments, the second DNA-binding domain comprises a zinc finger protein. In some embodiments, the zinc finger protein comprises a zinc finger motif having a zinc finger motif sequence provided herein, e.g., a sequence selected from the zinc finger motifs provided in Table 1. In some embodiments, the zinc finger protein comprises a zinc finger motif sequence provided in Table 1. In some embodiments, the epigenetic editing system comprises a first fusion protein or a first nucleic acid encoding the same, and a second fusion protein or a second nucleic acid encoding the same, wherein the first fusion protein comprises a first DNA-binding domain and a first DNMT domain, and the second fusion protein comprises a second DNA-binding domain and a transcriptional repressor domain. In some embodiments, the first fusion protein comprises a sequence of a fusion protein provided herein. In some embodiments, the second fusion protein comprises a sequence of a fusion protein provided herein. In some embodiments, the epigenetic editing system further comprises a third DNA-binding domain or a nucleic acid encoding the same, wherein the third DNA-binding domain binds to a third target region of the HBV genome. In some embodiments, the third target region is located in a region of the HBV genome within nucleotides 0-303, 1000-2448, or 2802-3182.In some embodiments, the third target region of the HBV genome is located in a CpG island. In some embodiments, the third target region of the HBV genome is located in a promoter. In some embodiments, the third target region of the HBV genome is located in a section of the HBV genome encoding a transcript selected from the group consisting of pgRNA, preCu mRNA, preS mRNA, S mRNA, and X mRNA. In some embodiments, the third DNA binding domain comprises a CRISPR-Cas protein. In some embodiments, the epigenetic editing system further comprises a third gRNA comprising a region complementary to a strand of the third target region. In some embodiments, the third gRNA comprises a sequence selected from the gRNA sequences provided herein, e.g., the gRNA sequences provided and / or disclosed in Tables 14 and / or 15. In some embodiments, the third DNA binding domain The epigenetic editing system comprises a zinc finger protein. In some embodiments, the zinc finger protein comprises a zinc finger motif having a sequence selected from the zinc finger motifs provided herein. In some embodiments, the zinc finger protein comprises the sequence of a zinc finger motif provided in Table 1. In some embodiments, the epigenetic editing system further comprises a second DNMT domain or a nucleic acid encoding same. In some embodiments, the second DNMT domain is a DNMT3A domain or a DNMT3L domain. In some embodiments, the epigenetic editing system comprises a third fusion protein or a nucleic acid encoding same, wherein the third fusion protein comprises a third DNA-binding domain and a second DNMT domain. In some embodiments, the third fusion protein comprises the sequence of a fusion protein provided herein. In some embodiments, the epigenetic editing system comprises a nucleic acid sequence provided in Table 20. In some embodiments, the reduction in the number of HBV viral episomes, replication of HBV genes or genomes, or expression of protein products encoded by HBV genes or genomes is at least about 20% compared to the number of HBV viral episomes, replication of HBV genes or genomes, or expression of protein products encoded by HBV genes or genomes measured or observed before contacting the HBV genome with the epigenetic editing system or before administering the epigenetic editing system to a subject.In some embodiments, the reduction in the number of HBV viral episomes, replication of HBV genes or genomes, or expression of protein products encoded by HBV genes or genomes is at least about 25%, at least about 50%, at least about 75%, at least about 80%, at least about 90%, at least about 95%, at least about 99%, at least about 99.5%, at least about 99.8%, at least about 99.9%, at least about 99.95%, at least about 99.99%, or greater than 99.99%, compared to the number of HBV viral episomes, replication of HBV genes or genomes, or expression of protein products encoded by HBV genes or genomes measured or observed before contacting the HBV genome with the epigenetic editing system or before administering the epigenetic editing system to a subject.

[0004]

[0005] Some aspects of the present disclosure provide an epigenetic editing system comprising a fusion protein or a nucleic acid encoding the fusion protein, wherein the fusion protein comprises (a) a DNA-binding domain that binds to a target region of an HBV gene or genome, (b) a first DNA methyltransferase (DNMT) domain, and (c) a transcriptional repressor domain. In some embodiments, the epigenetic editing system can reduce the number of HBV viral episomes, HBV replication, or expression of a gene product encoded by an HBV gene or genome, wherein the reduction is at least about 20% compared to contacting the HBV gene or genome with an appropriate control. In some embodiments, the HBV genome is a covalently closed circular DNA (cccDNA) or HBV integrated DNA. In some embodiments, the HBV genome comprises HBV genotype A, HBV genotype B, HBV genotype C, HBV genotype D, HBV genotype E, HBV genotype F, HBV genotype G, or HBV genotype H. In some embodiments, the HBV genome comprises a sequence having at least 80% identity to an HBV genome sequence provided herein. In some embodiments, the target region is located in a region of the HBV genome within nucleotides 0-303, 1000-2448, or 2802-3182 of an HBV genome sequence provided herein. In some embodiments, the target region of the HBV genome is located in a CpG island. In some embodiments, the target region of the HBV genome is located in a promoter. In some embodiments, the target region of the HBV genome is located in a section of the HBV genome encoding a transcript selected from the group consisting of pgRNA, preCure mRNA, preS mRNA, S mRNA, and X mRNA. In some embodiments, the DNA-binding domain comprises a CRISPR-Cas protein. In some embodiments, the epigenetic editing system further comprises a gRNA comprising a region complementary to a strand of the target region. In some embodiments, the gRNA comprises a sequence selected from the gRNA sequences provided herein, e.g., in Tables 14 and / or 15. In some embodiments, the DNA-binding domain comprises a zinc finger protein.In some embodiments, the zinc finger protein comprises a zinc finger motif having a sequence selected from the zinc finger motifs provided herein. In some embodiments, the zinc finger protein comprises the sequence of a zinc finger motif provided in Table 1. In some embodiments, the transcriptional repressor domain comprises the sequence of a transcriptional repressor provided herein. In some embodiments, the first DNMT domain is a DNMT3A domain or a DNMT3L domain. In some embodiments, the DNMT domain comprises the sequence of a DNMT domain provided herein. In some embodiments, the fusion protein further comprises a second DNMT domain. In some embodiments, the second DNMT domain is a DNMT3A domain or a DNMT3L domain. In some embodiments, the fusion protein further comprises a nuclear localization sequence (NLS). In some embodiments, the fusion protein comprises the sequence of a fusion protein provided herein. Some aspects of the present disclosure provide an epigenetic editing system comprising: a first fusion protein or a nucleic acid encoding the first fusion protein, wherein the first fusion protein comprises a first DNA-binding domain and a first DNMT domain, and the first DNA-binding domain binds to a first target region of the HBV genome; and a second fusion protein or a nucleic acid encoding the second fusion protein, wherein the second fusion protein comprises a second DNA-binding domain and a transcriptional repressor domain, and the second DNA-binding domain binds to a second target region of the HBV genome. In some embodiments, the epigenetic editing system can reduce the number of HBV viral episomes, HBV replication, or expression of a gene product encoded by the HBV genome by at least about 20% compared to contacting the HBV genome with an appropriate control. In some embodiments, the HBV genome is a covalently closed circular DNA (cccDNA) or HBV integrated DNA. In some embodiments, the HBV genome comprises HBV genotype A, HBV genotype B, HBV genotype C, HBV genotype D, HBV genotype E, HBV genotype F, HBV genotype G, or HBV genotype H.In some embodiments, the HBV genome comprises a sequence having at least 80% identity to the HBV genome provided herein. In some embodiments, the epigenetic editing system further comprises a third fusion protein or a nucleic acid encoding the third fusion protein, wherein the third fusion protein comprises a third DNA-binding domain and a second DNMT domain, and the third DNA-binding domain binds to a third target region of the HBV genome. In some embodiments, the first target region, the second target region, or the third target region is located in a region of the HBV genome within nucleotides 0-303, 1000-2448, or 2802-3182 of the HBV genome provided herein. In some embodiments, the first target region, the second target region, or the third target region of the HBV genome is located in a CpG island. In some embodiments, the first target region, the second target region, or the third target region of the HBV genome is located in a promoter. In some embodiments, the first target region, the second target region, or the third target region of the HBV genome is located in a section of the HBV genome that encodes a transcript selected from the group consisting of pgRNA, preCu mRNA, preS mRNA, S mRNA, and X mRNA. In some embodiments, the first DNA-binding domain, the second DNA-binding domain, or the third DNA-binding domain comprises a CRISPR-Cas protein. In some embodiments, the epigenetic editing system further comprises a first gRNA comprising a region complementary to a strand of the first target region, a second gRNA comprising a region complementary to a strand of the second target region, or a third gRNA comprising a region complementary to a strand of the third target region. In some embodiments, the first gRNA comprises a sequence selected from the gRNA sequences provided herein, e.g., as provided and / or disclosed in Tables 14 and / or 15, the second gRNA comprises a sequence selected from the gRNA sequences provided herein, e.g., as provided and / or disclosed in Tables 14 and / or 15, and / or the third gRNA comprises a sequence selected from the gRNA sequences provided herein, e.g., as provided and / or disclosed in Tables 14 and / or 15.In some embodiments, the first DNA-binding domain, the second DNA-binding domain, or the third DNA-binding domain comprises a zinc finger protein. In some embodiments, the zinc finger protein comprises a zinc finger motif having a sequence selected from the zinc finger motifs provided herein. In some embodiments, the zinc finger protein comprises the sequence of a zinc finger motif provided in Table 1. In some embodiments, the transcriptional repressor domain comprises ZIM3. In some embodiments, the first DNMT domain is a DNMT3A domain or a DNMT3L domain. In some embodiments, the first DNMT domain comprises the sequence of a DNMT provided herein. In some embodiments, the second DNMT domain is a DNMT3A domain or a DNMT3L domain. In some embodiments, the second DNMT domain comprises the sequence of a DNMT domain provided herein. In some embodiments, the first fusion protein comprises the sequence of a fusion protein provided herein. In some embodiments, the second fusion protein comprises the sequence of a fusion protein provided herein. In some embodiments, the third fusion protein comprises the sequence of a fusion protein provided herein. In some embodiments of any of the preceding methods, the epigenetic editing system comprises a nucleic acid sequence provided in Table 20. In some embodiments, the reduction in the number of HBV viral episomes, replication of HBV genes or genomes, or expression of protein products encoded by HBV genes or genomes is at least about 20% compared to the number of HBV viral episomes, replication of HBV genes or genomes, or expression of protein products encoded by HBV genes or genomes measured or observed before contacting the HBV genome with the epigenetic editing system or before administering the epigenetic editing system to the subject.In some embodiments, the reduction in the number of HBV viral episomes, replication of HBV genes or genomes, or expression of protein products encoded by HBV genes or genomes is at least about 25%, at least about 50%, at least about 75%, at least about 80%, at least about 90%, at least about 95%, at least about 99%, at least about 99.5%, at least about 99.8%, at least about 99.9%, at least about 99.95%, at least about 99.99%, or greater than 99.99%, compared to the number of HBV viral episomes, replication of HBV genes or genomes, or expression of protein products encoded by HBV genes or genomes measured or observed before contacting the HBV genome with the epigenetic editing system or before administering the epigenetic editing system to a subject.

[0005]

[0006] Some embodiments of the present disclosure provide methods of treating HDV infection in a subject, the method comprising administering to the subject an epigenetic editing system, wherein the epigenetic editing system comprises a first DNA-binding domain, a first DNMT domain, and a transcriptional repressor domain, or one or more nucleic acid molecules encoding them, wherein binding and contacting the first DNA-binding domain to a first target region of an HBV gene or genome results in a reduction in the number of HDV viral episomes, replication of the HDV gene or genome, or expression of a protein product encoded by the HDV gene or genome, wherein the reduction is at least about 20% compared to administering a suitable control. Some aspects of the present disclosure provide a method for inhibiting viral replication in cells infected with HDV, comprising administering an epigenetic editing system, wherein the epigenetic editing system comprises a first DNA-binding domain, a first DNMT domain, and a transcriptional repressor domain, or one or more nucleic acid molecules encoding them, wherein the first DNA-binding domain binds to a first target region of an HBV gene or genome, and the epigenetic editing system targets the target region of the HBV gene or genome, and the contacting results in a reduction in the number of HDV viral episomes or replication of the HDV gene or genome, the reduction being at least about 20% compared to administering a suitable control. In some embodiments, the first DNA-binding domain comprises a CRISPR-Cas protein. In some embodiments, the epigenetic editing system further comprises a first guide RNA (gRNA) comprising a region complementary to a strand of the first target region. In some embodiments, the gRNA comprises a sequence selected from the gRNAs provided herein, e.g., in Tables 14 and / or 15. In some embodiments, the first DNA-binding domain comprises a zinc finger protein. In some embodiments, the zinc finger protein comprises a zinc finger motif having a sequence selected from any of the zinc fingers or zinc finger motifs provided herein, e.g., in Table 1 or Table 20.In some embodiments, the zinc finger protein comprises the sequence of any of the zinc finger epigenetic repressors provided herein. In some embodiments, the transcriptional repressor domain comprises ZIM3. In some embodiments, the first DNMT domain is a DNMT3A domain or a DNMT3L domain. In some embodiments, the first DNMT domain comprises the sequence of a DNMT domain provided herein. In some embodiments, the epigenetic editing system further comprises a second DNMT domain or a nucleic acid encoding the same. In some embodiments, the second DNMT domain is a DNMT3A domain or a DNMT3L domain. In some embodiments, the second DNMT domain comprises the sequence of a DNMT domain provided herein. In some embodiments, the epigenetic editing system comprises a fusion protein or a nucleic acid encoding the same, wherein the fusion protein comprises a first DNA binding domain, a first DNMT domain, a repressor domain, and a second DNMT domain. In some embodiments, the fusion protein further comprises a nuclear localization sequence (NLS). In some embodiments, the fusion protein comprises the sequence of a fusion protein provided herein. In some embodiments, the first DNA-binding domain binds to a target region of the HBV gene or genome that encodes or controls expression of the S antigen. In some embodiments, the epigenetic editing system comprises a nucleic acid sequence provided in Table 20. In some embodiments, the reduction in the number of HBV viral episomes, replication of the HBV gene or genome, or expression of a protein product encoded by the HBV gene or genome is at least about 20% compared to the number of HBV viral episomes, replication of the HBV gene or genome, or expression of a protein product encoded by the HBV gene or genome measured or observed before contacting the HBV genome with the epigenetic editing system or before administering the epigenetic editing system to a subject.In some embodiments, the reduction in the number of HBV viral episomes, replication of HBV genes or genomes, or expression of protein products encoded by HBV genes or genomes is at least about 25%, at least about 50%, at least about 75%, at least about 80%, at least about 90%, at least about 95%, at least about 99%, at least about 99.5%, at least about 99.8%, at least about 99.9%, at least about 99.95%, at least about 99.99%, or greater than 99.99%, compared to the number of HBV viral episomes, replication of HBV genes or genomes, or expression of protein products encoded by HBV genes or genomes measured or observed before contacting the HBV genome with the epigenetic editing system or before administering the epigenetic editing system to a subject.

[0006]

[0007] Other features, objects, and advantages of the present invention will be apparent from the detailed description which follows. It should be understood, however, that the detailed description, while indicating embodiment(s) of the present invention, is given by way of illustration only and not by way of limitation. Various changes and modifications within the scope of the invention will become apparent to those skilled in the art from the detailed description. [Brief explanation of the drawings]

[0007] [Figure 1]

[0008] Figure 1 is a schematic diagram showing an exemplary structure of a circular HBV genome. HBV genes and CpG islands are indicated. Target sites for CRISPR-based epigenetic repressors (red arrows) and zinc finger-based epigenetic repressors (green arrows) are also indicated. [Figure 2]

[0009] Figure 2 is a heatmap showing the conservation of guide RNA target domains across different HBV genotypes. [Figure 3]

[0010] FIG. 3 is a bar graph showing the geographic distribution of different HBV genotypes. [Figure 4]

[0011] Figure 4A is a schematic diagram describing the experimental timeline for testing different CRISPR-based epigenetic repressors in HepAD38 cells expressing HPV in a doxycycline-inducible manner. Figure 4B is a schematic diagram showing the suppression of HBV by various CRISPR-based epigenetic repressors (#1.1–3.2). Control: UT: untransfected control; GFP: transfection control without repressor; HBV-KO: CRISPR nuclease-mediated knockout; sgRNA-scramble: CRISPR-based repressor and sgRNA not targeting HBV; B2M: CRISPR-based repressor and sgRNA targeting B2M. [Figure 5]

[0012] Figure 5A is a schematic diagram describing the experimental timeline for testing different CRISPR-based epigenetic repressors in the HepG2-NTCP infection model (see, e.g., Methods Mol Biol. 2017;1540:1-14). Figure 5B is a schematic diagram showing HBeAg expression (by ELISA) at different times after treatment of HBV-infected Hep2G-NTCP cells with different doses of CRISPR-based epigenetic repressor (ETR) or different doses of HBV-targeting Cas9 clease (Cas9), normalized and plotted against the HBeAg expression value measured for the negative control (blank). [Figure 6]

[0013] FIG. 6 is a schematic diagram describing the experimental timeline for guide RNA screening testing different CRISPR-based epigenetic repressor systems in the HePG2-NTCP infection model, along with ELISA readouts for HBe and HBsAg on day 6. [Figure 7]

[0014] FIG. 7 is a diagram showing the QC results from different LNP batches used in the guided screening. [Figure 8]

[0015] Figure 8 is a bar graph showing HBe and HBs expression for an exemplary CRISPR-based epigenetic repressor (#3.2), calculated as a percentage of the expression of each antigen measured for a non-targeting control. [Figure 9]

[0016] Figure 9 is a diagram showing the HBe expression values ​​(calculated as a percentage of the HBe expression measured for the non-targeting control) measured in a guide RNA screen for different guides. Each guide / repressor combination is represented by a dot. The 50% repression cutoff is shown as a horizontal line. The location of each guide RNA (shown at the bottom of the graph) within the HBV genome is mapped on the X-axis. The location and measured modulation of HBe expression for exemplary guide RNA #3.2 is shown by a red line. [Figure 10]

[0017] Figure 10 is a diagram showing the HBs expression values ​​(calculated as a percentage of the HBs expression measured for the non-targeting control) measured in a guide RNA screen for different guides. Each guide / repressor combination is represented by a dot. The 50% repression cutoff is shown as a horizontal line. The location of each guide RNA (shown at the bottom of the graph) within the HBV genome is mapped on the X-axis. The location and measured modulation of HBs expression for exemplary guide RNA #3.2 is shown by a red line. [Figure 11]

[0018] Figure 11 is a schematic diagram showing the correlation between HBs and HBe expression for the guides tested. The graph on the right shows the HBe and HBs suppression efficiency for 25 exemplary guides. [Figure 12]

[0019] FIG. 12A is a schematic diagram describing the experimental timeline for a guide RNA assay testing CRISPR-off single construct epigenetic editors in combination with individual exemplary gRNAs in a HepG2-NTCP infection model, along with ELISA readouts for HBe and HBs antigen at day 6; FIG. 12B is a graph summarizing the percentage reduction in HBV antigen relative to non-targeting controls at day 6. [Figure 13]

[0020] FIG. 13A is a schematic describing the experimental timeline for a guide RNA assay testing CRISPR-off single construct epigenetic editors in combination with individual exemplary gRNAs in a PLC / PRF / 5 cell model, along with an ELISA readout for HBsAg at day 4; FIG. 13B is a graph summarizing the percentage reduction in HBsAg relative to non-targeting controls at day 4. [Figure 14A]

[0021] Figure 14A is a diagram describing the experimental timeline for a guide RNA assay testing CRISPR-off single construct epigenetic editors in combination with individual exemplary gRNAs in the PXB cell model, along with ELISA readouts for HBe and HBs antigen at day 6; Figure 14B is a graph summarizing the percentage reduction in HBV antigen relative to non-targeting controls at day 6. Figure 14C is a diagram describing the experimental timeline for a guide RNA assay testing CRISPR-off single construct epigenetic editors in combination with individual exemplary gRNAs in the PXB cell model, along with ELISA readouts for HBe and HBs antigen at day 12. Figure 14D is a graph summarizing the percentage reduction in HBV antigen relative to non-targeting controls at day 12. Bars represent mean ± SEM; N=5. EE1 = PLA002 and gRNA#007, EE2 = PLA002 and gRNA#008, EE3 = PLA002 and gRNA#009, EE4 = PLA002 and gRNA#015, and EE5 = PLA002 and gRNA#011. [Figure 14B] FIG. 14B is a graph summarizing the percentage reduction in HBV antigen relative to non-targeted controls on day 6. [Figure 14C] Figure 14C is a schematic diagram describing the experimental timeline for a guide RNA assay testing CRISPR-off single construct epigenetic editors in combination with individual exemplary gRNAs in the PXB cell model, along with ELISA readouts for HBe and HBsAg at day 12. [Figure 14D] FIG. 14D is a graph summarizing the percentage reduction in HBV antigen at day 12 relative to non-targeted controls. [Figure 15]

[0022] Figure 15A is a schematic diagram describing the experimental timeline for zinc finger assays testing ZF-off single-construct epigenetic editors containing individual exemplary zinc finger motifs in a HepG2-NTCP infection model, along with ELISA readouts for HBe and HBs antigens at day 6; Figure 15B is a graph summarizing the percentage reduction in HBV antigen relative to a non-targeting control at day 6. "N" indicates a non-targeting control, "P" indicates a positive control, and individual numbers on the x-axis indicate exemplary constructs tested in the experiment, e.g., "1" represents the "mRNA0001" construct and "20" represents the "mRNA0020" construct. [Figure 16A]

[0023] Figure 16A is a graph summarizing the results of the top 10 ZF-off constructs from Figure 15B. Figure 16B is a schematic diagram showing the HBsAg (top) and HbeAg (middle) expression values ​​measured in the ZF-off screen (calculated as a percentage of HBsAg or HBeAg expression measured for a non-targeting control—top and middle, respectively). Each ZF-off construct is represented by a dot. The 50% and 60% suppression cutoffs are shown as horizontal lines. The position of each guide RNA (bottom) within the HBV genome is mapped on the X-axis. [Figure 16B]Figure 16B is a schematic diagram showing HBsAg (top) and HbeAg (middle) expression values ​​measured in the ZF-off screen (calculated as a percentage of HBsAg or HBeAg expression measured for the non-targeting control—top and middle, respectively). [Figure 17]

[0024] Figure 17 shows an experimental timeline for dose-response testing (top) and two graphs showing the dose-response of %HbsAg (bottom left) and %HbeAg (bottom right) in HepG2-NTCP cells upon administration of ZF fusion proteins. The mRNA corresponding to the ZF motif for each fusion protein is shown. [Figure 18]

[0025] Figure 18 shows an experimental timeline for testing durable silencing of HBsAg (top) and a graph (bottom) demonstrating the durability of HBsAg silencing by ZF fusion proteins. The mRNA corresponding to the ZF motif for each fusion protein is shown. [Figure 19]

[0026] Figure 19 shows an experimental timeline (top) for testing HBsAg silencing in the PLC / PRF / 5 in vitro model, and a graph showing the % HBsAg relative to the control at 14 days after administration of ZF fusion proteins. The mRNA corresponding to the ZF motif for each fusion protein is shown. Information on the % match to the target for each construct is also shown. [Figure 20A]

[0027] Figure 20A is a volcano plot showing differentially expressed (DE) genes for an exemplary ZF specificity assay. DE genes are indicated by dots. Figure 20B is a volcano plot showing DE for CRISPR-off and gRNA epigenetic editors. The dots represent genes by their change in expression (x-axis) and the statistical significance of that change (y-axis). EE1 = PLA002 and gRNA#007, EE2 = PLA002 and gRNA#008, EE3 = PLA002 and gRNA#009, EE4 = PLA002 and gRNA#015, and EE5 = PLA002 and gRNA#011. Results for low-specificity and host-target gene controls are also shown. Figures 20C-20D are scatter plots showing methylation levels between treatments (y-axis) and controls (x-axis) for 935,000 CpG sites in the human genome. The line represents the threshold for methylation changes considered significant (absolute [methylation difference] >= 0.2). DMRs are labeled on each figure. Results for the host target (PCSK9, next to the last panel) and for the low-specificity control (last panel) are also shown. Figure 20C shows results for effector alone. Figure 20D shows results for no treatment. EE1 = PLA002 and gRNA#007, EE2 = PLA002 and gRNA#008, EE3 = PLA002 and gRNA#009, EE4 = PLA002 and gRNA#015, EE5 = PLA002 and gRNA#011, EE6 = PLA002 and gRNA#003, and EE7 = PLA002 and gRNA#016. [Figure 20B] Figure 20B is a volcano plot showing the DE for CRISPR-off and gRNA epigenetic editors. [Figure 20C] 20C-20D are scatter plots showing methylation levels between treatments (y-axis) and controls (x-axis) for 935,000 CpG sites in the human genome. [Figure 20D] 20C-20D are scatter plots showing methylation levels between treatments (y-axis) and controls (x-axis) for 935,000 CpG sites in the human genome. [Figure 21]

[0028] FIG. 21 is an illustration of an experimental scheme for in vivo studies of multiplexed ZF fusion protein effectors. DETAILED DESCRIPTION OF THE INVENTION

[0008]

[0029] The present disclosure provides epigenetic editors for regulating HBV expression, as well as strategies and methods for using such epigenetic editors. By altering the expression of HBV, particularly by suppressing the expression of HBV, for example, of a gene contained in the HBV genome or a gene product encoded by the HBV genome, the compositions and methods described herein are useful for inhibiting viral function in infected cells, for example, in the context of treating HBV infection in a human subject or in the context of treating CHB.

[0009]

[0030] The structure and biological properties of HBV and HBV-associated diseases have been reported (see, e.g., Yuen, MF., Chen, DS., Dusheiko, G., et al., Hepatitis B virus infection. Nat Rev Dis Primers 4, 18035 (2018), the entire contents of which are incorporated herein by reference).

[0010]

[0031] Exemplary HBV sequences can be found in various NCBI database entries, for example, representative sequences can be found under accession numbers NC_00397 and U95551, which are incorporated by reference herein in their entirety, and these sequences are provided elsewhere herein.

[0011]

[0032] Although numerous treatment options for HBV have been reported, there remains a need for effective treatments for HBV infection. Gene editing approaches that target the HBV genome with genomic DNA cleavage carry the risk of off-target cleavage and genomic translocation. The present epigenetic editor and related methods of use offer several advantages over other genome manipulation methods, including increased efficiency, reduced risk of translocation, and durable silencing of HBV.

[0012]

[0033] Hepatitis D virus (HDV) is the smallest pathogen known to infect humans. Because HDV relies on HBV functions for most of its functions, including viral packaging, infectivity, transmission, and inhibition of host immunity, HDV infection is only seen in patients infected with HBV. Approximately 5% of patients with HBV infection also have HDV infection. HDV uses HBV S antigen (HBsAg) as a capsid protein, and therefore HDV infection depends on HBV S antigen production. Reducing HBV S antigen expression also reduces HDV infectivity. The structure and biological properties of HDV have been reported (see, e.g., Asselah and Rizzetto, Hepatitis D Virus Infection, The New England Journal of Medicine (389;1; July 6, 2023), which is incorporated herein by reference in its entirety). In some embodiments of the present disclosure, HDV infection is addressed by methods targeting HBV genes or genomes.

[0013]

[0034] In some embodiments, the epigenetic editors described herein may comprise one or more fusion proteins, each comprising a DNA-binding domain linked to one or more effector domains for epigenetic modification. In certain embodiments where the DNA-binding domain is a polynucleotide-guided DNA-binding domain, the epigenetic editor may further comprise one or more guide polynucleotides. The DNA-binding domain, effector domain, and guide polynucleotide of the epigenetic editors described herein may be selected in any functional combination, for example, from the following:

[0014]

[0035] The epigenetic editors described herein can be transiently expressed in host cells or integrated into the genome of host cells, and such cells and their progeny are also contemplated by the present disclosure. Both the transiently expressed epigenetic editor or its components and the integrated epigenetic editor or its components can effect stable epigenetic modification. For example, when the epigenetic editors described herein are introduced into host cells, target genes in the host cells can be stably or permanently suppressed or silenced. For example, in some embodiments provided herein, a transiently expressed epigenetic editor comprising a DNMT3A domain, a DNMT3L domain, and a KRAB domain can effect stable epigenetic modification. For example, in some embodiments provided herein, a constitutively expressed epigenetic editor comprising a DNMT3A domain and a DNMT3L domain can effect stable epigenetic modification. In some embodiments, expression of the target gene is reduced or silenced for at least 1 week, at least 2 weeks, at least 3 weeks, at least 4 weeks, at least 5 weeks, at least 6 weeks, at least 7 weeks, at least 2 months, at least 3 months, at least 4 months, at least 5 months, at least 6 months, at least 1 year, at least 2 years, or over the entire lifespan of the cell or a subject harboring the cell, compared to expression levels in the absence of the epigenetic editor. The epigenetic modification may be inherited by progeny of the host cell into which the epigenetic editor was introduced.

[0015]

[0036] The epigenetic editor can be introduced into a patient (e.g., a human patient) in need of the epigenetic editor, for example, into the patient's hepatocytes, biliary epithelial cells (cholangiocytes), stellate cells, Kupffer cells, and liver sinusoidal endothelial cells. I. DNA-binding domain

[0037] The epigenetic editors described herein may include one or more DNA binding domains that direct the effector domain of the epigenetic editor to a target sequence within the HBV genome. The DNA binding domains described herein may be, for example, polynucleotide-guided DNA binding domains, zinc finger protein (ZFP) domains, transcription activator-like effector (TALE) domains, meganuclease DNA binding domains, etc. Examples of DNA binding domains can be found in U.S. Patent No. 11,162,114, the entire contents of which are incorporated herein by reference.

[0016]

[0038] In some embodiments, the DNA-binding domains described herein are encoded by their native coding sequences, while in other embodiments, the DNA-binding domains are encoded by nucleotide sequences that have been codon-optimized for optimal expression in human cells.

[0017] A. Polynucleotide-guided DNA binding domain

[0039] In some embodiments, the DNA binding domain herein can be a protein domain that is directed to a target site in the HBV genome by a guide nucleic acid sequence (e.g., a guide RNA sequence).In certain embodiments, the protein domain can be derived from a CRISPR-associated nuclease, for example, class I or II CRISPR-associated nuclease.In some embodiments, the protein domain can be derived from a Cas nuclease, for example, type II, type IIA, type IIB, type IIC, type V, or type VI Cas nuclease. In certain embodiments, the protein domain is Cas1, Cas1B, Cas2, Cas3, Cas4, Cas5, Cas6, Cas7, Cas8, Cas9, Cas10, Cas14a, Cas14b, Cas14c, CasX, CasY, CasPhi, C2c4, C2c8, C2c9, C2c10, Csy1, Csy2, Csy3, Cse1, Cse2, Csc1, Csc2 The Class II Cas nuclease may be derived from a Class II Cas nuclease selected from Csa5, Csn2, Csm2, Csm3, Csm4, Csm5, Csm6, Cmr1, Cmr3, Cmr4, Cmr5, Cmr6, Csb1, Csb2, Csb3, Csx17, Csx14, Csx10, Csx16, CsaX, Csx3, Csx1, Csx1S, Csf1, Csf2, CsO, Csf4, and homologs and modified versions thereof. "Derived from" is used to mean that the protein domain comprises the complete polypeptide sequence of the parent protein or a variant thereof (e.g., having amino acid residue deletions, insertions, and / or substitutions). The variant retains the desired function of the parent protein (e.g., the ability to form a complex with a guide nucleic acid sequence and target DNA).

[0018]

[0040] In some embodiments, the CRISPR-associated protein domain may be a Cas9 domain described herein. Cas9 may refer to, for example, a polypeptide having at least about 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity and / or sequence similarity to a wild-type Cas9 polypeptide described herein. In some embodiments, the wild-type polypeptide is Cas9 from Streptococcus pyogenes (NCBI reference number NC_002737.2 (SEQ ID NO: 1)) and / or UniProt reference number Q99ZW2 (SEQ ID NO: 2). In some embodiments, the wild-type polypeptide is Cas9 from Staphylococcus aureus (SEQ ID NO: 3). In some embodiments, the CRISPR-associated protein domain is a Cpf1 domain or protein, or a polypeptide having at least about 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity and / or sequence similarity to a wild-type Cpf1 polypeptide described herein (e.g., Cpf1 from Francisella novicida (UniProt Reference No. U2UMQ6 or SEQ ID NO: 4)). In certain embodiments, the CRISPR-associated protein domain can be a modified form of the wild-type protein containing one or more amino acid residue changes, such as deletions, insertions, or substitutions; a fusion, or chimera; or a combination thereof.

[0019]

[0041] Cas9 sequences and structures of variant Cas9 orthologs have been described for a variety of organisms. Exemplary organisms from which the Cas9 domains herein may be derived include Streptococcus pyogenes, Streptococcus thermophilus, Streptococcus sp., Staphylococcus aureus, Listeria innocua, Lactobacillus gasseri, Francisella novicida, Wolinella succinogenes, Sutterella wadsworthensis, Gamma proteobacterium, Neisseria meningitidis, Campylobacter jejuni, Pasteurella multocida, and the like. multocida, Fibrobacter succinogenes, Rhodospirillum rubrum, Nocardiopsis dassonvillei, Streptomyces pristinaespiralis, Streptomyces viridochromogenes, Streptomyces viridochromogenes, Streptosporangium roseum, Alicyclobacillus acidocaldarius, Bacillus pseudomycoides, Bacillus selenitireducens selenitireducens, Exiguobacterium sibiricum, Lactobacillus delbrueckiidelbrueckii, Lactobacillus salivarius, Lactobacillus buchneri, Treponema denticola, Microscilla marina, Burkholderiales bacterium, Polar omonas naphthalenivorans, Polar omonas sp., Crocosphaera watsonii, Cyanothece sp., Microcystis aeruginosa, Synechococcus sp. sp., Acetohalobium arabaticum, Ammonifex degensii, Caldicelulosiruptor becscii, Candidatus Desulforudis, Clostridium botulinum, Clostridium difficile, Finegoldia magna, Natranaerobius thermophilus, Pelotomaculum thermopropionium, Acidithiobacillus caldas caldus, Acidithiobacillus ferrooxidans, Allochromatium vinosum, Marinobacter sp., Nitrosococcus halophilus, Nitrosococcus watsoniiwatsoni, Pseudoalteromonas haloplanktis, Ktedonobacter racemifer, Methanohalobium evestigatum, Anabaena variabilis, Nodularia spumigena, Nostoc sp., Arthrospira maxima, Arthrospira platensis, Arthrospira sp., Lyngbya sp., Microcoleus chthonoplastes, Oscillatoria sp. Examples of suitable Cas9 sequences include, but are not limited to, Bacillus sp., Petrotoga mobilis, Thermosipho africanus, Streptococcus pasteurianus, Neisseria cinerea, Campylobacter lari, Parvibaculum lavamentivorans, Corynebacterium diphtheriae, and Acaryochloris marina. Cas9 sequences are also included from the organisms and loci disclosed in Chylinski et al., RNA Biol. (2013) 10(5):726-37.

[0020]

[0042] In some embodiments, the Cas9 domain is from Streptococcus pyogenes. In some embodiments, the Cas9 domain is from Staphylococcus aureus.

[0043] Other Cas domains are also contemplated for use in the epigenetic editors herein, including, for example, those from CasX (Cas12E) (e.g., SEQ ID NO: 5), CasY (Cas12d) (e.g., SEQ ID NO: 6), Casφ (Casphi) (e.g., SEQ ID NO: 7), Cas12f1 (Cas14a) (e.g., SEQ ID NO: 8), Cas12f2 (Cas14b) (e.g., SEQ ID NO: 9), Cas12f3 (Cas14c) (e.g., SEQ ID NO: 10), and C2c8 (e.g., SEQ ID NO: 11).

[0021]

[0044] For epigenetic editing, the protein domain from the nuclease (e.g., Cas9 or Cpf1 domain) may have reduced or no nuclease activity due to mutation, so that the protein domain does not cleave DNA or has reduced DNA cleavage activity, but retains the ability to complex with guide nucleic acid sequences (e.g., guide RNA) and target DNA. For example, the nuclease activity may be reduced by at least 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 99% compared to the wild-type domain. In some embodiments, the CRISPR-associated protein domain described herein is catalytically inactive ("deactivated"). Examples of such domains include dCas9 ("deactivated" Cas9), dCpf1, ddCpf1, dCasphi, ddCas12a, dLbCpf1, and dFnCpf1. The dCas9 protein domain can contain one, two, or more mutations that eliminate its nuclease activity, for example, compared to wild-type Cas9. It is known that the DNA cleavage domain of Cas9 contains two subdomains: the HNH nuclease subdomain and the RuvC1 subdomain. The HNH subdomain cleaves the strand complementary to the gRNA, whereas the RuvC1 subdomain cleaves the non-complementary strand. Mutations within these subdomains can silence the nuclease activity of Cas9. For example, the mutations D10A (in RuvC1) and H840A (in HNH) completely inactivate the nuclease activity of SpCas9. SaCas9 can similarly be inactivated by the mutations D10A and N580A. In some embodiments, dCas9 contains at least one mutation in the HNH subdomain and / or RuvC1 subdomain that reduces or eliminates nuclease activity. In some embodiments, dCas9 contains only the RuvC1 subdomain or only the HNH subdomain.It should be understood that any mutation that inactivates the RuvC1 and / or HNH domain can be included in the dCas9 herein, for example, an insertion, deletion, or single or multiple amino acid substitution in the RuvC1 domain and / or HNH domain.

[0022]

[0045] In some embodiments, the dCas9 protein herein comprises a mutation at a position corresponding to position D10 (e.g., D10A), position corresponding to position H840 (e.g., H840A), or both of the wild-type SpCas9 sequence as numbered in the sequence provided in UniProt Accession No. Q99ZW2 (SEQ ID NO: 2). In certain embodiments, the dCas9 comprises the amino acid sequence of dSpCas9 (D10A and H840A) (SEQ ID NO: 12).

[0023]

[0046] In some embodiments, the dCas9 proteins described herein comprise a mutation at a position corresponding to position D10 (e.g., D10A), at a position corresponding to position N580 (e.g., N580A), or both, of the wild-type SaCas9 sequence (e.g., SEQ ID NO: 9). In certain embodiments, the dCas9 comprises the amino acid sequence of dSaCas9 (D10A and N580A) (SEQ ID NO: 13).

[0024]

[0047] Additional suitable mutations that inactivate Cas9 will be apparent to those skilled in the art based on this disclosure and knowledge in the art and are within the scope of this disclosure. Such mutations may include, but are not limited to, D839A, N863A, and / or K603R in SpCas9. The present disclosure contemplates any mutation that reduces or eliminates the nuclease activity of any Cas9 described herein (e.g., a mutation corresponding to any of the Cas9 mutations described herein).

[0025]

[0048] The dCpf1 protein domain can contain one, two, or more mutations that reduce or eliminate its nuclease activity compared to wild-type Cpf1. The Cpf1 protein has a RuvC-like endonuclease domain similar to the RuvC domain of Cas9, but does not have the HNH endonuclease domain, and the N-terminus of Cpf1 does not have the alpha-helical recognition lobe of Cas9. In some embodiments, dCpf1 contains one or more mutations D917A, E1006A, or D1255A, which correspond to the numbered positions in the sequence of the Francisella novicida Cpf1 protein (FnCpf1; SEQ ID NO: 4). In certain embodiments, the dCpfl protein comprises a mutation corresponding to D917A, E1006A, D1255A, D917A / E1006A, D917A / D1255A, E1006A / D1255A, or D917A / E1006A / D1255A, or a corresponding mutation in any of the Cpfl amino acid sequences described herein. In some embodiments, dCpfl comprises a D917A mutation. In certain embodiments, dCpfl comprises the amino acid sequence of dFnCpfl (SEQ ID NO: 14).

[0026]

[0049] Additional nuclease-inactive CRISPR-associated protein domains contemplated herein include, for example, those from dNmeCas9 (e.g., SEQ ID NO: 15), dCjCas9 (e.g., SEQ ID NO: 16), dSt1Cas9 (e.g., SEQ ID NO: 17), dSt3Cas9 (e.g., SEQ ID NO: 18), dLbCpf1 (e.g., SEQ ID NO: 19), dAsCpf1 (e.g., SEQ ID NO: 20), denAsCpf1 (e.g., SEQ ID NO: 21), dHFAsCpf1 (e.g., SEQ ID NO: 22), dRVRAsCpf1 (e.g., SEQ ID NO: 23), dRRAsCpf1 (e.g., SEQ ID NO: 24), dCasX (e.g., SEQ ID NO: 25), and dCasphi (e.g., SEQ ID NO: 26).

[0027]

[0050] In some embodiments, the Cas9 domains described herein can be high-fidelity Cas9 domains that contain one or more mutations that, for example, reduce the electrostatic interactions between the Cas9 domain and the sugar-phosphate backbone of DNA, resulting in increased target binding specificity. In certain embodiments, the high-fidelity Cas9 domains can be nuclease-inactive, as described herein.

[0028]

[0051] The CRISPR-associated protein domains described herein can recognize protospacer adjacent motif (PAM) sequences within target genes. A "PAM" sequence is typically a 2-6 bp DNA sequence immediately following the sequence targeted by the CRISPR-associated protein domain. The PAM sequence is required for CRISPR protein binding and cleavage but is not part of the target sequence. CRISPR-associated protein domains can recognize naturally occurring or canonical PAM sequences or can have altered PAM specificity. CRISPR-associated protein domains that bind to non-canonical PAM sequences have been described in the art. For example, Cas9 domains that bind to non-canonical PAM sequences are described in Kleinstiver et al., Nature (2015) 523(7561):481-5 and Kleinstiver et al., Nat Biotechnol. (2015) 33:1293-8. Such Cas9 domains can include, for example, those from "VRER" SpCas9, "EQR" SpCas9, "VQR" SpCas9, "SpG Cas9," "SpRY Cas9," and "KKH" SaCas9. Nuclease-inactive versions of these Cas9 domains are also contemplated, such as nuclease-inactive VRER SpCas9 (e.g., SEQ ID NO: 27), nuclease-inactive EQR SpCas9 (e.g., SEQ ID NO: 28), nuclease-inactive VQR SpCas9 (e.g., SEQ ID NO: 29), nuclease-inactive SpG Cas9 (e.g., SEQ ID NO: 30), nuclease-inactive SpRY Cas9 (e.g., SEQ ID NO: 31), and nuclease-inactive KKH SaCas9 (e.g., SEQ ID NO: 32). Another example is Francisella novicida Cas9 engineered to recognize 5'-YG-3' (where "Y" is a pyrimidine).

[0029]

[0052] Additional suitable CRISPR-associated proteins, orthologs, and variants, including nuclease-inactive variants and sequences, will be apparent to those of skill in the art based on the present disclosure.

[0030]

[0053] Guide RNAs that can be used with the CRISPR-associated protein domains herein are further described in Section II below. B. Zinc finger protein domain

[0054] In some embodiments, the DNA-binding domain of the epigenetic editors described herein comprises a zinc finger protein (ZFP) domain (or "ZF domain" as used herein). A ZFP is a protein with at least one zinc finger that binds to DNA in a sequence-specific manner. A "zinc finger" (ZF) or "zinc finger motif" (ZF motif) refers to a polypeptide domain that contains a beta-beta-alpha (ββα) protein fold stabilized by zinc ions. A ZF binds two to four base pairs of nucleotides, usually three or four base pairs (contiguous or noncontiguous). Each ZF typically contains approximately 30 amino acids. A ZFP domain can contain multiple ZFs, which contact their target nucleic acid sequences in tandem. Tandem arrays of ZFs can be engineered to generate artificial ZFPs that bind to desired nucleic acid targets. ZFPs can be rationally designed using databases that contain triplet (or quadruplet) nucleotide sequences and individual ZF amino acid sequences, with each triplet or quadruplet nucleotide sequence associated with one or more amino acid sequences of ZFs that bind to a particular triplet or quadruplet sequence. See, e.g., U.S. Patent Nos. 6,453,242, 6,534,261, and 8,772,453.

[0031]

[0055] ZFPs are widespread in eukaryotic cells and can belong to, for example, the C2H2 class, CCHC class, PHD class, or RING class. An exemplary motif that characterizes one class of these proteins (the C2H2 class) is -Cys-(X) 2~4 -Cys-(X) 12 -His-(X) 3~5-His- (SEQ ID NO: 1091), where X is any independently selected amino acid. In some embodiments, the ZFP domain herein can comprise a ZF array comprising a series of C2H2-ZFs, each of which contacts a series of three or more nucleotides. Additional configurations are also possible, for example, as described in Paschon et al., Nat. Commun. 10, 1133 (2019).

[0032]

[0056] The ZFP domains of the epigenetic editors described herein can comprise 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, or more ZFs. The ZFP domains can comprise an array of two-finger or three-finger units, e.g., 3, 4, 5, 6, 7, 8, 9, or 10 or more units, where each unit binds to a subsite in the target sequence. In some embodiments, a ZFP domain comprising at least three ZFs recognizes a target DNA sequence of 9 or 10 nucleotides. In some embodiments, a ZFP domain comprising at least four ZFs recognizes a target DNA sequence of 12-14 nucleotides. In some embodiments, a ZFP domain comprising at least six ZFs recognizes a target DNA sequence of 18-21 nucleotides.

[0033]

[0057] In some embodiments, the ZFs in the ZFP domains described herein are connected via a peptide linker. The peptide linker can be, for example, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or more amino acids in length. In some embodiments, the linker contains 5 or more amino acids. In some embodiments, the linker contains 7 to 17 amino acids. The linker can be flexible or rigid.

[0034]

[0058] In some embodiments, the zinc finger array comprises the sequence:

[0035] [ka]

[0036] or a sequence at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical thereto, where "XXXXXX" represents amino acids of the ZF recognition helix that confer DNA-binding specificity to the zinc finger, and each X may be independently selected. The italicized "XX" in the above sequence may be TR, LR, or LK, and "[linker]" represents a linker sequence. In some embodiments, the linker sequence is TGSQKP (SEQ ID NO: 1085), which may be used when the ZF-targeted subsites are adjacent. In some embodiments, the linker sequence is TGGGGSQKP (SEQ ID NO: 1086), which may be used when there are bases between the zinc finger-targeted subsites. The two shown linkers may be the same or different.

[0037]

[0059] A ZFP domain herein can contain an array of two or more adjacent ZFs, either directly adjacent to each other (e.g., separated by a short (canonical) linker sequence) or separated by a longer, flexible or structured polypeptide sequence. In some embodiments, directly adjacent fingers bind to contiguous nucleic acid sequences, i.e., adjacent trinucleotides / triplets. In some embodiments, adjacent fingers cross-link with each other's respective target triplets, which can serve to strengthen or enhance target sequence recognition and lead to binding of overlapping sequences. In some embodiments, distal ZFs within a ZFP domain can recognize (or bind to) non-contiguous nucleotide sequences.

[0038]

[0060] The amino acid sequences of the ZF DNA recognition helices of exemplary ZFP domains herein, and their HBV target sequences, are shown in Table 1 below.

[0039] [Table 1-1]

[0040] [Table 1-2]

[0041] [Table 1-3]

[0042] [Table 1-4]

[0043]

[0061] In some embodiments, the ZFP domain of the epigenetic editor binds to a target sequence provided herein. In further embodiments, the ZFP domain comprises, in order, the F1-F6 amino acid sequences of any one of the zinc finger proteins as set forth in Table 1 and Table 20. The F1-F6 amino acid sequences can be located within the ZF framework sequence of SEQ ID NO: 1084 or any other ZF framework known in the art.

[0044] C.TALE

[0062] In some embodiments, the DNA-binding domain of the epigenetic editors described herein comprises a transcription activation-like effector (TALE) domain. TALE DNA-binding domains contain a highly conserved sequence of approximately 33-34 amino acids, with a repeating variable dinucleotide (RVD) at positions 12 and 13 that is central to the recognition of specific nucleotides. TALEs can be engineered to actually bind to any desired DNA sequence. Methods for programming TALEs are known in the art. For example, such methods are described in Carroll et al., Genet Soc Amer. (2011) 188(4):773-82; Miller et al., Nat Biotechnol. (2007) 25(7):778-85; Christian et al., Genetics (2008) 186(2):757-61; Li et al., Nucl Acids Res. (2010) 39(1):359-72; and Moscou et al., Science (2009) 326(5959):1501.

[0045] D. Other DNA-binding domains

[0063] Other DNA-binding domains are contemplated for the epigenetic editors described herein. In some embodiments, the DNA-binding domain comprises an Argonaute protein domain, such as the Argonaute protein domain from Natronobacterium gregoryi (NgAgo). NgAgo is a ssDNA-guided endonuclease that is guided to its target site by 5'-phosphorylated ssDNA (gDNA), where it creates a double-strand break. In contrast to Cas9, the NgAgo-gDNA system does not require a protospacer adjacent motif (PAM). Therefore, the use of nuclease-inactive NgAgo (dNgAgo) can significantly expand the range of bases that can be targeted. The characterization and use of NgAgo is described, for example, in Gao et al., Nat Biotechnol. (2016) 34(7):768-73; Swarts et al., Nature (2014) 507(7491):258-61; and Swarts et al., Nucl Acids Res. (2015) 43(10):5120-9.

[0046]

[0064] In some embodiments, the DNA-binding domain comprises an inactivated nuclease, e.g., an inactivated meganuclease. Additional non-limiting examples of DNA-binding domains include a tetracycline-controlled repressor (tetR) DNA-binding domain, a leucine zipper, a helix-loop-helix (HLH) domain, a helix-turn-helix domain, a beta-sheet motif, a steroid receptor motif, a bZIP domain, a homeodomain, and an AT-hook.

[0047] II. Guide Polynucleotide

[0065] The epigenetic editor described herein, which includes a polynucleotide-guided DNA binding domain, can also include a guide polynucleotide that can form a complex with the DNA binding domain.The guide polynucleotide can include RNA, DNA, or a mixture of both.For example, if the polynucleotide-guided DNA binding domain is a CRISPR-associated protein domain, the guide polynucleotide can be a guide RNA (gRNA)."Guide RNA" or "gRNA" refers to a nucleic acid that can hybridize to a target sequence and direct the binding of a CRISPR-Cas complex to the target sequence.Methods for using guide polynucleotide sequences with programmable DNA binding proteins (e.g., CRISPR-associated protein domains) for site-specific targeting (e.g., for modifying genomes) are known in the art.

[0048]

[0066] A guide polynucleotide sequence (e.g., a gRNA sequence) can contain two parts: 1) a nucleotide sequence containing a "targeting sequence" that is complementary to a target nucleic acid sequence ("target sequence"), e.g., a nucleic acid sequence contained in a genomic target site, and 2) a nucleotide sequence that binds to a DNA-binding domain (e.g., a CRISPR-Cas protein domain) guided by the polynucleotide. The nucleotide sequence in 1) can contain a targeting sequence that is 100% complementary to a genomic nucleic acid sequence, e.g., a nucleic acid sequence contained in a genomic target site, and therefore can hybridize to the target nucleic acid sequence. The nucleotide sequence in 1) can be referred to, for example, as a crispr RNA or crRNA. The nucleotide sequence in 2) can be referred to as a scaffold sequence of the guide nucleic acid, e.g., a tracrRNA, or an activation region of the guide nucleic acid, and can contain a stem-loop structure. The above parts 1) and 2) can be fused to form one single guide (e.g., a single guide RNA or sgRNA) or can be on two separate nucleic acid molecules. In some embodiments, the guide polynucleotide comprises portions 1) and 2) connected by a linker. In some embodiments, the guide polynucleotide comprises portions 1) and 2) connected by a non-nucleic acid linker, such as a peptide linker or a chemical linker.

[0049]

[0067] Portion 2 (scaffold sequence) of the guide polynucleotide described herein can be as described, for example, in Jinek et al., Science (2012) 337:816-21; U.S. Patent Application Publication No. 2016 / 0208288; or U.S. Patent Application Publication No. 2016 / 0200779. Variants of portion 2) are also contemplated by the present disclosure. For example, the tetraloop and stem-loop of the gRNA scaffold (tracrRNA) sequence can be modified to include an RNA aptamer that can be bound by a specific protein domain. In some embodiments, such modified gRNAs can be used to facilitate the recruitment of repression or activation domains fused to protein-interacting RNA aptamers.

[0050]

[0068] The gRNAs provided herein typically comprise a targeting domain and a binding domain. The targeting domain (also referred to as a "targeting sequence") may comprise a nucleic acid sequence that binds to a target site, for example, a genomic nucleic acid molecule in a cell. The target site may be a double-stranded DNA sequence that contains not only a target sequence but also a PAM sequence, where the target sequence is located on the same strand as the PAM sequence and is directly adjacent to the PAM sequence. The targeting domain of the gRNA may comprise an RNA sequence corresponding to the target sequence, i.e., it resembles the sequence of the target domain, which may have one or more mismatches but usually comprises an RNA sequence rather than a DNA sequence. Thus, the targeting domain of the gRNA can base-pair (fully or partially complementary) with the sequence of the double-stranded target site that is complementary to the target sequence, and thus with the strand complementary to the strand containing the PAM sequence. Of course, the targeting domain of the gRNA typically does not comprise a sequence that resembles the PAM sequence. It should be further understood that the location of the PAM can be 5' or 3' of the target sequence, depending on the nuclease used. For example, the PAM is usually 3' of the target sequence for Cas9 nuclease and 5' of the target sequence for Casl2a nuclease. For examples of the location of the PAM and the mechanism of gRNA binding to the target site, see, for example, Figure 1 in Vanegas et al., Fungal Biol Biotechnol. (2019) 6:6, which is incorporated herein by reference. For additional examples and explanations of the mechanism of gRNA targeting of RNA-guided nucleases to the target site, see, for example, Fu et al., Nat Biotechnol (2014) 32(3):279-84 and Sternberg et al., Nature (2014) 507(7490):62-7, each of which is incorporated herein by reference.

[0051]

[0069] In some embodiments, the targeting domain sequence contains between 17 and 30 nucleotides and corresponds perfectly to the target sequence (i.e., without any mismatched nucleotides). However, in some embodiments, the targeting domain sequence may contain one or more, but usually no more than four, mismatches, e.g., one, two, three, or four mismatches. Because the targeting domain is part of a gRNA, which is an RNA molecule, it will usually contain ribonucleotides, while a DNA-targeting domain will contain deoxyribonucleotides.

[0052]

[0070] An exemplary illustration of a Cas9 target site comprising a 22-nucleotide targeting domain and a NGG PAM sequence, and of a gRNA comprising a targeting domain that corresponds perfectly to the target sequence (and thus base-pairs with perfect complementarity to the DNA strand complementary to the strand comprising the target sequence and PAM) is provided below:

[0053] [ka]

[0054]

[0071] An exemplary illustration of a Casl2a target site comprising a 22-nucleotide targeting domain and a TTN PAM sequence, and of a gRNA comprising a targeting domain that corresponds perfectly to the target sequence (and thus base-pairs with perfect complementarity to the DNA strand complementary to the strand comprising the target sequence and PAM), is provided below:

[0055] [ka]

[0056]

[0072] Without wishing to be bound by theory, it is believed that, at least in some embodiments, the length of the targeting domain and its complementarity with the target sequence contribute to the specificity of the interaction between the gRNA / Cas9 molecular complex and the target nucleic acid. In some embodiments, the targeting domain of the gRNA provided herein is 5-50 nucleotides in length. In some embodiments, the targeting domain is 15-25 nucleotides in length. In some embodiments, the targeting domain is 18-22 nucleotides in length. In some embodiments, the targeting domain is 19-21 nucleotides in length. In some embodiments, the targeting domain is 15 nucleotides in length. In some embodiments, the targeting domain is 16 nucleotides in length. In some embodiments, the targeting domain is 17 nucleotides in length. In some embodiments, the targeting domain is 18 nucleotides in length. In some embodiments, the targeting domain is 19 nucleotides in length. In some embodiments, the targeting domain is 20 nucleotides in length. In some embodiments, the targeting domain is 21 nucleotides in length. In some embodiments, the targeting domain is 22 nucleotides in length. In some embodiments, the targeting domain is 23 nucleotides in length. In some embodiments, the targeting domain is 24 nucleotides in length. In some embodiments, the targeting domain is 25 nucleotides in length. In certain embodiments, the targeting domain corresponds completely to the target sequence provided herein or a portion thereof, without any mismatches. In some embodiments, the targeting domain of the gRNA provided herein contains one mismatch compared to the target sequence provided herein. In some embodiments, the targeting domain contains two mismatches compared to the target sequence. In some embodiments, the targeting domain contains three mismatches compared to the target sequence.

[0057]

[0073] The method for designing, selecting and validating gRNA is described herein and known in the art.Software tools can be used to optimize the gRNA corresponding to target DNA sequence, for example, to minimize the total off-target activity throughout the genome.For example, DNA sequence search algorithms can be used to identify the target sequence in the crRNA of gRNA for use with Cas9.Exemplary gRNA design tools include those described in Bae et al., Bioinformatics (2014) 30:1473-5.

[0058]

[0074] The guide polynucleotides (e.g., gRNAs) described herein can be of various lengths. In some embodiments, the length of the spacer or targeting sequence depends on the CRISPR-associated protein components of the epigenetic editor system used. For example, Cas proteins from different bacterial species have different optimal targeting sequence lengths. Thus, the spacer sequence can include, for example, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, or more than 50 nucleotides in length. In some embodiments, the spacer comprises 10-24, 11-20, 11-16, 18-24, 19-21, or 20 nucleotides in length. In some embodiments, the guide polynucleotide (e.g., gRNA) is 15-100 (e.g., 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50) nucleotides in length. and a spacer sequence of at least 10 (e.g., 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50) contiguous nucleotides complementary to the target sequence. In some embodiments, the guide polynucleotides described herein can be shortened by, for example, 1, 2, 3, 4, 5, 10, 15, 20, 25, 30, 40, 50, or more nucleotides.

[0059]

[0075] In certain embodiments, the 3' end of the HBV target sequence is immediately adjacent to a PAM sequence (e.g., a canonical PAM sequence, e.g., NGG of SpCas9). The degree of complementarity between the targeting sequence of the guide polynucleotide (e.g., the spacer sequence of a gRNA) and the target sequence can be at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%. In certain embodiments, the targeting sequence and the target sequence can be 100% complementary. In other embodiments, the targeting sequence and the target sequence can contain, for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 mismatches.

[0060]

[0076] Guide polynucleotides (e.g., gRNAs) can be modified, for example, by chemical and synthetic modifications. Modified gRNAs can include, for example, modifications or replacements of one or both of the non-linked phosphate oxygens and / or one or more of the linking phosphate oxygens in the phosphodiester backbone linkages; modifications of the ribose sugar (e.g., of the 2' hydroxyl on the ribose sugar); modifications of the phosphate moiety; modifications or replacements of naturally occurring nucleobases; modifications or replacements of the ribose-phosphate backbone; modifications of the 3' and / or 5' ends of the oligonucleotide; replacement of a terminal phosphate group; or conjugation of a moiety, cap, or linker; or any combination thereof.

[0061]

[0077] In some embodiments, one or more ribose groups of the gRNA may be modified. Examples of chemical modifications to the ribose group include, but are not limited to, 2'-O-methyl (2'-OMe), 2'-fluoro (2'-F), 2'-deoxy, 2'-O-(2-methoxyethyl) (2'-MOE), 2'-NH2,2'-O-allyl, 2'-O-ethylamine, 2'-O-cyanoethyl, 2'-O-acetal ester, or bicyclic nucleotides, such as locked nucleic acids (LNA), 2'-5(constrained ethyl (S-cEt)), constrained MOE, or 2'-O,4'-C-aminomethylene bridged nucleic acid (2',4'-BNANC). 2'-O-methyl and / or 2'-fluoro modifications may increase the binding affinity and / or nuclease stability of the gRNA oligonucleotide.

[0062]

[0078] In some embodiments, one or more phosphate groups of the gRNA may be chemically modified. Examples of chemical modifications to phosphate groups include, but are not limited to, phosphorothioate (PS), phosphonoacetate (PACE), thiophosphonoacetate (thioPACE), amide, triazole, phosphonate, and phosphotriester modifications. In some embodiments, the guide polynucleotides described herein may contain one, two, three, or more PS linkages at or near the 5'-end and / or 3'-end, and the PS linkages may be consecutive or discontinuous.

[0063]

[0079] In some embodiments, the gRNAs herein comprise a mixture of ribonucleotides and deoxyribonucleotides and / or one or more PS linkages.

[0080] In some embodiments, one or more nucleobases of the gRNA can be chemically modified. Examples of chemically modified nucleobases include, but are not limited to, 2-thiouridine, 4-thiouridine, N6-methyladenosine, pseudouridine, 2,6-diaminopurine, inosine, thymidine, 5-methylcytosine, 5-substituted pyrimidine, isoguanine, isocytosine, and nucleobases with halogenated aromatic groups. Chemical modifications can be made in the spacer region, the tracr RNA region, the stem loop, or any combination thereof.

[0064]

[0081] Table 2 below lists exemplary target sequences for epigenetic modifications of HBV, as well as the coordinates of the start and end positions of the targeted sites on the HBV genome.

[0065] [Table 2-1]

[0066] [Table 2-2]

[0067] [Table 2-3]

[0068] [Table 2-4]

[0069]

[0082] The target domains identified above, adjacent to a PAM sequence, e.g., the S. pyogenes Cas9 PAM sequence, can be targeted by a CRISPR-based epigenetic repressor, e.g., an epigenetic repressor containing a dCas9 DNA binding domain. For example, target sites 1-143 are suitable for dCas9-based epigenetic repressor targeting.

[0070]

[0083] A gRNA suitable for targeting any of the target domain sequences will, in some embodiments, comprise a target domain sequence that is the RNA equivalent of the provided DNA sequence of the targeting domain sequence (i.e., the RNA nucleotides of that sequence, rather than the provided DNA nucleotides, have uracil instead of thymine), and an appropriate tracr RNA sequence.

[0071]

[0084] Any tracr sequence known in the art is contemplated for the gRNA described herein. In some embodiments, the gRNA described herein has a tracr sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to the tracr sequence shown in Table 3 below, or the tracr sequence (SEQ ID NO:) shown below.

[0072] [Table 3]

[0073]

[0085] In some embodiments, the gRNA herein is provided directly to cells (for example, by RNP complexes together with CRISPR-associated protein domains). In some embodiments, the gRNA is provided to cells by an expression vector (for example, a plasmid vector or a viral vector) that is introduced into cells, and then the cells express the gRNA from the expression vector. Methods for introducing gRNA and expression vectors into cells are known in the art.

[0074] III. Effector Domain

[0086] The epigenetic editors described herein contain one or more effector protein domains (also referred to herein as "epigenetic effector domains" or "effector domains") that effect epigenetic modification of a target gene. Epigenetic editors with one or more effector domains can modulate the expression of a target gene without altering its nucleic acid sequence. In some embodiments, the effector domains described herein can suppress or silence the expression of HBV or HBV genes, for example, by suppressing transcription or by modifying or remodeling HBV chromatin. Such effector domains are also referred to herein as "repression domains," "repressor domains," "epigenetic repressor domains," or "epigenetic repression domains." Non-limiting examples of chemical modifications that can be mediated by effector domains include methylation, demethylation, acetylation, deacetylation, phosphorylation, sumoylation, and / or ubiquitination of DNA or histone residues.

[0075]

[0087] In some embodiments, the effector domain of an epigenetic editor described herein can modify a histone tail, for example, by adding or removing an activity mark to the histone tail.

[0076]

[0088] In some embodiments, the effector domain of an epigenetic editor described herein may comprise or recruit a transcription-associated protein domain, e.g., a transcription repressor. The transcription-associated protein may be endogenous or exogenous.

[0077]

[0089] In some embodiments, the effector domain of an epigenetic editor described herein can include, for example, a protein that directly or indirectly prevents a transcription factor from reaching a gene of interest that harbors a target sequence.

[0078]

[0090] The effector domain can be a full-length protein or a fragment thereof ("functional domain") that retains epigenetic effector function. Functional domains capable of modulating (e.g., suppressing) gene expression can be derived from larger proteins. For example, functional domains capable of reducing target gene expression can be identified based on the sequence of a repressor protein. The amino acid sequence of a gene expression-modulating protein can be obtained from available genome browsers, such as the UCSD Genome Browser or the Ensembl Genome Browser. Protein annotation databases, such as UniProt or Pfam, can be used to identify functional domains within the complete protein sequence. As a starting point, the largest sequence encompassing all regions identified by different databases can be tested for gene expression modulation activity. Various truncations can then be tested to identify the smallest functional unit.

[0079]

[0091] Variants of the effector domains described herein are also contemplated by the present disclosure. A variant may refer to, for example, a polypeptide having at least about 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity and / or sequence similarity to the wild-type effector domains described herein. In certain embodiments, a variant retains at least about 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% of the epigenetic effector function of the wild-type effector domain.

[0080]

[0092] In some embodiments, an epigenetic editor described herein may comprise one effector domain, two effector domains, three effector domains, four effector domains, five effector domains, six effector domains, seven effector domains, eight effector domains, nine effector domains, ten effector domains, or more. In certain embodiments, an epigenetic editor comprises one or more fusion proteins (e.g., one, two, or three fusion proteins), each having one or more effector domains (e.g., one, two, or three effector domains) linked to a DNA-binding domain. In some embodiments, the effector domain may induce a combination of epigenetic modifications, for example, transcriptional repression and DNA methylation, DNA methylation and histone deacetylation, DNA methylation and histone demethylation, DNA methylation and histone methylation, DNA methylation and histone phosphorylation, DNA methylation and histone ubiquitination, or DNA methylation and histone sumoylation.

[0081]

[0093] In certain embodiments, the effector domains described herein (e.g., DNMT3A and / or DNMT3L) are encoded by nucleotide sequences as found in the effector domain's native genome (e.g., human or mouse). In other embodiments, the effector domains described herein are encoded by nucleotide sequences that have been codon-optimized for optimal expression in human cells.

[0082]

[0094] Effector domains described herein can include, for example, transcriptional repressors, DNA methyltransferases, and / or histone modifiers, as further detailed below.

[0083] A. Transcriptional repressors

[0095] In some embodiments, the epigenetic effector domains described herein mediate the suppression of target gene expression (e.g., transcription). The effector domain may include, for example, a Krüppel-associated box (KRAB) repression domain, a repressor element silencing transcription factor (REST) ​​repression domain, a KRAB-associated protein 1 (KAP1) domain, a MAD domain, an FKHR (forkhead in rhabdomyosarcoma gene) repressor domain, an EGR-1 (early growth response gene product-1) repressor domain, an ets2 repressor factor repressor domain (ERD), a MAD smSIN3-interacting domain (SID), a WRPW motif of the hairy-related basic helix-loop-helix (bHLH) repressor protein, an HP1 alpha chromoshadow repression domain, an HP1 beta repression domain, or any combination thereof. The effector domain may recruit one or more protein domains that suppress target gene expression, for example, via a scaffolding protein. In some embodiments, the effector domain may recruit or interact with a scaffold protein domain that recruits a PRMT protein, HDAC protein, SETDB1 protein, or NuRD protein domain.

[0084]

[0096] In some embodiments, the effector domain comprises a functional domain derived from a zinc finger repressor protein, such as a KRAB domain. KRAB domains are found in approximately 400 human ZFP-based transcription factors. Descriptions of KRAB domains can be found, for example, in Ecco et al., Development (2017) 144 (15): 2719-29 and Lambert et al., Cell (2018) 172: 650-65.

[0085]

[0097] In certain embodiments, the effector domain comprises a repression domain (e.g., KRAB) derived from KOX1 / ZNF10, KOX8 / ZNF708, ZNF43, ZNF184, ZNF91, HPF4, HTF10, or HTF34. In some embodiments, the effector domain is selected from the group consisting of ZIM3, ZNF436, ZNF257, ZNF675, ZNF490, ZNF320, ZNF331, ZNF816, ZNF680, ZNF41, ZNF189, ZNF528, ZNF543, ZNF554, ZNF140, ZNF610, ZNF264, ZNF350, ZNF8, ZNF582, ZNF30, ZNF324, ZNF98, ZNF669, ZNF677, ZNF596, ZNF214, ZNF37, ZNF34, ZNF The repression domain may comprise a repression domain (e.g., KRAB) derived from F250, ZNF547, ZNF273, ZNF354, ZFP82, ZNF224, ZNF33, ZNF45, ZNF175, ZNF595, ZNF184, ZNF419, ZFP28-1, ZFP28-2, ZNF18, ZNF213, ZNF394, ZFP1, ZFP14, ZNF416, ZNF557, ZNF566, ZNF729, ZIM2, ZNF254, ZNF764, ZNF785, or any combination thereof. For example, the repression domain may be a KRAB domain derived from KOX1, ZIM3, ZFP28, or ZN627. In certain embodiments, the repression domain is a ZIM3 KRAB domain. In further embodiments, the effector domain is derived from a human protein, such as human ZIM3, human KOX1, human ZFP28, or human ZN627.

[0086]

[0098] Exemplary effector domains that can reduce or silence target gene expression are provided in Table 4 below (SEQ: SEQ ID NO: see Table 20 for exemplary effector domain sequences). Further examples of repressors and transcriptional repressor domains can be found, for example, in PCT Patent Publication WO2021 / 226077 and Tycko et al., Cell (2020) 183(7):2020-35, each of which is incorporated herein by reference in its entirety.

[0087]

Table 4-1

[0088]

Table 4-2

[0089]

Table 4-3

[0090]

Table 4-4

[0091]

Table 4-5

[0092]

Table 4-6

[0093]

Table 4-7

[0094]

[0099] Functional analogs of any one of the above-listed proteins, i.e., molecules that have the same or substantially the same biological function (e.g., retain 70% or more, 80% or more, 90% or more, 95% or more, or 98% or more of the protein's transcription factor function), are encompassed by the present disclosure. For example, functional analogs can be isoforms or variants of the above-listed proteins, e.g., containing portions of the proteins with or without additional amino acid residues and / or containing mutations compared to the above proteins. In some embodiments, functional analogs have at least 75, 80, 85, 90, 95, 98, or 99% sequence identity to one of the sequences listed in Table 4. Homologs, orthologs, and variants of the above-listed proteins are also contemplated.

[0095]

[0100] In certain embodiments, the epigenetic editors described herein include a KRAB domain derived from KOX1, ZIM3, ZFP28, or ZN627, and / or an effector domain derived from KAP1, MECP2, HP1a, HP1b, CBX8, CDYL2, TOX, TOX3, TOX4, EED, EZH2, RBBP4, RCOR1, or SCML2, optionally wherein the parent protein is a human protein. In certain embodiments, the epigenetic editors described herein include a domain derived from KOX1, ZIM3, ZFP28, and / or ZN627, optionally wherein the parent protein is a human protein. In certain embodiments, the epigenetic editor may include a KRAB domain derived from KOX1 (ZNF10), e.g., human KOX1. In certain embodiments, the epigenetic editor may include a KRAB domain derived from ZIM3 (ZNF657 or ZNF264), e.g., human ZIM3. In certain embodiments, an epigenetic editor may comprise a KRAB domain derived from ZFP28, e.g., human ZFP28. In certain embodiments, an epigenetic editor may comprise a KRAB domain derived from ZN627, e.g., human ZN627. In certain embodiments, an epigenetic editor described herein may comprise a CDYL2, e.g., human CDYL2, and / or a TOX domain (e.g., human TOX domain), in combination with a KOX1 KRAB domain (e.g., human KOX1 KRAB domain).

[0096]

[0101] In certain embodiments, the epigenetic effectors described herein comprise a repression domain derived from ZNF10 (SEQ ID NO: 1024). For example, the repression domain can comprise the sequence of SEQ ID NO: 1024, or a sequence at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 1024.

[0097] B. DNA methyltransferase

[0102] In some embodiments, the effector domain of the epigenetic editor described herein alters target gene expression through DNA modifications such as methylation. Highly methylated areas of DNA tend to be less transcriptionally active than less methylated areas. DNA methylation occurs primarily at CpG sites (short for "C-phosphate-G-" or "cytosine-phosphate-guanine" sites). Many mammalian genes have promoter regions near or containing CpG islands (nucleic acid regions with a high CpG dinucleotide frequency).

[0098]

[0103] The effector domain described herein may be, for example, a DNA methyltransferase (DNMT) or its catalytic domain, or may be capable of recruiting a DNA methyltransferase. DNMTs include enzymes that catalyze the transfer of methyl groups to DNA nucleotides, such as canonical cytosine-5 DNMTs (e.g., DNMT1, DNMT3A, DNMT3B, and DNMT3C), which catalyze the addition of methyl groups to genomic DNA. The term also encompasses non-canonical family members that do not themselves catalyze methylation but recruit (activate) catalytically active DNMTs; a non-limiting example of such a DNMT is DNMT3L. See, e.g., Lyko, Nat Review (2018) 19:81-92. Unless otherwise indicated, a DNMT domain refers to a polypeptide domain derived from a catalytically active DNMT (e.g., DNMT1, DNMT3A, and DNMT3B) or from a catalytically inactive DNMT (e.g., DNMT3L). DNMTs can repress the expression of target genes by recruiting inhibitory regulatory proteins. In some embodiments, the methylation is present in a CG (or CpG) dinucleotide sequence. In some embodiments, the methylation is present in a CHG or CHH sequence, where H is any one of A, T, or C. In some embodiments, the DNMT in the epigenetic editor may include, for example, DNMT1, DNMT3A, DNMT3B, and / or DNMT3C. In some embodiments, the DNMT is a mammalian (e.g., human or mouse) DNMT. In certain embodiments, the DNMT is DNMT3A (e.g., human DNMT3A). In certain embodiments, the epigenetic editor described herein includes a DNMT3A domain comprising SEQ ID NO: 1028, or a sequence at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 1028.In certain embodiments, the epigenetic editor described herein comprises a DNMT3A domain comprising SEQ ID NO: 1029, or a sequence at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 1029. In some embodiments, the DNMT3A domain can have, for example, a mutation at position H739 (e.g., H739A or H739E), a mutation at position R771 (e.g., R771L), and / or a mutation at position R836 (e.g., R836A or R836Q), or any combination thereof (numbering according to SEQ ID NO: 1028).

[0099]

[0104] In some embodiments, the effector domain described herein can be a DNMT-like domain. As used herein, a "DNMT-like domain" refers to a regulator of DNA methyltransferase that can activate or recruit other DNMT domains but does not itself have methylation activity. In some embodiments, the DNMT-like domain is a mammalian (e.g., human or mouse) DNMT-like domain. In certain embodiments, the DNMT-like domain is DNMT3L, which can be, for example, human DNMT3L or mouse DNMT3L. In certain embodiments, the epigenetic editor described herein comprises a DNMT3L domain comprising SEQ ID NO: 1032, or a sequence at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 1032. In certain embodiments, an epigenetic editor herein comprises a DNMT3L domain comprising SEQ ID NO: 1033, or a sequence at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 1033. In certain embodiments, an epigenetic editor described herein comprises a DNMT3L domain comprising SEQ ID NO: 1034, or a sequence at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 1034. In certain embodiments, an epigenetic editor described herein comprises a DNMT3L domain comprising SEQ ID NO: 1035, or a sequence at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 1035. In some embodiments, the DNMT3L domain can have, for example, a corresponding mutation at position D226 (e.g., D226V), a corresponding mutation at position Q268 (e.g., Q268K), or both (numbering according to SEQ ID NO: 1032).

[0100]

[0105] In certain embodiments, the epigenetic editors described herein may include both a DNMT effector domain and a DNMT-like effector domain. For example, the epigenetic editor may include a DNMT3A-3L domain in which DNMT3A and DNMT3L are covalently linked. In other embodiments, the epigenetic editors described herein may include an effector domain that includes only a DNMT3A domain (e.g., human DNMT3A) or only a DNMT-like domain (e.g., DNMT3L, which may be human or mouse DNMT3L).

[0101]

[0106] Table 5 below provides exemplary methyltransferases from which the effector domains of the epigenetic editors described herein may be derived. For the sequences of these exemplary methyltransferases, see Table 20.

[0102] [Table 5-1]

[0103] [Table 5-2]

[0104]

[0107] Functional analogs of any one of the above-listed proteins, i.e., molecules that have the same or substantially the same biological function (e.g., retain 70% or more, 80% or more, 90% or more, 95% or more, or 98% or more of the protein's DNA methylation or recruitment function), are encompassed by the present disclosure. For example, a functional analog can be an isoform or variant of the above-listed protein, e.g., containing a portion of the protein with or without additional amino acid residues and / or containing mutations compared to the above protein. In some embodiments, a functional analog has at least 75, 80, 85, 90, 95, 98, or 99% sequence identity to one of the sequences listed in Table 5. In some embodiments, an effector domain herein comprises only a functional domain (or a functional analog thereof) of a above-listed protein, e.g., a catalytic domain or a recruitment domain.

[0105]

[0108] As used herein, a DNMT domain (e.g., a DNMT3A domain or a DNMT3L domain) refers to a protein domain that is identical to a parent protein (e.g., human or mouse DNMT3A or DNMT3L), or a functional analog thereof (e.g., having a functional fragment of the parent protein, e.g., a catalytic or recruitment fragment; and / or having a mutation that improves the activity of the DNMT protein).

[0106]

[0109] The epigenetic editor herein can, for example, cause methylation of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500, 600, 700, 800, 900, or 1000 or more CpG dinucleotide sequences in target gene or chromosome.CpG dinucleotide sequence can be located in or near target gene in CpG island, or can be located in non-CpG island region.CpG island generally refers to nucleic acid sequence or chromosome region with high CpG dinucleotide frequency.For example, CpG island can contain at least 50% GC content. CpG islands may have a high observed-to-predicted CpG ratio, for example, an observed-to-predicted CpG ratio of at least 60%. As used herein, the observed-to-predicted CpG ratio is determined by the number of CpGs x (sequence length) / (number of Cs x number of Gs). In some embodiments, CpG islands have an observed-to-predicted CpG ratio of at least 60%, 70%, 80%, 90% or more. CpG islands may be, for example, sequences or regions of at least 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, or 800 nucleotides. In some embodiments, only 1, or fewer than 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 30, 40, or 50 CpG dinucleotides are methylated by the epigenetic editor.

[0107]

[0110] In some embodiments, the epigenetic editors herein induce methylation in hypomethylated nucleic acid sequences, i.e., sequences that may lack a methyl group at a 5-methylcytosine nucleotide (e.g., in a CpG) compared to a standard control. Hypomethylation can occur, for example, in senescent cells or in cancer (e.g., early stages of neoplasia) compared to younger cells or non-cancerous cells, respectively.

[0108]

[0111] In some embodiments, the epigenetic editors described herein induce methylation in hypermethylated nucleic acid sequences.

[0112] In some embodiments, methylation can be introduced by an epigenetic editor at sites other than CpG dinucleotides. For example, the target gene sequence can be methylated at the C nucleotide of a CpA, CpT, or CpC sequence. In some embodiments, the epigenetic editor includes a DNMT3A domain and induces methylation at CpG, CpA, CpT, CpC sequences, or any combination thereof. In some embodiments, the epigenetic editor includes a DNMT3A domain that lacks the regulatory subdomain and maintains only the catalytic domain. In some embodiments, an epigenetic editor including a DNMT3A catalytic domain induces methylation exclusively at CpG sequences. In some embodiments, an epigenetic editor including a DNMT3A domain containing a mutation, for example, an R836A or R836Q mutation (numbered according to SEQ ID NO: 1028), has higher methylation activity for CpA, CpC, and / or CpT sequences compared to an epigenetic editor including a wild-type DNMT3A domain.

[0109] C. Histone Modifiers

[0113] In some embodiments, the effector domain of the epigenetic editor herein mediates histone modification. Histone modification plays a structural and biochemical role in gene transcription, for example, by forming or destroying nucleosome structures that bind to histones and prevent gene transcription. Histone modifications can include, for example, acetylation, deacetylation, methylation, phosphorylation, ubiquitination, sumoylation, etc., for example, at their N-terminus ("histone tail"). These modifications maintain or specifically transform chromatin structure, thereby controlling responses occurring in chromosomal DNA, such as gene expression, DNA replication, DNA repair, etc. Post-translational modification of histones is an epigenetic regulatory mechanism and is considered to be essential for gene regulation in eukaryotic cells. Recent studies have revealed that chromatin remodeling factors, such as SWI / SNF, RSC, NURF, and NRD, which modify nucleosome structure to facilitate the access of transcription factors to DNA; histone acetyltransferases (HATs), which regulate the acetylation status of histones; and histone deacetylases (HDACs) are important regulatory factors.

[0110]

[0114] Specifically, the unstructured N-terminus of histones can be modified by acetylation, deacetylation, methylation, ubiquitination, phosphorylation, sumoylation, ribosylation, citrullination, O-GluNAcylation, crotonylation, or any combination thereof. For example, histone acetyltransferase (HAT) utilizes acetyl-CoA as a cofactor to catalyze the transfer of an acetyl group to the epsilon-amino group of a lysine side chain. This neutralizes the positive charge of the lysine, weakening the interaction between the histone and DNA, thus opening the chromosome for transcription factors to bind and initiate transcription. Acetylation of the K14 and K9 lysines of histone H3 by histone acetyltransferase enzymes may be associated with transcriptional competence in humans. Lysine acetylation may directly or indirectly create binding sites for chromatin-modifying enzymes that regulate transcriptional activation. Meanwhile, histone methylation of lysine 9 of histone H3 may be associated with heterochromatin, or transcriptionally silent chromatin.

[0111]

[0115] In certain embodiments, the effector domain of the epigenetic editor described herein comprises a histone methyltransferase domain. The effector domain may comprise, for example, a DOT1L domain, a SET domain, a SUV39H1 domain, a G9a / EHMT2 protein domain, an EZH1 domain, an EZH2 domain, a SETDB1 domain, or any combination thereof. In certain embodiments, the effector domain comprises a histone-lysine-N-methyltransferase SETDB1 domain.

[0112]

[0116] In some embodiments, the effector domain comprises a histone deacetylase protein domain. In certain embodiments, the effector domain comprises an HDAC family protein domain, such as HDAC1, HDAC3, HDAC5, HDAC7 or HDAC9 protein domain. In certain embodiments, the effector domain comprises a nucleosome remodeling deacetylase complex (NURD), which removes acetyl groups from histones.

[0113] D. Other Effector Domains

[0117] In some embodiments, the effector domain comprises a tripartite motif-containing protein (TRIM28, TIF1-beta, or KAP1). In certain embodiments, the effector domain comprises one or more KAP1 proteins. The KAP1 protein in the epigenetic editor herein may form a complex with one or more other effector domains of the epigenetic editor or with one or more proteins involved in modulating gene expression in a cellular environment. For example, KAP1 may be recruited by the KRAB domain of a transcriptional repressor. The KAP1 protein domain may interact with or recruit one or more protein complexes that reduce or silence gene expression. In some embodiments, KAP1 interacts with or recruits histone deacetylase proteins, histone-lysine methyltransferase proteins, chromatin remodeling proteins, and / or heterochromatin proteins. For example, the KAP1 protein domain can interact with or recruit heterochromatin protein 1 (HP1) protein, SETDB1 protein, HDAC protein, and / or NuRD protein complex components. In some embodiments, the KAP1 protein domain interacts with or recruits ZFP90 protein (e.g., ZFP90 isoform 2) and / or FOXP3 protein. An exemplary KAP1 amino acid sequence is set forth in SEQ ID NO: 1062.

[0114]

[0118] In some embodiments, the effector domain comprises a protein domain that interacts with or recruits one or more DNA epigenetic marks. For example, the effector domain may comprise a methyl-CpG-binding protein 2 (MECP2) protein, which interacts with methylated DNA nucleotides in a target gene (which may or may not be in a CpG island of the target gene). The MECP2 protein domain in the epigenetic editors described herein may induce a condensed chromatin structure, thereby reducing or silencing the expression of the target gene. In some embodiments, the MECP2 protein domain in the epigenetic editors described herein may interact with histone deacetylases (e.g., HDACs), thereby repressing or silencing the expression of the target gene. In some embodiments, the MECP2 protein domain in the epigenetic editors described herein may prevent transcription factors or transcriptional activators from reaching the target sequence, thereby repressing or silencing the expression of the target gene. An exemplary MECP2 amino acid sequence is set forth in SEQ ID NO: 1063.

[0115]

[0119] Examples of effector domains of the epigenetic editors described herein include, for example, a chromo shadow domain, a ubiquitin-2-like Rad60 SUMO-like (Rad60-SLD / SUMO) domain, a chromatin organization modifier domain (chromo domain), a Yaf2 / RYBP C-terminal binding motif domain (YAF2_RYBP), a CBX family C-terminal motif domain (CBX7_C), a zinc finger C3HC4 type (RING finger) domain (ZF-C3HC4_2), a cytochrome b5 domain (Cyt-b5), a helix-loop-helix domain (HLH), a helix-hairpin-helix motif domain (e.g., HHH_3), a high mobility group box domain (HMG-box), a basic leucine zipper domain (e.g., bZIP_1 or bZIP_2), a Myb_DNA binding domain, a homeodomain, an FCS sequence-containing MYM-type zinc finger domain (MYM-type Zinc finger with FCS sequence domain (ZF-FCS), interferon regulatory factor 2-binding protein zinc finger domain (IRF-2BP1_2), SSX repression domain (SSXRD), B-box type zinc finger domain (ZF-B_box), CXXC zinc finger domain (ZF-CXXC), chromosome condensation regulator 1 domain (RCC1), SRC homology 3 domain (SH3_9), sterile alpha motif domain (SAM_1), sterile alpha motif domain (SAM_2), sterile alpha motif / Pointed domain (SAM_PNT), vestibules / Tondu family domain (Vg_Tdu), LIM domain, RNA recognition motif domain (RRM_1), paired amphipathic helix domain (PAH), proteasome ATPase OB C-terminal domain (Prot_ATP_ID_OB), and Nervy homology 2 (nervy homology 2). 2) domain (NHR2), hinge domain of cleavage stimulatory factor subunit 2 (CSTF2_hinge), PPAR gamma N-terminal region domain (PPAR gamma_N), CDC48N-terminal domain (CDC48_2), WD40 repeat domain (WD40), Fip1 motif domain (Fip1), PDZ domain (PDZ_6), von Willebrand factor type C domain (VWC), NAB conserved region 1 domain (NCD1), S1 RNA binding domain (S1), HNF3 C-terminal domain (HNF_C), Tudor domain (Tudor_2), histone-like transcription factor (CBF / NF-Y) and archaeal histone domain (CBFD_NFYB_HMF), zinc finger protein domain (DUF3669), EGF-like domain (cEGF), GATA zinc finger domain (GATA), TEA / ATTS domain (TEA), phorbol ester / diacylglycerol binding domain (C1-1), polycomb-like MTF2 factor 2 domain (Mtf2_C), transactivation domain of the FOXO protein family (FOXO-TAD), homeobox KN domain Also contemplated are the nucleotide sequences of ...

[0116]

[0120] In some embodiments, the effector domain is a protein domain comprising a YAF2_RYBP domain or a homeodomain, or any combination thereof. In certain embodiments, the homeodomain of the YAF2_RYBP domain is a PRD domain, an NKL domain, a HOXL domain, or a LIM domain. In certain embodiments, the YAF2_RYBP domain may comprise a 32-amino acid Yaf2 / RYBP C-terminal binding motif domain (32aa RYBP).

[0117]

[0121] In some embodiments, the effector domain comprises a protein domain selected from the group consisting of a SUMO3 domain, a chromodomain of M-phase phosphoprotein 8 (MPP8), a chromoshadow domain from chromobox 1 (CBX1), and a SAM_1 / SPM domain from Scm polycomb group protein homolog 1 (SCMH1).

[0118]

[0122] In some embodiments, the effector domain comprises an HNF3 C-terminal domain (HNF_C). The HNF_C domain can be from FOXA1 or FOXA2. In certain embodiments, the HNF_C domain comprises an EH1 (Engrailed Homology 1) motif.

[0119]

[0123] In some embodiments, the effector domain may comprise an interferon regulatory factor 2 binding protein zinc finger domain (IRF-2BP1_2), a Cyt-b5 domain from the DNA repair factor HERC2 E3 ligase, a variant SH3 domain (SH3_9) from bridging integrator 1 (BIN1), an HMG box domain from the transcription factor TOX or a ZF-C3HC4_2 RING finger domain from the polycomb component PCGF2, a chromodomain-helicase-DNA binding protein 3 (CHD3) domain, or a ZNF783 domain.

[0120] IV. Epigenetic Editor

[0124] Provided herein is an epigenetic editor, also referred to herein as an epigenetic editing system, which uses, for example, any combination of one or more DNA binding domains described herein and one or more effector domains (e.g., epigenetic repression domains) described herein to direct epigenetic modification to a target sequence within a gene of interest. The DNA binding domain (in the case where the DNA binding domain is a polynucleotide-guided DNA binding domain, in cooperation with a guide polynucleotide such as those described herein) directs the effector domain to epigenetically modify the target sequence, resulting in gene suppression or silencing that can be permanent and heritable over cell generations. In some embodiments, the epigenetic editors described herein can reversibly or irreversibly suppress or silence genes in cells.

[0121]

[0125] In certain embodiments, the epigenetic editors described herein comprise one or more fusion proteins, each comprising (1) a DNA-binding domain and (2) an effector domain. The effector domain may be present on one or more fusion proteins included in the epigenetic editor. For example, a single fusion protein may comprise all of the effector domains together with the DNA-binding domain. Alternatively, the effector domains, or subsets thereof, may be present on separate fusion proteins, each with a DNA-binding domain (which may be the same or different). The fusion proteins described herein may further comprise one or more linkers (e.g., peptide linkers), detectable tags, nuclear localization signals (NLSs), or any combination thereof. As used herein, "fusion protein" refers to a chimeric protein in which two or more coding sequences (e.g., for the DNA-binding domain and / or the effector domain) are linked, either covalently or non-covalently, directly or indirectly.

[0122]

[0126] In some embodiments, the epigenetic editors described herein comprise two, three, four, five, six, seven, eight, nine, ten, or more effector (e.g., repression) domains, which may be the same or different. In certain embodiments, two or more of the effector domains function synergistically. The combination of effector domains may include a DNA methylation domain, a histone deacetylase domain, a histone methylation domain, and / or a scaffolding domain that recruits any of the above. For example, the epigenetic editors described herein may comprise one or more transcriptional repressor domains (e.g., KRAB domains, e.g., KOX1, ZIM3, ZFP28, or ZN627 KRAB) in combination with one or more DNA methylation domains (e.g., DNMT domains) and / or recruiter domains (e.g., DNMT3L domains). Such epigenetic editors may, for example, comprise a KRAB domain, a DNMT3A domain, and a DNMT3L domain. The epigenetic editor may comprise a DNMT3A domain and a DNMT3L domain, and preferably may further comprise a KRAB domain. In some embodiments, the epigenetic editor further comprises an additional effector domain (e.g., a KAP1, MECP2, HP1b, CBX8, CDYL2, TOX, TOX3, TOX4, EED, RBBP4, RCOR1, or SCML2 domain). In some embodiments, the additional effector domain is a CDYL2, TOX, TOX3, TOX4, or HP1a domain. For example, the epigenetic editor described herein may comprise a CDYL2 and / or TOX domain in combination with a KRAB domain (e.g., a KOX1 KRAB domain).

[0123] A. Linker

[0127] The fusion proteins described herein can include one or more linkers connecting the components of the epigenetic editor. The linkers can be peptide or non-peptide linkers.

[0124]

[0128] In some embodiments, one or more linkers utilized in the epigenetic editors provided herein are peptide linkers, i.e., linkers comprising a peptide moiety. Peptide linkers can be of any length applicable to the epigenetic editor fusion proteins described herein. In some embodiments, the linker can comprise a peptide of between 1 and 200 amino acids (e.g., between 1 and 80). In some embodiments, the linker can be of a length of 1 to 5, 1 to 10, 1 to 20, 1 to 30, 1 to 40, 1 to 50, 1 to 60, 1 to 80, 1 to 100, 1 to 150, 1 to 200, 5 to 10, 5 to 20, 5 to 30, 5 to 40, 5 to 60, 5 to 80, 5 to 100, 5 to 150, 5 to 200, 10 to 20, 10 to 30, 10 to 40, 10 to 50, 10 to 60, 10 to 80, 1 0-100, 10-150, 10-200, 20-30, 20-40, 20-50, 20-60, 20-80, 20-100, 20-150, 20-200, 30-40, 30-50, 30-60, 30-80, 30-100, 30-150, 30-200, 40-50, 40-60, 40-80, 40-100, 40-150, 40-200, 50-60 The peptide linker may comprise 50-80, 50-100, 50-150, 50-200, 60-80, 60-100, 60-150, 60-200, 80-100, 80-150, 80-200, 100-150, 100-200, or 150-200 amino acids. Longer or shorter linkers are also contemplated. In some embodiments, the peptide linker is 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 25, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or 100 amino acids in length. For example, the peptide linker can be 4, 5, 16, 20, 24, 27, 32, 40, 64, 92, or 104 amino acids in length. The peptide linker can be flexible or rigid. In certain embodiments, the peptide linker comprises the amino acid sequence of any one of SEQ ID NOs: 1064-1068, or a sequence at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical thereto.

[0125]

[0129] In certain embodiments, the peptide linker is an XTEN linker. Such a linker can include a portion of the XTEN sequence (Schellenberger et al., Nat Biotechnol (2009) 27(1):1186-90), an unstructured, hydrophilic polypeptide consisting only of residues G, S, P, T, E, and A. The term "XTEN," as used herein, refers to a recombinant peptide or polypeptide lacking hydrophobic amino acid residues. XTEN linkers are typically unstructured and contain a limited set of naturally occurring amino acids. Fusion of XTEN to a protein alters its hydrodynamic properties, reducing the clearance and degradation rate of the fusion protein. These XTEN fusion proteins are produced using recombinant technology without the need for chemical modification and are degraded by natural pathways. XTEN linkers can be, for example, 5, 10, 16, 20, 26, or 80 amino acids in length. In some embodiments, the XTEN linker is 16 amino acids in length. In some embodiments, the XTEN linker is 80 amino acids in length. In certain embodiments, the XTEN linker can be XTEN10, XTEN16, XTEN20, or XTEN80. In certain embodiments, the XTEN linker can comprise the amino acid sequence of any one of SEQ ID NOs: 1069-1073 and 1092, or a sequence at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical thereto. In some embodiments, the XTEN linker can be XTEN10, XTEN16, XTEN20, or XTEN80.

[0126]

[0130] In some embodiments, one or more linkers used in the epigenetic editors provided herein are non-peptide linkers.For example, the linker can be a carbon bond, a disulfide bond, or a carbon-heteroatom bond.In certain embodiments, the linker is a carbon-nitrogen bond of an amide linkage.In certain embodiments, the linker is a cyclic or acyclic, substituted or unsubstituted, or branched or unbranched aliphatic or heteroaliphatic linker.

[0127]

[0131] In some embodiments, one or more linkers utilized in the epigenetic editors provided herein are polymeric (e.g., polyethylene, polyethylene glycol, polyamide, polyester, etc.). The linker may include, for example, an aminoalkanoic acid monomer, dimer, or polymer; an aminoalkanoic acid (e.g., glycine, ethanoic acid, alanine, beta-alanine, 3-aminopropionic acid, 4-aminobutanoic acid, 5-pentanoic acid, etc.); an aminohexanoic acid (Ahx) monomer, dimer, or polymer; or a polyethylene glycol moiety (PEG); or an aryl or heteroaryl moiety. In certain embodiments, the linker may be based on a carboxylic acid moiety (e.g., cyclopentane or cyclohexane) or a phenyl ring. The linker may include a functionalized moiety to facilitate attachment of a nucleophile (e.g., thiol, amino) from the peptide to the linker. Any electrophile can be used as part of the linker. Exemplary electrophiles include, but are not limited to, activated esters, activated amides, alkyl halides, aryl halides, acyl halides, and isothiocyanates.

[0128]

[0132] A variety of linker lengths and flexibilities can be utilized between any two components of an epigenetic editor (e.g., between an effector domain (e.g., a repression domain) and a DNA-binding domain (e.g., a Cas9 domain), between a first effector domain and a second effector domain, etc.). Linkers can range from very flexible linkers, e.g., glycine / serine-rich linkers, to more rigid linkers to achieve an optimal length for effector domain activity for a particular application. In some embodiments, a more flexible linker is a glycine / serine-rich linker (GS-rich linker), in which more than 45% (e.g., more than 48, 50, 55, 60, 70, 80, or 90%) of the residues are glycine or serine residues. Non-limiting examples of GS-rich linkers are (GGGGS)n (SEQ ID NO: 485), (G)n, and the W linker (SEQ ID NO: 486). In some embodiments, more rigid linkers are of the form (EAAAK)n (SEQ ID NO:487), (SGGS)n (SEQ ID NO:488), and (XP)n (SEQ ID NO:489). In the above formulas for flexible and rigid linkers, n can be any integer between 1 and 30. In some embodiments, n is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15. In some embodiments, the linker comprises a (GGS)n motif, where n is 1, 3, or 7 (SEQ ID NO:490). In some embodiments, the linker comprises a (GGGGS)n motif, where n is 4 (SEQ ID NO:491).

[0129]

[0133] In some embodiments, a linker in an epigenetic editor described herein comprises a nuclear localization signal, e.g., having the amino acid sequence of any one of SEQ ID NOs: 1074-1079. In some embodiments, a linker in an epigenetic editor described herein comprises an expression tag, e.g., a detectable tag, e.g., green fluorescent protein.

[0130] B. Nuclear localization signal

[0134] The fusion proteins described herein may contain one or more nuclear localization signals, and in certain embodiments, may contain two or more nuclear localization signals. For example, a fusion protein may contain one, two, three, four, or five nuclear localization signals. As used herein, a "nuclear localization signal" (NLS) is an amino acid sequence that directs a protein to the nucleus. In certain embodiments, the NLS may be an SV40 NLS. A fusion protein may contain an NLS at its N-terminus, C-terminus, or both, and / or the NLS may be embedded in the center of the fusion protein (e.g., at the N- or C-terminus of a DNA-binding domain or effector domain). In certain embodiments, the NLS comprises the amino acid sequence of any one of SEQ ID NOs: 1074-1079, or a sequence at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to a selected sequence. Additional NLSs are known in the art.

[0131] C. Tag

[0135] The epigenetic editors provided herein can include one or more additional sequences ("tags") for tracking, detecting, and localizing the editor. In some embodiments, the epigenetic editor includes 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more detectable tags. Each of the detectable tags can be the same or different.

[0132]

[0136] For example, epigenetic editor fusion proteins can include a cytoplasmic localization sequence, a transport sequence, such as a nuclear export sequence, or other localization sequence, as well as a sequence tag that is useful for solubilizing, purifying, or detecting the fusion protein. Suitable protein tags provided herein include, but are not limited to, biotin carboxylase carrier protein (BCCP) tags, myc tags, calmodulin tags, FLAG tags, hemagglutinin (HA) tags, polyhistidine tags (also called histidine tags or His tags), maltose-binding protein (MBP) tags, nus tags, glutathione-S-transferase (GST) tags, green fluorescent protein (GFP) tags, thioredoxin tags, S tags, Softag (e.g., Softag 1 or Softag 3), strep tags, biotin ligase tags, FlAsH tags, V5 tags, and SBP tags. Additional suitable sequences will be apparent to those skilled in the art. Sequences disclosed herein that present included tag sequences are also contemplated without the tag sequence presented, and similarly, sequences disclosed herein that lack a tag sequence are also contemplated, including the addition of appropriate sequences apparent to one of skill in the art.

[0133] D. Fusion Protein Conjugation

[0137] The epigenetic editor fusion proteins described herein can have their components structured in different arrangements. For example, the DNA binding domain can be at the C-terminus, the N-terminus, or between two or more epigenetic editor domains or additional domains. In some embodiments, the DNA binding domain is at the C-terminus of the epigenetic editor. In some embodiments, the DNA binding domain is at the N-terminus of the epigenetic editor. In some embodiments, the DNA binding domain is linked to one or more nuclear localization signals. In some embodiments, the DNA binding domain is flanked on both sides by epigenetic effector domains and / or additional domains. In some embodiments, where "DBD" represents the DNA binding domain and "ED" represents the effector domain, the epigenetic editor is - N']-[ED1]-[DBD]-[ED2]-[C' - N']-[ED1]-[DBD]-[ED2]-[ED3]-[C' - N']-[ED1]-[ED2]-[DBD]-[ED3]-[C' or - N']-[ED1]-[ED2]-DBD]-[ED3]-[ED4]-[C' Includes placement of.

[0134]

[0138] In some embodiments, the epigenetic editor comprises a DNA binding domain (DBD), a DNA methyltransferase (DNMT) domain, and a transcriptional repressor ("repressor") domain that suppresses or silences expression of a target gene. The DBD, DNMT, and transcriptional repressor domains can be any of those described herein in any combination. For example, the epigenetic editor can comprise a DBD, a DNMT3A domain, and a DNMT3L domain. The epigenetic editor can comprise a DBD, a DNMT3A domain, a DNMT3L domain, and preferably further comprises a KRAB domain. In some embodiments, the epigenetic editor can comprise: N']-[DNA methyltransferase domain]-[DBD]-[repressor domain]-[C' N']-[repressor domain]-[DBD]-[DNA methyltransferase domain]-[C' N']-[DNA methyltransferase domain]-[repressor domain]-[DBD-[C' or N']-[repressor domain]-[DNA methyltransferase domain]-[DBD]-[C' The present invention includes fusion proteins having the following arrangement:

[0135]

[0139] In some embodiments, the connecting structure "]-[" in any one of the epigenetic editor structures is a linker, e.g., a peptide linker; a detectable tag; a peptide bond; a nuclear localization signal; and / or a promoter or regulatory sequence. Multiple connecting structures "]-[" in the epigenetic editor structure may be the same or different linkers, tags, NLSs, or peptide bonds. In certain embodiments, the DNA methyltransferase domain comprises DNMT3A, DNMT3L, or both. In certain embodiments, the DBD is a DNA binding domain (e.g., dCas9) guided by a catalytically inactive polynucleotide, or a ZFP domain. In certain embodiments, the repressor domain is a KRAB domain.

[0136]

[0140] In some embodiments, the epigenetic editor comprises: N']-[DNMT3A-DNMT3L]-[DBD]-[KRAB]-[C' N']-[KRAB]-[DBD]-[DNMT3A-DNMT3L]-[C' N']-[KRAB]-[DBD]-[DNMT3A]-[C' N']-[DNMT3A]-[DBD]-[KRAB]-[C' N']-[KRAB]-[DBD]-[DNMT3A]-[DNMT3L]-[C' N']-[DNMT3A]-[DNMT3L]-[DBD]-[KRAB]-[C' N']-[DNMT3A]-[DBD]-[C' N']-[DBD]-[DNMT3A]-[C' N']-[DNMT3L]-[DBD]-[C' N']-[DBD]-[DNMT3L]-[C' wherein [DNMT3A-DNMT3L] indicates that the DNMT3A and DNMT3L domains are directly fused by a peptide bond, and the connecting structure]-[ is any one of the linkers described herein, a detectable tag, an affinity domain, a peptide bond, a nuclear localization signal, a promoter, and / or a regulatory sequence. The DBD, KRAB, DNMT3A, and DNMT3L domains can be any of those described herein in any combination. In certain embodiments, the DBD is a CRISPR-associated protein domain (e.g., dCas9) or a ZFP domain; the KRAB domain is derived from KOX1, ZIM3, ZFP28, or ZN627; the DNMT3A domain is a human DNMT3A domain; and the DNMT3L domain is a human or mouse DNMT3L domain; any combination of these components is also contemplated by the present disclosure.

[0137]

[0141] In some embodiments, the epigenetic editor comprises: N']-[DNMT3A]-[DBD]-[SETDB1]-[C' N']-[DNMT3A]-[DNMT3L]-[DBD]-[SETDB1]-[C' N']-[DNMT3A-DNMT3L]-[DBD]-[SETDB1]-[C' N']-[SETDB1]-[DBD]-[DNMT3A]-[DNMT3L]-[C' N']-[SETDB1]-[DBD]-[DNMT3A]-[C' wherein [DNMT3A-DNMT3L] indicates that the DNMT3A and DNMT3L domains are directly fused by a peptide bond, and the connecting structure]-[ is any one of the linkers described herein, a detectable tag, an affinity domain, a peptide bond, a nuclear localization signal, a promoter, and / or a regulatory sequence. The DBD, SETDB1, DNMT3A, and DNMT3L domains can be any of those described herein in any combination. In certain embodiments, the DBD is a CRISPR-associated protein domain (e.g., dCas9) or a ZFP domain; the SETDB1 domain is derived from human SETDB1, ZIM3, ZFP28, or ZN627; the DNMT3A domain is a human DNMT3A domain; and the DNMT3L domain is a human or mouse DNMT3L domain; any combination of these components is also contemplated by the present disclosure.

[0138]

[0142] Particular constructs contemplated herein include: DNMT3A-DNMT3L-XTEN80-NLS-dCas9-NLS-XTEN16-KOX1 KRAB (arrangement 1), and DNMT3A-DNMT3L-XTEN80-NLS-ZFP domain-NLS-XTEN16-KOX1 KRAB (arrangement 2) In certain embodiments, DNMT3L and DNMT3A are both derived from human parent proteins. In certain embodiments, DNMT3L and DNMT3A are derived from human and mouse parent proteins, respectively. In certain embodiments, DNMT3L and DNMT3A are derived from mouse and human parent proteins, respectively. In certain embodiments, DNMT3L and DNMT3A are both derived from mouse parent proteins. In some embodiments, dCas9 is dSpCas9. In some embodiments, KOX1 is human KOX1.

[0139]

[0143] In certain embodiments, the fusion proteins described herein may have Configuration 1 and may include SEQ ID NO: 1080, or a sequence at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical thereto. In SEQ ID NO: 1080 below, the XTEN linker is underlined, the NLS sequence is in bold, the DNMT3A sequence is in italics, the DNMT3L sequence is underlined and italics, the dCas9 domain is in bold and italics, and the KOX1 KRAB domain is underlined and bold:

[0140] [ka]

[0141]

[0144] In certain embodiments, the fusion protein described herein may have configuration 2 and include SEQ ID NO: 1081, or a sequence at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical thereto. In SEQ ID NO: 1081 below, the XTEN linker is underlined, the NLS sequence is bold and underlined, the DNMT3A sequence is italicized, the DNMT3L sequence is underlined and italicized, the ZFP domain is bold, and the KOX1 KRAB domain is underlined and bold. The variable amino acids represented by X are amino acids of the DNA recognition helix of the zinc finger, and the italicized XX can be either TR, LR, or LK.

[0142] [ka]

[0143] In certain embodiments, the six "XXXXXXX" regions in SEQ ID NO: 1081 comprise, in order, the F1-F6 amino acid sequences shown in Table 1. [Linker] represents a linker sequence. In some embodiments, one or both linker sequences may be TGSQKP (SEQ ID NO: 1085). In some embodiments, one or both linker sequences may be TGGGGSQKP (SEQ ID NO: 1086). In some embodiments, one linker sequence may have the amino acid sequence of SEQ ID NO: 1085, and the other linker sequence may have the amino acid sequence of SEQ ID NO: 1086.

[0144]

[0145] Multiple epigenetic editors can be used to activate or suppress a target gene or multiple target genes. For example, an epigenetic editor fusion protein comprising a DNA binding domain (e.g., dCas9 domain) and an effector domain can be co-delivered with two or more guide polynucleotides (e.g., gRNA), each targeting a different target DNA sequence. The target sites for two of the DNA binding domains can be the same, or can be adjacent to each other, or can be separated by, for example, about 100 base pairs, about 200 base pairs, about 300 base pairs, about 400 base pairs, about 500 base pairs, or about 600 or more base pairs. In addition, when targeting double-stranded DNA, such as an endogenous gene locus, the guide polynucleotides can target the same or different strands (one or more for the plus strand and / or one or more for the minus strand).

[0145] V. Target Sequence

[0146] The epigenetic editor herein can be directed to an HBV target sequence to effect epigenetic modification of HBV or an HBV gene. As used herein, a "target sequence," "target site," or "target region" is a nucleic acid sequence present within a genome or gene of interest, e.g., within an HBV genome or HBV gene; in some cases, the target sequence may be outside but near the gene of interest, and methylation of the target sequence or binding by a repressor represses gene expression. In some embodiments, the target sequence may be a hypomethylated or hypermethylated nucleic acid sequence.

[0146]

[0147] The target sequence can be located in any part of the target gene. In some embodiments, the target sequence is part of or near the non-coding sequence of the gene. In some embodiments, the target sequence is part of an exon of the gene. In some embodiments, the target sequence is part of or near the transcriptional regulatory sequence of the gene, for example, a promoter or repressor. In some embodiments, the target sequence is adjacent to, overlaps with, or encompasses a CpG island, for example, a CpG island identified in the HBV genome. In some embodiments, the target sequence is outside the CpG island. In certain embodiments, the target sequence is within about 3000, 2900, 2800, 2700, 2600, 2500, 2400, 2300, 2200, 2100, 2000, 1900, 1800, 1700, 1600, 1500, 1400, 1300, 1200, 1100, 1000, 900, 800, 700, 600, 500, 400, 300, 200, or 100 base pairs (bp) adjacent to the HBV TSS. In certain embodiments, the target sequence is within 500 bp adjacent to the HBV TSS. In certain embodiments, the target sequence is within 1000 bp adjacent to the HBV TSS.

[0147]

[0148] In some embodiments, the target sequence may hybridize to a guide polynucleotide sequence (e.g., gRNA) complexed with a fusion protein comprising a polynucleotide-guided DNA-binding domain (e.g., a CRISPR protein, e.g., dCas9) and an effector domain. The guide polynucleotide sequence may be designed to be complementary to the target sequence or identical to the opposite strand of the target sequence. In some embodiments, the guide polynucleotide comprises a spacer sequence that is about 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the protospacer sequence in the target sequence. In certain embodiments, the guide polynucleotide comprises a spacer sequence that is 100% identical to the protospacer sequence in the target sequence.

[0148]

[0149] In some embodiments, when the DNA binding domain of an epigenetic editor described herein is a zinc finger array, the target sequence can be recognized by said zinc finger array.

[0149]

[0150] In some embodiments, when the DNA binding domain of the epigenetic editor described herein is a TALE, the target sequence can be recognized by said TALE.

[0150]

[0151] The target sequence described herein can be specific to one genotype of HBV, one copy of HBV target gene, or one allele of HBV target gene.However, in some embodiments, the target sequence can be conserved across two or more HBV genotypes, across two or more copies of HBV gene, and across alleles of HBV gene.Therefore, epigenetic modification and its expression modulation can be specific to one copy or one allele of target gene, or in other embodiments, can be universal across different HBV genotypes, or HBV gene copies or alleles.

[0151]

[0152] In some embodiments, the target sequence is comprised in the following sequence:

[0152] [ka]

[0153]

[0153] In some embodiments, the target sequence is comprised in the following sequence:

[0154] [ka]

[0155] VI. Epigenetic Modifications The epigenetic editors described herein can make sequence-specific epigenetic modifications (e.g., altered chemical modifications) of target genes harboring the target sequence. Such epigenetic modulation is safer and more easily reversible than modulation resulting from gene editing, e.g., using the generation of DNA double-strand breaks. In some embodiments, the epigenetic modulation can reduce or silence the target gene. In some embodiments, the modification is directed to a specific site in the target sequence. In some embodiments, the modification is directed to a specific allele of the target sequence. Thus, the epigenetic modification can result in modulating (e.g., reducing) expression of copies of the target gene harboring a specific allele, but not modulating (e.g., not reducing) expression of other copies of the target gene. In some embodiments, the specific allele is associated with a disease, condition, or disorder.

[0156] In some embodiments, the epigenetic modification reduces or eliminates transcription of a target gene harboring the target sequence. In some embodiments, the epigenetic modification reduces or eliminates transcription of a copy of the target gene harboring a specific allele recognized by the epigenetic editor. In some embodiments, the epigenetic editor reduces or eliminates the level of expression of a protein encoded by the target gene. In some embodiments, the epigenetic editor reduces or eliminates the level of expression of a protein encoded by a copy of the target gene harboring a specific allele recognized by the epigenetic editor. The target HBV gene can be epigenetically modified in vitro, ex vivo, or in vivo.

[0157] The effector domain of the epigenetic editors described herein can alter (e.g., accumulate or remove) chemical modifications to nucleotides of a target gene or to histones associated with the target gene. Chemical modifications to a single nucleotide or a single histone can be altered, or chemical modifications to 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 1500, 2000, 2500, 3000 or more nucleotides can be altered.

[0158] In some embodiments, the effector domain of an epigenetic editor described herein can alter a CpG dinucleotide within a target gene. In some embodiments, all CpG dinucleotides within a 2000, 1500, 1000, 500, or 200 bp range adjacent to the target sequence (e.g., at the alteration site described herein) are altered according to a modification type described herein, compared to the original state of the gene or the gene in a comparable cell not contacted with the epigenetic editor. In some embodiments, at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, 50, 60, 70, 80, 90, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700 or more CpG dinucleotides are altered compared to the original state of the gene or the gene in a comparable cell not contacted with the epigenetic editor. In some embodiments, at least 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% of the CpG dinucleotides are altered compared to the original state of the gene or the gene in a comparable cell not contacted with an epigenetic editor, hi some embodiments, a single CpG dinucleotide is altered compared to the original state of the gene or the gene in a comparable cell not contacted with an epigenetic editor.

[0159] The effector domain of the epigenetic editor described herein can alter the histone modification state of histones associated with or bound to a target gene. For example, the effector domain can cause a modification to accumulate on one or more lysine residues of the histone tails of the histones associated with the target gene. In some embodiments, the effector domain can cause deacetylation of one or more histone tails of the histones associated with the target gene, thereby reducing or silencing expression of the target gene. In some embodiments, the histone modification state is a methylation state. For example, the effector domain can cause H3K9, H3K27, or H4K20 methylation (e.g., one or more of H3K9me2, H3K9me3, H3K27me2, H3K27me3, and H4K20me3 methylation) on one or more histone tails associated with the target gene, thereby reducing or silencing expression of the target gene.

[0160] In some embodiments, all histone tails attached to DNA nucleotides within a 2000, 1500, 1000, 500, or 200 bp range flanking the target sequence are altered according to the modification types described herein, compared to the original state of the chromosome or a comparable cell not contacted with an epigenetic editor. In some embodiments, at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 105, 110, 115, 120, or more of the attached histone tails are altered, compared to the original state of the chromosome or a comparable cell not contacted with an epigenetic editor. In some embodiments, at least 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% of the histone tails of the bound histones are altered compared to the original state of the chromosome or a comparable cell not contacted with an epigenetic editor. For example, a single histone tail of the bound histones may be altered compared to the original state of the chromosome or a comparable cell not contacted with an epigenetic editor. As another example, a single bound histone octamer may be altered compared to the original state of the chromosome or a comparable cell not contacted with an epigenetic editor.

[0161] The chemical modifications deposited on target gene DNA nucleotides or histone residues can be at or immediately adjacent to a target sequence within the target gene. In some embodiments, the effector domain of an epigenetic editor described herein alters the chemical modification state of a nucleotide or histone tail attached to a nucleotide 100-200, 200-300, 300-400, 400-55, 500-600, 600-700, or 700-800 nucleotides 5' or 3' to a target sequence within the target gene. In some embodiments, the effector domain alters the chemical modification state of nucleotides or histone tails attached to nucleotides within 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 1100, 1200, 1300, 1400, 1500, 1600, 1700, 1800, 1900, or 2000 nucleotides adjacent to the target sequence. As used herein, "adjacent" refers to nucleotide positions 5' to the 5' end and 3' to the 3' end of a particular sequence, e.g., a target sequence.

[0162] In some embodiments, the effector domain mediates or induces a change in a chemical modification of a nucleotide or histone tail attached to a nucleotide distal to the target sequence. Such a modification may be initiated near the target sequence and then propagated to one or more nucleotides within the target gene distal to the target sequence. For example, the effector domain may initiate an alteration in the chemical modification state of one or more nucleotides or one or more histone residues bound to one or more nucleotides within a range of 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500 nucleotides adjacent to the target sequence, and the alteration in the chemical modification state may propagate from the target sequence within the target gene to one or more nucleotides either upstream or downstream of the target sequence at least 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 1100, 1200, 1300, 1400, 1500, 1600, 1700, 1800, 1900, 2000, 2500, 3000, or more nucleotides. In certain embodiments, the chemical modification may begin at less than 2, 3, 5, 10, 20, 30, 40, 50, or 100 nucleotides within the target gene and propagate to at least 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 2000, or more nucleotides within the target gene. In some embodiments, the chemical modification propagates to the entire gene. Additional proteins or transcription factors, such as transcription repressors, methyltransferases, or transcriptional regulatory scaffold proteins, may also be involved in the propagation of the chemical modification. Alternatively, only epigenetic editors may be involved.

[0163] In some embodiments, the epigenetic editors described herein reduce expression of a target gene by at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 95%, at least about 99%, or more compared to a control cell, control tissue, or control subject (e.g., in the absence of the epigenetic editor), as measured by transcription of the target gene copy in a cell, tissue, or subject. In some embodiments, the epigenetic editors described herein reduce expression of a copy of the target gene by at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 95%, at least about 99%, or more compared to a control cell, control tissue, or control subject, as measured by transcription of a copy of the target gene in a cell, tissue, or subject. In certain embodiments, the copy of the target gene harbors a specific sequence or allele that is recognized by the epigenetic editor. In certain embodiments, the epigenetically modified copy encodes a functional protein, and thus, the epigenetic editors disclosed herein can reduce or eliminate protein expression and / or function. For example, the epigenetic editors described herein may reduce the expression and / or function of a protein encoded by a target gene by at least 3-fold, at least 4-fold, at least 5-fold, at least 6-fold, at least 7-fold, at least 8-fold, at least 9-fold, at least 10-fold, at least 11-fold, at least 12-fold, at least 13-fold, at least 14-fold, at least 15-fold, at least 20-fold, at least 25-fold, at least 30-fold, at least 35-fold, at least 40-fold, at least 45-fold, at least 50-fold, at least 60-fold, at least 70-fold, at least 80-fold, at least 90-fold, or at least 100-fold in a cell, tissue, or subject compared to a control cell, control tissue, or control subject.

[0164] Modulation of target gene expression can be assayed by determining parameters that are either indirectly or directly affected by expression of the target gene, such as changes in RNA or protein levels; changes in protein activity; changes in product levels; changes in downstream gene expression; changes in transcription or activity of reporter genes such as luciferase, CAT, beta-galactosidase, or GFP; changes in signal transduction; changes in phosphorylation and dephosphorylation; changes in receptor-ligand interactions; changes in second messengers such as cGMP, cAMP, IP3, and Ca2 + These include changes in the concentration of proteins, such as proteins; changes in cell growth; changes in angiogenesis; and / or changes in any functional effects of gene expression. Measurements can be performed in vitro, in vivo, and / or ex vivo, and can be performed by conventional methods, such as measuring RNA or protein levels, measuring RNA stability, and / or identifying downstream or reporter gene expression. Readouts can be, for example, changes in intracellular second messengers, such as cGMP and inositol triphosphate (IP3), changes in intracellular calcium levels, cytokine release, etc., via chemiluminescence, fluorescence, colorimetric reactions, antibody binding, inducible markers, or ligand binding assays.

[0165] Methods for determining the expression level of a gene, e.g., a target of an epigenetic editor, can include determining the transcript level of the gene by, for example, reverse transcription PCR, quantitative RT-PCR, droplet digital PCR (ddPCR), Northern blot, RNA sequencing, DNA sequencing (e.g., sequencing of complementary deoxyribonucleic acid (cDNA) obtained from RNA), next-generation (Next-Gen) sequencing, nanopore sequencing, pyrosequencing, or nanostring sequencing. The level of protein expressed from the gene can be determined by, for example, Western blotting, enzyme-linked immunoabsorbance assay, mass spectrometry, immunohistochemistry, or flow cytometry analysis. Gene expression product levels can be normalized to an internal standard, e.g., total messenger ribonucleic acid (mRNA), or the expression level of a specific gene, e.g., a housekeeping gene.

[0166] In some embodiments, the effect of an epigenetic editor in modulating target gene expression can be investigated using a reporter system. For example, an epigenetic editor can be designed to target a reporter gene encoding a reporter protein, such as a fluorescent protein. Expression of the reporter gene in such a model system can be monitored, for example, by flow cytometry, fluorescence-activated cell sorting (FACS), or fluorescence microscopy. In some embodiments, a population of cells can be transfected with a vector harboring a reporter gene. The vector can be constructed such that the reporter gene is expressed when the vector is transfected into cells. Suitable reporter genes include genes encoding fluorescent proteins, such as green, yellow, cherry, cyan, or orange fluorescent proteins. A population of cells with a reporter system can be transfected with DNA, mRNA, or a vector encoding an epigenetic editor targeted to the reporter gene.

[0167] VII. Pharmaceutical Compositions Another aspect of the present disclosure is a pharmaceutical composition comprising, as an active ingredient (or sole active ingredient), one or more epigenetic editors described herein or their component parts (e.g., fusion proteins and / or guide polynucleotides), or nucleic acid molecules encoding said epigenetic editors or their component parts. For example, a pharmaceutical composition may contain a nucleic acid molecule encoding a fusion protein (and, if applicable, a guide polynucleotide) of an epigenetic editor described herein. In some embodiments, separate pharmaceutical compositions comprise the fusion protein and the guide polynucleotide. In some embodiments, multiple pharmaceutical compositions, each comprising an epigenetic editor, are administered simultaneously. A pharmaceutical composition may also contain cells that have undergone epigenetic modifications mediated or introduced by the epigenetic editors provided herein.

[0168]

[0167] Generally, the epigenetic editors or components thereof described herein of the present disclosure, or nucleic acid molecules encoding said epigenetic editors or components thereof, are suitable for administration as a formulation in association with one or more pharmaceutically acceptable excipients, for example, as described below.

[0169] The term "excipient" is used herein to describe any ingredient other than the compound of the present disclosure. The choice of excipient will largely depend on factors such as the particular mode of administration, the excipient's effect on solubility and stability, and the nature of the dosage form. As used herein, "pharmaceutically acceptable excipient" includes any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonicity and absorption delaying agents, and the like that are physiologically compatible. Some examples of pharmaceutically acceptable excipients are water, saline, phosphate-buffered saline, dextrose, glycerol, ethanol, and the like, and combinations thereof. In many cases, it will be preferable to include an isotonic agent, for example, a sugar, a polyalcohol such as mannitol, sorbitol, or sodium chloride in the composition. Additional examples of pharmaceutically acceptable substances are wetting agents, or minor amounts of auxiliary substances that enhance the shelf life or effectiveness of the antibody, such as wetting or emulsifying agents, preservatives, or buffers.

[0170] Pharmaceutical composition formulations suitable for parenteral administration typically include the active ingredient in combination with a pharmaceutically acceptable carrier, such as sterile water or sterile isotonic saline. Such formulations may be prepared, packaged, or sold in a form suitable for bolus administration or for continuous administration. In some embodiments, the epigenetic editor or its components are introduced into target cells in the form of a nucleic acid molecule encoding the epigenetic editor or its components; therefore, the pharmaceutical compositions herein comprise a nucleic acid molecule. Such nucleic acid molecules can be, for example, DNA, RNA, or mRNA, and / or modified nucleic acid sequences (e.g., having a chemical modification, a 5' cap, or one or more 3' modifications). In some embodiments, the nucleic acid molecule can be delivered as naked DNA or RNA, for example, by transfection or electroporation, or can be conjugated to a molecule (e.g., N-acetylgalactosamine) that facilitates uptake by target cells. In some embodiments, the nucleic acid molecule may be within a nucleic acid expression vector, which may include expression control sequences, such as promoters, enhancers, transcription signal sequences, transcription termination sequences, introns, polyadenylation signals, Kozak consensus sequences, internal ribosome entry sites (IRES), etc. Such expression control sequences are well known in the art. The vector may also include a sequence encoding a signal peptide (e.g., for nuclear localization, nucleolar localization, or mitochondrial localization) associated with (e.g., inserted into or fused to) the protein-encoding sequence.

[0171] Examples of vectors include, but are not limited to, plasmid vectors; viral vectors based on vaccinia virus, poliovirus, adenovirus, adeno-associated virus, SV40, herpes simplex virus, human immunodeficiency virus, retrovirus (e.g., murine leukemia virus, or spleen necrosis virus); vectors derived from retroviruses, such as Rous sarcoma virus, Harvey sarcoma virus, avian leukosis virus, lentivirus, human immunodeficiency virus, myeloproliferative sarcoma virus, and mammary tumor virus; and other recombinant vectors. In certain embodiments, the vector is a plasmid or viral vector. Viral particles can also be used to deliver nucleic acid molecules encoding the epigenetic editors described herein or their components. For example, "empty" viral particles can be assembled to contain any appropriate cargo. Viral vectors and viral particles can also be engineered to incorporate targeting ligands to alter target tissue specificity.

[0172] In certain embodiments, the epigenetic editors or components thereof described herein are encoded by nucleic acid sequences present in one or more viral vectors or by suitable capsid proteins of any viral vector. Examples of viral vectors include adeno-associated viral vectors (e.g., derived from AAV3, AAV3b, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAVrh8, AAV10, and / or variants thereof); retroviral vectors (e.g., Moloney murine leukemia virus, MML-V), adenoviral vectors (e.g., AD100), lentiviral vectors (e.g., HIV- and FIV-based vectors), and herpesviral vectors (e.g., HSV-2).

[0173] In some embodiments, delivery involves an adeno-associated virus (AAV) vector. AAV vector delivery can be particularly useful when the DNA-binding domain of the epigenetic editor fusion protein is a zinc finger. While not wishing to be bound by any one, the smaller size of zinc finger arrays compared to larger DNA-binding domains, such as Cas protein domains, can allow such fusion proteins to be conveniently packaged into viral vectors, such as AAV vectors.

[0174] Any AAV serotype, e.g., a human AAV serotype, can be used in the AAV vectors described herein, including, but not limited to, AAV serotype 1 (AAV1), AAV serotype 2 (AAV2), AAV serotype 3 (AAV3), AAV serotype 4 (AAV4), AAV serotype 5 (AAV5), AAV serotype 6 (AAV6), AAV serotype 7 (AAV7), AAV serotype 8 (AAV8), AAV serotype 9 (AAV9), AAV serotype 10 (AAV10), and AAV serotype 11 (AAV11), and variants thereof. In some embodiments, the AAV variant has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% amino acid sequence identity to wild-type AAV. In certain embodiments, AAV variants can be engineered so that their capsid proteins have reduced immunogenicity or enhanced transduction ability in humans. In some cases, one or more regions of at least two different AAV serotype viruses are shuffled and reassembled to generate chimeric variants. For example, chimeric AAVs can contain inverted terminal repeats (ITRs) of a heterologous serotype compared to the serotype of the capsid. The resulting chimeric AAV can have different antigen reactivity or recognition compared to its parent serotype. In some embodiments, chimeric AAV variants contain amino acid sequences from two, three, four, five, or more different AAV serotypes.

[0175] Non-viral systems are also contemplated for delivery as described herein. Non-viral systems include, but are not limited to, nucleic acid transfection methods, including electroporation, sonoporation, calcium phosphate transfection, microinjection, DNA biolistic methods, lipid-mediated transfection, heat shock transfection, compacted DNA-mediated transfection, lipofection, cationic drug-mediated transfection, and transfection using liposomes, immunoliposomes, or cationic facial amphiphiles (CFAs). In certain embodiments, one or more mRNAs encoding the epigenetic editor fusion proteins described herein can be co-electroporated with one or more guide polynucleotides (e.g., gRNAs) described herein. One important category of non-viral nucleic acid vectors is nanoparticles, which can be organic (e.g., lipid) or inorganic (e.g., gold) nanoparticles. For example, organic (eg, lipid and / or polymer) nanoparticles may be suitable for use as delivery vehicles in certain embodiments of the present disclosure.

[0176] In some embodiments, delivery is accomplished using lipid nanoparticles (LNPs). LNP compositions are typically on the order of micrometers or smaller in size and may comprise a lipid bilayer. In some embodiments, LNP refers to any particle having a diameter of less than 1000 nm, 500 nm, 250 nm, 200 nm, 150 nm, 100 nm, 75 nm, 50 nm, or 25 nm. Nanoparticle compositions include lipid nanoparticles (LNPs), liposomes (e.g., lipid vesicles), and lipoplexes.

[0177] The LNPs described herein can be prepared from cationic, anionic, or neutral lipids. In some embodiments, LNPs can include a neutral lipid, such as the fusogenic phospholipid 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE) or the membrane component cholesterol, as a helper lipid to enhance transfection activity and nanoparticle stability. In some embodiments, LNPs can include hydrophobic lipids, hydrophilic lipids, or both hydrophobic and hydrophilic lipids. Any lipid or lipid combination known in the art can be used to generate LNPs. Lipids can be combined in any molar ratio to generate LNPs. In some embodiments, LNPs are liver-targeted (e.g., preferentially or specifically target the liver) LNPs.

[0178]

[0177] LNP formulations and LNP delivery methods that can be used will be apparent to those skilled in the art based on this disclosure and the current state of the art. Non-limiting, exemplary compositions and methods include Shah, R., Eldridge, D., Palombo, E., and Harding, I., Lipid Nanoparticles: Production, Characterization and Stability, Springer, 2015, ISBN-13 978-3319107103; Ziegler, S., Lipid Nanoparticles: Advances in Research and Applications, Nova Science Pub., Inc., ISBN-13 978-1536186536; Mitchell, MJ, Billingsley, MM, Haley, RM et al., Engineering precision nanoparticles for drug delivery, Nat Rev Drug Discov 20, 101-124 (2021); Hou, X., Zaks, T., Langer, R. et al., Lipid nanoparticles for mRNA delivery. Nat Rev Mater 6, 1078~1094(2021); Lipid-Nanoparticle-Based Delivery of CRISPR / Cas9 Genome-Editing Components, Pardis Kazemian, Si-Yue Yu, Sarah B. Thomson, Alexandra Birkenshaw, Blair R. Leavitt, and Colin JDRoss., Molecular Pharmaceutics 2022 19(6), 1669~1686; Cullis PR, Hope MJ., Lipid Nanoparticle Systems for Enabling Gene Therapies, Mol Ther. 2017 Jul 5; 25(7):1467~1475; Hatit, MZC, Lokugamage, MP, Dobrowolski, CNet al., "Species-dependent in vivo mRNA delivery and cellular responses to nanoparticles," Nat. Nanotechnol. 17, 310 - 318 (2022); Lam, K., Schreiner, P., Leung, A., Stainton, P., Reid, S., Yaworski, E., Lutwyche, P. and Heyes, J. (2023), "Optimizing Lipid Nanoparticles for Delivery in Primates," Adv. Mater; Dilliard, S.A., Siegwart, D.J., "Passive, active and endogenous organ-targeted lipid and polymer nanoparticles for delivery of genetic drugs," Nat Rev Mater (2023); Kasiewicz, L.N. et al., "Lipid nanoparticles incorporating a GalNAc ligand enable in vivo liver ANGPTL3 editing in wild-type and somatic LDLR knockout non-human primates," bioRxiv 2021.11.08.467731, doi:https: / / doi.org / 10.1101 / 2021.11.08.467731; Tombacz, I. et al., "Highly efficient CD4+ T cell targeting and genetic recombination using engineered CD4+ cell-homing mRNA-LNPs," Molecular Therapy, Vol. 29, No. 11, 2021, 3293 - 3304; Cheng, Q., Wei, T., Farbiak, L. et al., "Selective organ targeting (SORT) nanoparticles for tissue-specific mRNA delivery and CRISPR-Cas gene editing," Nat. Nanotechnol. 15, 313 - 320 (2020); Zhang, Y.et al., Lipids and Lipid Derivatives for RNA Delivery, Chemical Reviews 2021 121(20);Lam, K. et al., Unsaturated,Trialkyl Ionizable Lipids are Versatile Lipid-Nanoparticle Components for Therapeutic and Vaccine Applications, Adv.Mater.2023, 35;Han, X., Zhang, H., Butowska, K. et al., An ionizable lipid toolbox for RNA delivery, Nat Commun 12, 7233(2021);U.S. Patent No. 9,364,435;U.S. Patent No. 8,058,069;U.S. Patent No. 8,822,668;U.S. Patent No. 8,492,359;U.S. Patent No. 11,141,378;U.S. Patent No. 9,518,272;U.S. Patent No. 9,404,127;U.S. Patent No. 9,006,417;U.S. Patent No. 7,901,708;U.S. Patent No. 9,005,654;U.S. Patent No. 9,878,042;U.S. Patent No. 9,682,139;U.S. Patent No. Nos. 8,642,076; 9,593,077; 9,415,109; 9,701,623; 10,369,226; 9,999,673; 9,301,923; 10,342,761; 10,137,201; and International Publication No. WO2016081029A1, each of which is incorporated herein by reference in its entirety. One of ordinary skill in the art would be able to identify suitable LNPs and delivery methods based on this disclosure and the current state of the art. The disclosure is not limited in this respect.

[0179]

[0178] Other methods of delivery to target cells will be known to those of skill in the art and can be used with the compositions of the present disclosure. Any type of cell can be targeted for delivery of the epigenetic editors or components thereof described herein. For example, the cells can be eukaryotic or prokaryotic. In some embodiments, the cells are mammalian (e.g., human) cells. Human cells can include, for example, hepatocytes, biliary epithelial cells (cholangiocytes), stellate cells, Kupffer cells, and hepatic sinusoidal endothelial cells.

[0180] In some embodiments, the epigenetic editors described herein or their components are delivered to host cells for transient expression, for example, by a transient expression vector. Transient expression of the epigenetic editors or their components can result in persistent or permanent epigenetic modifications of the target gene. For example, the epigenetic modifications can be stable for at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 weeks or more, or for 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 months or more after introduction of the epigenetic editor into the host cell. The epigenetic modifications can be maintained after one or more mitotic and / or meiotic events of the host cell. In certain embodiments, the epigenetic modifications are maintained transgenerationally in progeny arising from or derived from the host cell.

[0181] VIII. Therapeutic Uses of Epigenetic Editors

[0181] The present disclosure also provides methods for treating or preventing a condition in a subject, the methods comprising administering to the subject an epigenetic editor or pharmaceutical composition described herein. The epigenetic editor effects epigenetic modification of a target polynucleotide sequence within a target gene associated with a disease, condition, or disorder in the subject, thereby modulating expression of the target gene to treat or prevent the disease, condition, or disorder. In some embodiments, the epigenetic editor reduces expression of the target gene to an extent sufficient to achieve a desired effect, e.g., a therapeutically relevant effect, such as prevention or treatment of the disease, condition, or disorder.

[0182]

[0182] In some embodiments, a subject is administered a system for modulating (e.g., inhibiting) expression of HBV or an HBV gene, the system comprising (1) a fusion protein and, optionally, a guide polynucleotide of an epigenetic editor described herein, or (2) a nucleic acid molecule encoding the fusion protein and, optionally, a guide polynucleotide.

[0183] "Treate," "treating," and "treatment" refer to a method of alleviating or eliminating a biological disorder and / or at least one of its attendant symptoms. As used herein, "alleviating" a disease, disorder, or condition means reducing the severity and / or frequency of occurrence of the symptoms of the disease, disorder, or condition. Furthermore, references herein to "treatment" include references to curative, palliative, and preventative treatment. In some embodiments, alleviating symptoms compared to comparable untreated controls can include at least a 3%, 5%, 10%, 20%, 40%, 50%, 60%, 80%, 90%, 95%, 98%, 99%, 99.5%, 99.9%, or 100% reduction in symptoms as measured by any standard technique.

[0184] In some embodiments, the subject may be a mammal, e.g., a human, hi some embodiments, the subject is selected from a non-human primate, e.g., a chimpanzee, a cynomolgus monkey, or a macaque, as well as other ape and monkey species.

[0185]

[0185] In some embodiments, the human patient has a condition characterized by HBV infection. In some embodiments, the patient has hepatitis B.

[0186] In some embodiments, a patient to be treated with an epigenetic editor of the present disclosure has received prior treatment for the condition to be treated (e.g., HBV and / or HDV, or Hepatitis B). In other embodiments, the patient has not received such prior treatment. In some embodiments, the patient has failed (or is refractory to) a prior treatment for the condition (e.g., a prior HBV treatment).

[0186]

[0187] The epigenetic editors of the present disclosure can be administered in therapeutically effective amounts to patients with the conditions described herein. As used herein, "therapeutically effective amount" refers to the amount of therapeutic agent administered that will alleviate to some extent one or more symptoms of the disorder being treated and / or result in a clinical endpoint desired by a healthcare professional. A therapeutically effective amount can be measured by its ability to stabilize disease progression and / or ameliorate symptoms, and preferably halt disease progression, in a patient. The ability of the epigenetic editors of the present disclosure to reduce or silence HBV expression can be assessed by in vitro assays, such as those described herein, or in appropriate animal models predictive of efficacy in humans. An appropriate dosage regimen will be selected to provide the optimal therapeutic response in each particular situation and will be administered, for example, as a single bolus or as a continuous infusion, with possible adjustment of dosage as indicated by the urgent needs of each care.

[0187]

[0188] The epigenetic editor of the present disclosure can be administered without additional therapeutic treatment, i.e., as a sole therapy (monotherapy). Alternatively, treatment with the epigenetic editor of the present disclosure can include at least one additional therapeutic treatment (combination therapy). In some embodiments, the additional therapeutic agent is any known in the art for HBV and / or HDV. In some embodiments, the therapeutic agent includes, but is not limited to, an antiviral agent such as entecavir, tenofovir, lamivudine, telbivudine, bictegravir, emtricitabine, or defovir, and an immunomodulator such as pegylated interferon and interferon alpha.

[0188]

[0189] The epigenetic editors of the present disclosure, or components thereof (or nucleic acid molecules encoding the epigenetic editors or components thereof), can be administered by any method accepted in the art (e.g., parenterally, intravenously, intradermally, or intramuscularly).

[0189]

[0190] An epigenetic editor or component thereof (or a nucleic acid molecule encoding an epigenetic editor or component thereof) of the present disclosure may be administered to a subject one, two, three, or four, five, six, seven, eight, nine, ten, or more times. In some embodiments, the one, two, three, or four, five, six, seven, eight, nine, ten, or more administrations of an epigenetic editor or component thereof (or a nucleic acid molecule encoding an epigenetic editor or component thereof) are closely spaced in time (e.g., within one, two, three, four, five, six days, one week, two weeks, four weeks, one month, or two months of each other). In some embodiments, a subject is re-administered an epigenetic editor or component thereof (or a nucleic acid molecule encoding an epigenetic editor or component thereof) of the present disclosure at least one more time following the initial administration. In some cases, the subject is administered a subsequent dose of an epigenetic editor or component thereof (or a nucleic acid molecule encoding the epigenetic editor or component thereof) of the present disclosure that targets a DNA region of the HBV genome that is different from the DNA region of the HBV genome targeted by the epigenetic editor or component thereof that the subject received during the initial administration. In some cases, the subject is administered multiple doses (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, or more) of an epigenetic editor or component thereof (or a nucleic acid molecule encoding the epigenetic editor or component thereof) of the present disclosure. In some embodiments, the subject is administered a single dose of a different epigenetic editor or component thereof (or a nucleic acid molecule encoding the epigenetic editor or component thereof) of the present disclosure, wherein at least two of the epigenetic editors or components thereof target different DNA regions of the HBV genome.In some cases, a subject is administered multiple doses (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, or more) of different epigenetic editors of the present disclosure or their component parts (or nucleic acid molecules encoding the epigenetic editors or their component parts), where at least two of the epigenetic editors or their component parts target different DNA regions of the HBV genome. In some embodiments, re-administration of an epigenetic editor of the present disclosure or its component parts (or nucleic acid molecules encoding the epigenetic editors or its component parts) has better therapeutic efficacy than a single administration thereof, e.g., a stronger inhibition of HBV replication, or a more significant reduction in HBV DNA and / or HBV antigens (e.g., HBsAg, HBeAg, and / or HBV core antigen (HBcAg)) present in the subject, e.g., in the subject's circulatory system and / or liver.

[0190] XII.Definitions

[0191] The term "nucleic acid," as used herein, refers to any oligonucleotide or polynucleotide containing nucleotides (e.g., deoxyribonucleotides or ribonucleotides) in either single-stranded or double-stranded form, including DNA and RNA. A "nucleotide" contains the sugar deoxyribose (DNA) or ribose (RNA), a base, and a phosphate group, linked together by the phosphate group. "Base" includes naturally occurring compounds, such as purines and pyrimidines, including adenine, thymine, guanine, cytosine, uracil, inosine, and natural analogs; as well as synthetic derivatives of purines and pyrimidines, including, but not limited to, modified versions that introduce new reactive groups, such as amines, alcohols, thiols, carboxylic acids, alkyl halides, etc. Nucleic acids can contain known nucleotide analogs and / or modified backbone residues or linkages, which may be synthetic, naturally occurring, or non-naturally occurring. Such nucleotide analogs, modified residues, and modified linkages are known in the art and may confer on nucleic acid molecules enhanced cellular uptake, reduced immunogenicity, and / or increased stability in the presence of nucleases.

[0191]

[0192] As used herein, an "isolated" or "purified" nucleic acid molecule is a nucleic acid molecule that exists away from its native environment. For example, an "isolated" or "purified" nucleic acid molecule (1) is separated from the nucleic acid of its original source, genomic DNA or cellular RNA, and / or (2) is not found in nature. In some embodiments, an "isolated" or "purified" nucleic acid molecule is a recombinant nucleic acid molecule.

[0192]

[0193] It will be understood that in addition to the specific proteins and nucleic acid molecules mentioned herein, the present disclosure also contemplates the use of variants, derivatives, homologs, and fragments thereof. A variant of any given sequence may have a specific sequence of residues (whether amino acid or nucleic acid residues) modified in such a way that the polypeptide or polynucleotide in question substantially retains at least one of its intrinsic functions. A variant sequence may be obtained by adding, deleting, substituting, modifying, replacing, and / or varying at least one residue (in some embodiments, up to 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, or 20 residues) present in the naturally occurring sequence. For certain proteins described herein (e.g., the KRAB, dCas9, DNMT3A, and DNMT3L proteins described herein), the present disclosure also contemplates any naturally occurring form of the protein or a variant or homolog that retains at least one of its endogenous functions (e.g., at least 50%, 60%, 70%, 80%, 90%, 85%, 96%, 97%, 98%, or 99% of that function compared to the particular protein described).

[0193]

[0194] As used herein, any polypeptide or nucleic acid sequence homolog contemplated herein includes a sequence having a certain homology with wild-type amino acid and nucleic acid sequences. Homologous sequences can include sequences, such as amino acid sequences, that are at least 50%, 55%, 65%, 75%, 85%, 90%, 91%, 92%<93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the target sequence. The term "percent identical" in the context of amino acid or nucleotide sequences refers to the percentage of residues in two sequences that are the same when aligned for maximum matching. In some embodiments, the length of the reference sequence aligned for comparison purposes is at least 30% (e.g., at least 40, 50, 60, 70, 80, or 90%, or 100%) of the reference sequence. Sequence identity can be measured using sequence analysis software (e.g., the Sequence Analysis Software Package, BLAST, BESTFIT, GAP, or PILEUP / PRETTYBOX programs of the Genetics Computer Group, University of Wisconsin Biotechnology Center, 1710 University Avenue, Madison, Wis. 53705). Such software matches identical or similar sequences by assigning degrees of homology to various substitutions, deletions, and / or other modifications. In an exemplary approach to determining the degree of identity, the BLAST program can be used, in which a probability score between e-3 and e-100 indicates closely related sequences.

[0194]

[0195] The percent identity of two nucleotide or polypeptide sequences is determined, for example, by BLAST® (available from the U.S. National Library of Medicine's National Center for Biotechnology Information website) using default parameters. In some embodiments, the length of a reference sequence aligned for comparison purposes is at least 30% (e.g., at least 40, 50, 60, 70, 80, or 90%) of the reference sequence.

[0195]

[0196] Of course, the numbering of specific positions or residues in a polypeptide sequence will depend on the particular protein and numbering scheme used. Numbering may differ, for example, in precursors to the mature protein and the mature protein itself, and sequence differences between species may affect numbering. One skilled in the art will be able to identify each residue in any homologous protein and in the respective encoding nucleic acid by methods well known in the art, for example, by sequence alignment and determination of homologous residues.

[0196]

[0197] The terms "modulate" or "alter" refer to a change in the amount, degree, or extent of a function. For example, the epigenetic editors described herein can modulate the activity of a promoter sequence by binding to a motif within the promoter, thereby inducing, enhancing, or suppressing transcription of a gene operably linked to the promoter sequence. As another example, the epigenetic editors described herein can prevent RNA polymerase from transcribing a gene or inhibit translation of an mRNA transcript. The terms "inhibit," "suppress," "repress," "silence," and the like, when used with respect to the epigenetic editors described herein or their components, refer to reducing or preventing the activity (e.g., transcription) of a nucleic acid sequence (e.g., a target gene) or protein compared to the activity of the nucleic acid sequence or protein in the absence of the epigenetic editor or its components. The terms can include partially or completely blocking activity, or preventing or delaying activity. The inhibited activity may be, for example, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% less than that of the control, or may be at least 1.5-, 2-, 3-, 4-, 5-, or 10-fold less than that of the control.

[0197]

[0198] The term "about" or "approximately" means within an acceptable error range for a particular value, as determined by one of ordinary skill in the art, which will depend in part on how the value is measured or determined, e.g., the limitations of the measurement system. For example, "about" can mean within one standard deviation about a given value, or it can mean more than one standard deviation, depending on the implementation. When a particular value is described in this application or claims, unless otherwise stated, the term "about" should be assumed to mean an acceptable error range for the particular value.

[0198]

[0199] Ranges provided herein are understood to be shorthand for all of the values ​​within the range. For example, a range of 1 to 50 is understood to include any number, combination of numbers, or subrange from the group consisting of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50, as well as all intervening decimal values ​​between the aforementioned integers, such as 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, and 1.9. With respect to subranges, "nested subranges" extending from both endpoints of the range are specifically contemplated. For example, nested subranges of the exemplary range of 1 to 50 could include 1 to 10, 1 to 20, 1 to 30, and 1 to 40 in one direction, or 50 to 40, 50 to 30, 50 to 20, and 50 to 10 in the opposite direction.

[0199]

[0200] Unless otherwise defined herein, scientific and technical terms used in connection with the present disclosure shall have the meanings commonly understood by those of ordinary skill in the art. Exemplary methods and materials are described below, although methods and materials similar or equivalent to those described herein can also be used in the practice or testing of the present disclosure. In the case of conflict, the present specification, including definitions, will control. Furthermore, unless the context requires otherwise, singular terms shall include pluralities and plural terms shall include the singular. Throughout this specification and the embodiments, the words "have" and "comprise," or variations such as "has," "having," "comprises," or "comprising," shall imply the inclusion of a stated integer or group of integers, but not the exclusion of any other integer or group of integers. The recitation of a list of elements herein includes any of the elements alone or in any combination. The recitation of an embodiment herein includes that embodiment as a single embodiment or in combination with any other embodiment herein. All publications, patents, patent applications, and other references mentioned herein are incorporated herein by reference in their entirety. In the event that a reference incorporated by reference conflicts with the present disclosure contained herein, this specification is intended to supersede and / or take precedence over any such conflicting material. Although numerous documents are cited herein, this citation does not constitute an admission that any of these documents form part of the common general knowledge in the art.

[0200]

[0201] In order that the present disclosure may be better understood, the following examples are set forth, which are provided for illustrative purposes only and are not to be construed as limiting the scope of the disclosure in any way. [Example]

[0201] Example 1: Selection of target HBV sequences for epigenetic silencing

[0202] Target sequences were designed manually and computationally using representative HBV genome sequences (SEQ ID NOs: 1082, 1083) as references.

[0202]

[0203] Target site design focused on CpG islands identified within the HBV genome, but also considered target sites outside of HBV CpG islands.

[0204] Table 2 presents some representative target sites that have been identified as suitable for targeting by epigenetic repressors.

[0203]

[0205] The target domains identified above, adjacent to the PAM sequence, e.g., the Streptococcus pyogenes Cas9 PAM sequence, can be targeted by CRISPR-based epigenetic repressors, e.g., epigenetic repressors containing the dCas9 DNA binding domain. For example, target sites 1-143 are suitable for dCas9-based epigenetic repressor targeting. Figure 1 provides an overview of the locations of the identified target sites in the HBV genome.

[0204]

[0206] The target sites were analyzed for conservation across HBV genotypes A through E (Figures 2 and 3). Some target sites were identified that were well conserved across two or more, or in some cases, all, HBV genotypes. Targeting such conserved sites allows for silencing of different genotypes by the same epigenetic repressor.

[0205] Example 2: Guide RNA assay in HepAD38 HBV cells

[0207] The HepAD38 cell line expresses the HBV genome under a deoxycycline-inducible promoter (see, e.g., Ladner et al., Inducible expression of human hepatitis B virus (HBV) in stably transfected hepatoblastoma cells: a novel system for screening potential inhibitors of HBV replication. Antimicrob. Agents Chemother. 41:1715-1720 (1997), incorporated herein by reference).

[0206]

[0208] The results are shown in Figures 4A and B. Example 3: Guide RNA assay in HepG2-NTCP cells

[0209] HepG2 cells were engineered by lentiviral transduction to express the human NTCP receptor used by hepatitis B virus (HBV) to infect cells.

[0207]

[0210] HBV viral particles were produced using the HepAD38 cell line, a subclone derived from the HepG2 cell line that expresses the HBV genome (genotype D subtype ayw) under the transcriptional control of a tetracycline-responsive promoter in the TET-OFF system.

[0208]

[0211] Three combinations of engineered transcriptional repressors (ETRs), consisting of three plasmids expressing dCas9-KRAB, dCas9-DNMT3A, and dCas9-DNMT3L, were used in combination with one or more of the designed sgRNAs.

[0209]

[0212] LNPs were formulated using GENVOY ILM Lipid Mix (Precision Nanosystem) and the Spark Nanoassembly Formulator (Precision Nanosystem). The LNPs were formulated according to the manufacturer's recommendations, using a nitrogen:phosphate (NP) ratio equal to 6 and a flow rate ratio (FRR) of 2:1. The RNA payload was diluted to a final concentration of 350 ng / uL in PNI formulation buffer. ETR, dCas9-KRAB, dCas9-DNMT3A, dCas9-DNMT3L, and each of the 121 sgRNAs were mixed in a 1:1:1:4 ratio. The RNA mix, Genvoy Liquid Mix (25 mM), and PBS were each loaded into the dedicated chambers of the Spark cartridge and formulated. Packaged mRNA was quantified using the Quant-it™ RiboGreen RNA Assay Kit (Thermo Fisher), and LNPs were sized by Dynamic Light Scattering (Zetasizer, Malvern Panalytic) to assess the quality of the formulated LNPs.

[0210]

[0213] HepG2-NTCP cells were plated at 20,000 cells / well in collagen-coated 96-well plates. After 24 hours, cells were infected with HBV at a multiplicity of genome equivalents (MGE) of 5,000. After 16 hours, the viral inoculum was removed, cells were washed with PBS, and fresh medium was added. Three days after infection, LNPs were used to deliver each sgRNA and mRNA encoding each component of the triple ETR construct (dCas9-KRAB, dCas9-DNMT3A, and dCas9-DNMT3L). After 3 days, LNPs were removed, the medium was replaced, and cells were maintained in complete medium for 3 days.

[0211]

[0214] Six days after LNP removal, viral antigens HBeAg and HBsAg were quantified using an ELISA assay. Data were normalized to a non-targeting guide designed against mouse PCSK9, and the control 3.2 gRNA was used as a positive control. Cell viability assays were performed and normalized to the non-targeting control.

[0212]

[0215] The table below provides the amino acid sequences of exemplary epigenetic editors (ETR constructs) used in the gRNA screen:

[0213] [Table 6-1]

[0214] [Table 6-2]

[0215] [Table 6-3]

[0216]

[0216] The table below provides the amino acid and polynucleotide sequences of exemplary epigenetic editors.

[0217] [Table 7-1]

[0218] [Table 7-2]

[0219] [Table 7-3]

[0220] [Table 7-4]

[0221]

Table 7-5

[0222]

Table 7-6

[0223]

Table 7-7

[0224]

Table 7-8

[0225]

Table 7-9

[0226]

Table 7-10

[0227]

Table 7-11

[0228]

Table 7-12

[0229]

Table 7-13

[0230]

Table 7-14

[0231]

[0217] Table 8 below lists the components of the fusion polypeptide PLA001 and their corresponding amino acid positions in the fusion polypeptide sequence shown in Table 7 (SEQ ID NO:481).

[0232] [Table 8]

[0233]

[0218] Table 9 below lists the components of the polynucleotide encoding the fusion polypeptide PLA001 and their corresponding nucleotide positions in the polynucleotide sequence shown in Table 7 (SEQ ID NO:482).

[0234] [Table 9]

[0235]

[0219] Table 10 below lists the components of the fusion polypeptide PLA002 and their corresponding amino acid positions in the fusion polypeptide sequence shown in Table 7 (SEQ ID NO: 483).

[0236] [Table 10]

[0237]

[0220] Table 11 below lists the components of the polynucleotide encoding the fusion polypeptide PLA002 and their corresponding nucleotide positions in the polynucleotide sequence shown in Table 7 (SEQ ID NO:484).

[0238] [Table 11]

[0239] [Table 12]

[0240] [Table 13]

[0241]

[0221] Table 14 below provides the gRNA sequences tested.

[0242] [Table 14-1]

[0243] [Table 14-2]

[0244] [Table 14-3]

[0245] [Table 14-4]

[0246] [Table 14-5]

[0247] [Table 15-1]

[0248] [Table 15-2]

[0249] [Table 15-3]

[0250] In vitro silencing was observed in the HepG2-NTCP infection model using gRNAs targeting CpG islands and ETRs (Figures 5A-5B). Primary screening was performed using LNPs of quality within expected parameters and pilot experiments using single guides (Figures 6-8). Results demonstrated that 48 gRNAs showed less than 50% expression of HBeAg compared to non-targeting controls on day 6 (Figure 9), and 28 gRNAs showed less than 50% expression of HBsAg compared to non-targeting controls on day 6 (Figure 10). HBsAg and HBeAg expression were positively correlated, as shown in Figure 11.

[0251] Example 4: Zinc finger repressors for silencing HBV Zinc finger repressors were designed to target epigenetic target sites identified in the HBV genome. Table 1 above provides the amino acid sequences of the zinc fingers and their corresponding motif sequences, as well as the target sequences of the zinc fingers.

[0252]

[0224] The zinc finger repressors listed in Table 1 are tested in an HBV infection model, for example, in HepG2 as described herein, and efficient suppression of HBV is confirmed for the zinc finger repressors provided in Table 1.

[0253] Example 5: Further in vitro evaluation of gRNAs

[0225] A CRISPR-Off single construct encoding PLA002, consisting of KRAB, DNMT3A, DNMT3L and dCas9, was used in combination with one or more of the designed sgRNAs for the in vitro assays described in this example.

[0254] HepG2-NTCP cells were infected with HBV for 4 days according to a procedure similar to that in Example 3, and then transfected with CRISPR-off constructs and individual exemplary gRNAs (as shown in Table 13) formulated in research-grade LNPs. Six days after transfection, HBsAg and HBeAg protein expression in the supernatant was assessed by ELISA, as depicted in FIG. 12A. Results from this experiment are shown in FIG. 12B. All of the gRNAs tested resulted in a reduction in HBsAg and HBeAg levels in the supernatant. The positive control used in this experiment was a gRNA against the HBV genome, which was previously shown to reduce antigen by approximately 50%.

[0255] In another experiment, gRNA activity was evaluated using PLC / PRF / 5, an HBV-integrated cell line. PLC / PRF / 5 cells were transfected with CRISPR-off (PLA002) and individual gRNAs using a commercially available lipid-based transfection reagent. As depicted in Figure 13A, 4 days after transfection, HBsAg protein expression in the supernatant was assessed by ELISA. Results from this experiment are shown in Figure 13B. Target conservation was assessed in silico and defined as 100% gRNA-DNA match.

[0256] In a further experiment, primary human hepatocytes (PHH) derived from humanized mice were infected with HBV for 4 days and then transfected with CRISPR-off (PLA002) and individual gRNAs formulated in research-grade LNP, GenVoy LNP. As depicted in Figure 14A, HBsAg and HBeAg protein expression in the supernatant was assessed by ELISA 6 days after transfection. Results from this experiment are shown in Figure 14B. The positive control used in this experiment was an HBV gRNA previously shown to reduce antigen by approximately 50%. The data suggested strong in vitro silencing by certain gRNAs 6 days after transfection. In a second PHH experiment depicted in Figure 14C, post-infection HBsAg and HBeAg protein expression in the supernatant was assessed by ELISA 12 days after delivery of 100 ng of payload (1:1 effector-to-guide RNA ratio) in research-grade LNP. The epigenetic editor also suppresses HBsAg and HBeAg secretion in HBV-infected PHH cells at this time point, as shown in Figure 14D.

[0257]

[0229] Exemplary gRNA sequences tested in this example are listed in Table 13. Example 6: Evaluation of ZFPs in HepG2-NTCP cells In this example, ZF-off single constructs encoding fusion proteins consisting of KRAB, DNMT3A, DNMT3L, and selected exemplary zinc finger motifs were tested. The sequences of exemplary zinc fingers tested in this example are listed in Table 20, as are the sequences for the plasmids that generated a subset of the ZF-off single construct fusion proteins.

[0258] Certain exemplary ZF-off constructs were formulated into research-grade LNPs. HepG2-NTCP cells were infected with HBV for 4 days and then transfected with ZF-off-loaded LNPs. As depicted in Figure 15A, HBsAg and HBeAg protein expression in the supernatant was assessed by ELISA on day 6 post-infection. Figure 15B shows the results as measured by the percentage reduction of HBV antigen compared to a non-targeting control. The positive control used in this experiment is an HBV gRNA previously shown to reduce antigen by approximately 50%. Figure 16A shows the results of the top 10 ZF-off constructs that resulted in the greatest reduction of HBV antigen. Figure 16B shows the results for all constructs in the screen.

[0259]

[0232] Figures 16 and 17 below show the raw data from these experiments, listed with the mRNA numbers from which the zinc finger motifs occur.

[0260] [Table 16-1]

[0261] [Table 16-2]

[0262] [Table 17-1]

[0263] [Table 17-2]

[0264] Example 7. Dose-response test of viral antigen in Hepg2-NTCP cells In this example, the top ZF fusion proteins were tested in a five-point dose-response assay for HBsAg and HBeAg. The five dosage points were 200 ng, 150 ng, 100 ng, 50 ng, and 25 ng. A schematic of the experiment and the results are shown in Figure 17.

[0265] Example 8. Testing for durable suppression in HepG2.2.15 cells In this example, top ZF fusion proteins were tested for durable suppression of HBsAg. Active ZFPs demonstrated durable silencing with 50 ng of total treatment for up to 27 days. A schematic diagram of the experiment and results are shown in Figure 18.

[0266] Example 9. Testing silencing of HBsAg in a second model of int-HBV In this example, top ZF fusion proteins were tested for repression of HBsAg in PLC / PRF / 5 cells. A subset of ZFPs silenced HBsAg in this second model. A schematic diagram and results of the experiment are shown in Figure 19.

[0267] Example 10. Testing the specificity of CRISPR-off with ZF fusion proteins and guide RNAs In this example, ZF fusion proteins targeting HBV, which demonstrated significant silencing, were profiled for specificity in HepG2-NTCP at day 19. All comparisons were made to a non-targeting ZFP control. Exemplary results for a ZF fusion protein with an mRNA0001 zinc finger motif are shown in Figure 20A. CRISPR-off with guide RNA was also profiled. HepG2-NTCP cells were transfected with 100 ng of total payload using GenVoy™ LNP at a 1:1 gRNA:effector ratio. Cells were split every 3–4 days and collected at day 15 post-treatment for specificity assessment, including RNA-seq and methylation arrays. Differential gene expression was identified using DESeq2. As shown in Figure 20B, little or no changes above the selected thresholds (absolute [log2 [fold change]] > 1 and -log10 [adjusted p-value] > 5) were observed for effectors targeting HBV DNA, as expected. For methylation arrays, Infinium MethylationEPIC v2.0 arrays were used to identify DMRs in silico. EE3, EE4, and EE5 had DMR=0 results. The results are shown in Figures 20C-20D.

[0268] Example 11. Stable HBV silencing by epigenetic editing in a non-transgenic mouse model of persistent HBV infection We used a nontransgenic model of persistent HBV infection in immunocompetent mice (AAV-HBV) generated by administering an adeno-associated viral vector (AAV) containing HBV genotype D DNA to mice. Administration of the AAV-HBV vector resulted in the expression of hepatitis B surface antigen (HBsAg), hepatitis B e antigen (HBeAg), and high levels of serum HBV DNA in mice.

[0269] CRISPR-off and ZF-off constructs are tested. Constructs are delivered by IV administration of mRNA / gRNA(CRISPR-Off) or mRNA(ZF-Off) formulated in lipid nanoparticles (LNPs) at 2.5 mg / kg and 0.5 mg / kg for CRISPR-Off and ZF-Off, respectively. Some constructs are formulated in LNP compositions as described in US Patent Application Publication No. 20220402862A1 and / or US Patent Application Publication No. 20230203480A1. A subset of mice are re-administered two weeks after the initial administration, and a second subset is re-administered one month after the initial administration. Readouts are circulating viral DNA, HBsAg, and HBeAg assays using mouse plasma at one or more time points (e.g., 7, 14, 28, and 35 days). A durable and significant reduction in the levels of one or more of HBV DNA, HBsAg and HBeAg is observed for some constructs.

[0270] Longer-term persistence is tested over a 3-6 month period using HBV DNA, HBsAg, and HBeAg markers. A gradual and durable reduction in one or more of these markers is seen with delivery of some constructs. Mice are sacrificed and livers are collected for further analysis; durable silencing is confirmed by a 2-log or less reduction in HBsAg and HBV DNA.

[0271] Example 12: Stable HBV silencing by epigenetic editing in transgenic mice expressing viral HBV DNA

[0240] A transgenic mouse model of persistent HBV infection (Tg-HBV) was used, in which the genome was engineered to incorporate HBV genotype A DNA, resulting in the expression of HBsAg and HBeAg as well as circulating viral DNA in mice.

[0272] CRISPR-off and ZF-off constructs are tested. Constructs are delivered by IV administration of mRNA / gRNA(CRISPR-Off) or mRNA(ZF-Off) formulated in LNPs at 2.5 mg / kg and 0.5 mg / kg for CRISPR-Off and ZF-Off, respectively. Some constructs are formulated in LNP compositions as described in US Patent Application Publication No. 20220402862A1 and / or US Patent Application Publication No. 20230203480A1. A subset of mice are re-administered two weeks after the initial administration, and a second subset is re-administered one month after the initial administration. Readouts are circulating viral DNA, HBsAg, and HBeAg assays using mouse plasma at one or more time points (e.g., 7, 14, 28, and 35 days). A durable and significant reduction in the levels of one or more of HBV DNA, HBsAg and HBeAg is observed for some constructs.

[0273] Longer-term persistence is tested over a 3-6 month period using HBV DNA, HBsAg, and HBeAg markers. A gradual and durable reduction in one or more of these markers is seen with delivery of some constructs. Mice are sacrificed and livers are collected for further analysis; durable silencing is confirmed by a 2-log or less reduction in HBsAg and HBV DNA.

[0274] Example 13. CRISPR-Off guide RNA multiplexing studies in AAV-HBV and Tg-HBV mouse models AAV-HBV and Tg-HBV mice are injected with a single dose of 1.5 mg / kg of one, two, or three guide RNAs and CRISPR-Off fusion protein in LNP according to Table 18. Samples include CRISPR-Off from each of PLA002 and PLA003. HBV DNA, HBsAg, and HBeAg are assayed in plasma at one or more time points, and mouse livers are collected for further analysis. Durable silencing is confirmed by a 2-log or less reduction in HBsAg and HBV DNA.

[0275]

[0244]

[0276] [Table 18]

[0277] Example 14. Zinc finger protein multiplexing studies in AAV-HBV and Tg-HBV mouse models AAV-HBV and Tg-HBV mice are injected with a single dose of one, two, or three ZF fusion proteins in LNP according to Table 19 at 0.5 mg / kg (schematic, Figure 21). HBV DNA, HBsAg, and HBeAg are assayed in plasma at one or more time points, and mouse livers are collected for further analysis. Durable silencing is confirmed by a 2-log or less reduction in HBsAg and HBV DNA.

[0278] [Table 19]

[0279] array

[0246] The sequence numbers (SEQ) of the nucleotide (nt) and amino acid (aa) sequences set forth in this disclosure are listed in Table 20 below.

[0280] [Table 20-1]

[0281]

Table 20-2

[0282]

Table 20-3

[0283]

Table 20-4

[0284]

Table 20-5

[0285]

Table 20-6

[0286]

Table 20-7

[0287]

Table 20-8

[0288]

Table 20-9

[0289]

Table 20-10

[0290]

Table 20-11

[0291]

Table 20-12

[0292]

Table 20-13

[0293]

Table 20-14

[0294]

Table 20-15

[0295]

Table 20-16

[0296]

Table 20-17

[0297]

Table 20-18

[0298]

Table 20-19

[0299]

Table 20-20

[0300]

Table 20-21

[0301]

Table 20-22

[0302]

Table 20-23

[0303]

Table 20-24

[0304]

Table 20-25

[0305]

Table 20-26

[0306]

Table 20-27

[0307]

Table 20-28

[0308]

Table 20-29

[0309]

Table 20-30

[0310]

Table 20-31

[0311]

Table 20-32

[0312]

Table 20-33

[0313]

Table 20-34

[0314]

Table 20-35

[0315]

Table 20-36

[0316]

Table 20-37

[0317]

Table 20-38

[0318]

Table 20-39

[0319]

Table 20-40

[0320]

Table 20-41

[0321]

Table 20-42

[0322]

Table 20-43

[0323]

Table 20-44

[0324]

Table 20-45

[0325]

Table 20-46

[0326]

Table 20-47

[0327]

Table 20-48

[0328]

Table 20-49

[0329]

Table 20-50

[0330]

Table 20-51

[0331]

Table 20-52

[0332]

Table 20-53

[0333]

Table 20-54

[0334]

Table 20-55

[0335]

Table 20-56

[0336]

Table 20-57

[0337]

Table 20-58

[0338]

Table 20-59

[0339]

Table 20-60

[0340]

Table 20-61

[0341]

Table 20-62

Claims

1. 1. A method of modifying the epigenetic state of a Hepatitis B virus (HBV) gene or genome, comprising contacting the HBV gene or genome with an epigenetic editing system; The epigenetic editing system a first DNA-binding domain, a first DNMT domain, and a transcriptional repressor domain, or one or more nucleic acid molecules encoding them Including, the first DNA-binding domain binds to a first target region of the HBV gene or genome; the contacting step comprises: the number of HBV viral episomes, Replication of the HBV gene or genome, or Expression of the protein product encoded by the HBV gene or genome resulting in a reduction of The method, wherein the reduction is at least about 20% as compared to contacting the HBV gene or genome with a suitable control.

2. 1. A method of treating an HBV infection in a subject, comprising administering to the subject an epigenetic editing system; The epigenetic editing system a first DNA-binding domain, a first DNMT domain, and a transcriptional repressor domain, or one or more nucleic acid molecules encoding them Including, the first DNA-binding domain binds to a first target region of an HBV gene or genome; To bring into contact the number of HBV viral episomes, Replication of the HBV gene or genome, or Expression of the protein product encoded by the HBV gene or genome resulting in a reduction of wherein said reduction is at least about 20% as compared to administering a suitable control.

3. 1. A method of modulating expression of an HBV gene or genome, comprising contacting the HBV gene or genome with an epigenetic editing system; The epigenetic editing system a first DNA-binding domain, a first DNMT domain, and a transcriptional repressor domain, or one or more nucleic acid molecules encoding them Including, the first DNA-binding domain binds to a first target region of the HBV gene or genome; wherein said contacting step results in a reduction in expression of a gene product encoded by said HBV gene or genome, and optionally said gene product is a nucleic acid or a protein; The method, wherein the reduction is at least about 20% compared to contacting the HBV genome with a suitable control.

4. 1. A method of inhibiting viral replication in a cell infected with HBV, comprising administering an epigenetic editing system; The epigenetic editing system a first DNA-binding domain, a first DNMT domain, and a transcriptional repressor domain, or one or more nucleic acid molecules encoding them Including, the first DNA-binding domain binds to a first target region of an HBV gene or genome, and the epigenetic editing system targets the target region of the HBV gene or genome; the contacting results in a reduction in the number of HBV viral episomes or replication of said HBV genes or genome; wherein said reduction is at least about 20% as compared to administering a suitable control.

5. 5. The method of claim 1, wherein the HBV genome is a covalently closed circular DNA (cccDNA) or an HBV integrated DNA.

6. 6. The method of any one of claims 1 to 5, wherein the HBV genome comprises HBV genotype A, HBV genotype B, HBV genotype C, HBV genotype D, HBV genotype E, HBV genotype F, HBV genotype G, or HBV genotype H.

7. 7. The method of any one of claims 1 to 6, wherein the HBV genome comprises a sequence having at least 80% identity to the HBV genome sequence provided herein.

8. 8. The method of claim 7, wherein the first target region is located in a region of the HBV genome within nucleotides 0 to 303, 1000 to 2448, or 2802 to 3182 of the HBV genome provided herein.

9. 7. The method of claim 1, wherein the first target region of the HBV genome is located in a CpG island.

10. 7. The method of claim 1, wherein the first target region of the HBV genome is located in a promoter.

11. 7. The method of any one of claims 1 to 6, wherein the first target region of the HBV genome is located in a section of the HBV genome encoding a transcript selected from the group consisting of pgRNA, precure mRNA, preS mRNA, S mRNA, and X mRNA.

12. 12. The method of any one of claims 1 to 11, wherein the first DNA binding domain comprises a CRISPR-Cas protein.

13. 13. The method of Claim 12, wherein the epigenetic editing system further comprises a first guide RNA (gRNA) comprising a region complementary to a strand of the first target region.

14. 14. The method of claim 13, wherein the gRNA comprises a sequence selected from the gRNAs provided herein, e.g., in Tables 14 and / or 15.

15. 12. The method of any one of claims 1 to 11, wherein the first DNA binding domain comprises a zinc finger protein.

16. 16. The method of claim 15, wherein the zinc finger protein comprises a zinc finger motif having a sequence selected from any zinc finger or zinc finger motif provided herein, e.g., in Table 1.

17. The method of claim 15 or 16, wherein the zinc finger protein comprises the sequence of any of the zinc finger epigenetic repressors provided herein.

18. 18. The method of any one of claims 1 to 17, wherein the transcriptional repressor domain comprises ZIM3.

19. 19. The method of any one of claims 1 to 18, wherein the first DNMT domain is a DNMT3A domain or a DNMT3L domain.

20. 20. The method of claim 19, wherein the first DNMT domain comprises the sequence of a DNMT domain provided herein.

21. 21. The method of any one of claims 1 to 20, wherein the epigenetic editing system further comprises a second DNMT domain or a nucleic acid encoding same.

22. 22. The method of claim 21, wherein the second DNMT domain is a DNMT3A domain or a DNMT3L domain.

23. 23. The method of claim 22, wherein the second DNMT domain comprises the sequence of a DNMT domain provided herein.

24. 24. The method of any one of Claims 21 to 23, wherein the epigenetic editing system comprises a fusion protein or a nucleic acid encoding the same, and the fusion protein comprises the first DNA-binding domain, the first DNMT domain, the repressor domain, and the second DNMT domain.

25. 25. The method of claim 24, wherein the fusion protein further comprises a nuclear localization sequence (NLS).

26. 26. The method of claim 25, wherein the fusion protein comprises the sequence of a fusion protein provided herein.

27. 22. The method of any one of claims 1 to 21, wherein the epigenetic editing system further comprises a second DNA-binding domain or a nucleic acid encoding the same, and the second DNA-binding domain binds to a second target region of the HBV genome.

28. 28. The method of claim 27, wherein the second target region is located in a region of the HBV genome within nucleotides 0-303, 1000-2448, or 2802-3182.

29. 28. The method of claim 27, wherein the second target region of the HBV genome is located in a CpG island.

30. 28. The method of claim 27, wherein the second target region of the HBV genome is located in a promoter.

31. 28. The method of claim 27, wherein the second target region of the HBV genome is located in a section of the HBV genome encoding a transcript selected from the group consisting of pgRNA, precure mRNA, preS mRNA, S mRNA, and X mRNA.

32. 32. The method of any one of claims 27 to 31, wherein the two DNA binding domains comprise CRISPR-Cas proteins.

33. 33. The method of Claim 32, wherein the epigenetic editing system further comprises a second gRNA comprising a region complementary to a strand of the second target region.

34. 34. The method of Claim 33, wherein the gRNA comprises a sequence selected from the gRNA sequences provided herein, e.g., the sequences provided in Tables 14 and / or 15.

35. 32. The method of any one of claims 27 to 31, wherein the second DNA binding domain comprises a zinc finger protein.

36. 36. The method of claim 35, wherein the zinc finger protein comprises a zinc finger motif having a zinc finger motif sequence provided herein, e.g., a sequence selected from the zinc finger motifs provided in Table 1.

37. 37. The method of claim 35 or 36, wherein the zinc finger protein comprises a sequence of a zinc finger motif provided in Table 1.

38. the epigenetic editing system comprises a first fusion protein or a first nucleic acid encoding the first fusion protein, and a second fusion protein or a second nucleic acid encoding the second fusion protein; the first fusion protein comprises the first DNA-binding domain and the first DNMT domain; the second fusion protein comprises the second DNA-binding domain and the transcriptional repressor domain; 38. The method of any one of claims 27 to 37.

39. 39. The method of claim 38, wherein the first fusion protein comprises the sequence of a fusion protein provided herein.

40. 39. The method of claim 38, wherein the second fusion protein comprises the sequence of a fusion protein provided herein.

41. 41. The method of any one of claims 38 to 40, wherein the epigenetic editing system further comprises a third DNA-binding domain or a nucleic acid encoding same, wherein the third DNA-binding domain binds to a third target region of the HBV genome.

42. 42. The method of claim 41, wherein the third target region is located in a region of the HBV genome within nucleotides 0-303, 1000-2448, or 2802-3182.

43. 42. The method of claim 41, wherein the third target region of the HBV genome is located in a CpG island.

44. 42. The method of claim 41, wherein the third target region of the HBV genome is located in a promoter.

45. 42. The method of claim 41, wherein the third target region of the HBV genome is located in a section of the HBV genome encoding a transcript selected from the group consisting of pgRNA, precure mRNA, preS mRNA, S mRNA, and X mRNA.

46. 46. ​​The method of any one of claims 41 to 45, wherein the three DNA binding domains comprise CRISPR-Cas proteins.

47. 47. The method of Claim 46, wherein the epigenetic editing system further comprises a third gRNA comprising a region complementary to a strand of the third target region.

48. 48. The method of Claim 47, wherein the third gRNA comprises a sequence selected from a gRNA sequence provided herein, e.g., a sequence provided in Tables 14 and / or 15.

49. 46. ​​The method of any one of claims 41 to 45, wherein the third DNA binding domain comprises a zinc finger protein.

50. 50. The method of claim 49, wherein the zinc finger protein comprises a zinc finger motif having a sequence selected from the zinc finger motifs provided herein.

51. 51. The method of claim 49 or 50, wherein the zinc finger protein comprises a sequence of a zinc finger motif provided in Table 1.

52. 52. The method of any one of Claims 41 to 51, wherein the epigenetic editing system further comprises a second DNMT domain or a nucleic acid encoding same.

53. 53. The method of claim 52, wherein the second DNMT domain is a DNMT3A domain or a DNMT3L domain.

54. 54. The method of Claim 53, wherein the epigenetic editing system comprises a third fusion protein or a nucleic acid encoding same, wherein the third fusion protein comprises the third DNA-binding domain and the second DNMT domain.

55. 55. The method of claim 54, wherein the third fusion protein comprises the sequence of a fusion protein provided herein.

56. Fusion proteins or nucleic acids encoding said fusion proteins An epigenetic editing system comprising: the fusion protein (a) a DNA-binding domain that binds to a target region of an HBV gene or genome; (b) a first DNA methyltransferase (DNMT) domain; and (c) a transcriptional repressor domain; epigenetic editing systems, including

57. 57. The epigenetic system of claim 56, wherein the epigenetic editing system is capable of reducing the number of HBV viral episomes, HBV replication, or expression of gene products encoded by the HBV gene or genome, wherein the reduction is at least about 20% compared to contacting the HBV gene or genome with a suitable control.

58. 58. The epigenetic system of claim 56 or 57, wherein the HBV genome is a covalently closed circular DNA (cccDNA) or HBV integrated DNA.

59. 59. The epigenetic system of any one of claims 56 to 58, wherein the HBV genome comprises HBV genotype A, HBV genotype B, HBV genotype C, HBV genotype D, HBV genotype E, HBV genotype F, HBV genotype G or HBV genotype H.

60. 60. The epigenetic system of any one of claims 56 to 59, wherein the HBV genome comprises a sequence having at least 80% identity to the HBV genome sequence provided herein.

61. 61. The epigenetic system of any one of claims 56 to 60, wherein the target region is located in a region of the HBV genome within nucleotides 0 to 303, 1000 to 2448, or 2802 to 3182 of the HBV genome sequence provided herein.

62. 61. The epigenetic system of any one of claims 56 to 60, wherein the target region of the HBV genome is located in a CpG island.

63. 61. The epigenetic system of any one of claims 56 to 60, wherein the target region of the HBV genome is located in a promoter.

64. 61. The epigenetic system of any one of claims 56-60, wherein the target region of the HBV genome is located in a section of the HBV genome encoding a transcript selected from the group consisting of pgRNA, precure mRNA, preS mRNA, S mRNA, and X mRNA.

65. 65. The epigenetic system of claims 56 to 64, wherein the DNA binding domain comprises a CRISPR-Cas protein.

66. 66. The epigenetic system of Claim 65, wherein the epigenetic editing system further comprises a gRNA comprising a region complementary to a strand of the target region.

67. 67. The epigenetic system of Claim 66, wherein the gRNA comprises a sequence selected from the gRNA sequences provided herein, e.g., in Tables 14 and / or 15.

68. 65. The epigenetic system of any one of claims 56 to 64, wherein the DNA binding domain comprises a zinc finger protein.

69. 69. The epigenetic system of claim 68, wherein the zinc finger protein comprises a zinc finger motif having a sequence selected from the zinc finger motifs provided herein.

70. 70. The epigenetic system of claim 68 or 69, wherein the zinc finger protein comprises a sequence of a zinc finger motif provided in Table 1.

71. 71. The epigenetic system of any one of claims 56 to 70, wherein the transcriptional repressor domain comprises the sequence of a transcriptional repressor provided herein.

72. 72. The epigenetic system of any one of claims 56 to 71, wherein the first DNMT domain is a DNMT3A domain or a DNMT3L domain.

73. 73. The epigenetic system of claim 72, wherein the DNMT domain comprises the sequence of a DNMT domain provided herein.

74. 74. The epigenetic system of any one of claims 56 to 73, wherein the fusion protein further comprises a second DNMT domain.

75. 75. The epigenetic system of claim 74, wherein the second DNMT domain is a DNMT3A domain or a DNMT3L domain.

76. 76. The epigenetic system of any one of claims 56 to 75, wherein the fusion protein further comprises a nuclear localization sequence (NLS).

77. 77. The epigenetic system of claim 76, wherein the fusion protein comprises the sequence of a fusion protein provided herein.

78. a first fusion protein or a nucleic acid encoding the first fusion protein, wherein the first fusion protein comprises a first DNA-binding domain and a first DNMT domain, and the first DNA-binding domain binds to a first target region of the HBV genome; a second fusion protein or a nucleic acid encoding the second fusion protein, wherein the second fusion protein comprises a second DNA-binding domain and a transcriptional repressor domain, and the second DNA-binding domain binds to a second target region of the HBV genome; epigenetic editing systems, including

79. 79. The epigenetic system of claim 78, wherein the epigenetic editing system is capable of reducing the number of HBV viral episomes, HBV replication, or expression of gene products encoded by the HBV genome, wherein the reduction is at least about 20% compared to contacting the HBV genome with a suitable control.

80. 80. The epigenetic system of claim 78 or 79, wherein the HBV genome is a covalently closed circular DNA (cccDNA) or HBV integrated DNA.

81. 81. The epigenetic system of any one of claims 78 to 80, wherein the HBV genome comprises HBV genotype A, HBV genotype B, HBV genotype C, HBV genotype D, HBV genotype E, HBV genotype F, HBV genotype G or HBV genotype H.

82. 82. The epigenetic system of any one of claims 78 to 81, wherein the HBV genome comprises a sequence having at least 80% identity to the HBV genome provided herein.

83. 82. The epigenetic system of any one of claims 78 to 81, further comprising a third fusion protein or a nucleic acid encoding said third fusion protein, said third fusion protein comprising a third DNA-binding domain and a second DNMT domain, said third DNA-binding domain binding to a third target region of the HBV genome.

84. 84. The epigenetic system of claim 83, wherein the first target region, the second target region, or the third target region is located in a region of the HBV genome within nucleotides 0-303, 1000-2448, or 2802-3182 of the HBV genome provided herein.

85. 84. The epigenetic system of claim 83, wherein the first target region, the second target region, or the third target region of the HBV genome is located in a CpG island.

86. 84. The epigenetic system of claim 83, wherein the first target region, the second target region, or the third target region of the HBV genome is located in a promoter.

87. 84. The epigenetic system of claim 83, wherein the first target region, the second target region, or the third target region of the HBV genome is located in an area of ​​the HBV genome encoding a transcript selected from the group consisting of pgRNA, preCure mRNA, preS mRNA, S mRNA, and X mRNA.

88. 84. The epigenetic system of Claim 83, wherein the first DNA binding domain, the second DNA binding domain, or the third DNA binding domain comprises a CRISPR-Cas protein.

89. 89. The epigenetic system of Claim 88, wherein the epigenetic editing system further comprises a first gRNA comprising a region complementary to a strand of the first target region, a second gRNA comprising a region complementary to a strand of the second target region, or a third gRNA comprising a region complementary to a strand of the third target region.

90. 90. The epigenetic system of Claim 89, wherein the first gRNA comprises a sequence selected from the gRNA sequences provided herein, e.g., provided in Tables 14 and / or 15, the second gRNA comprises a sequence selected from the gRNA sequences provided herein, e.g., provided in Tables 14 and / or 15, and / or the third gRNA comprises a sequence selected from the gRNA sequences provided herein, e.g., provided in Tables 14 and / or 15.

91. 84. The epigenetic system of Claim 83, wherein the first DNA binding domain, the second DNA binding domain, or the third DNA binding domain comprises a zinc finger protein.

92. 92. The epigenetic system of claim 91, wherein the zinc finger protein comprises a zinc finger motif having a sequence selected from the zinc finger motifs provided herein.

93. 93. The epigenetic system of claim 91 or 92, wherein the zinc finger protein comprises a sequence of a zinc finger motif provided in Table 1.

94. 94. The epigenetic system of any one of claims 78 to 93, wherein the transcriptional repressor domain comprises ZIM3.

95. 95. The epigenetic system of any one of claims 78 to 94, wherein the first DNMT domain is a DNMT3A domain or a DNMT3L domain.

96. 96. The epigenetic system of claim 95, wherein the first DNMT domain comprises a sequence of a DNMT provided herein.

97. 84. The epigenetic system of claim 83, wherein the second DNMT domain is a DNMT3A domain or a DNMT3L domain.

98. 98. The epigenetic system of claim 97, wherein the second DNMT domain comprises the sequence of a DNMT domain provided herein.

99. 99. The epigenetic system of any one of claims 78 to 98, wherein the first fusion protein comprises the sequence of a fusion protein provided herein.

100. 100. The epigenetic system of any one of claims 78 to 99, wherein the second fusion protein comprises the sequence of a fusion protein provided herein.

101. 100. The epigenetic system of any one of claims 83 to 99, wherein the third fusion protein comprises the sequence of a fusion protein provided herein.

102. 56. The method of any one of Claims 1-55, wherein the epigenetic editing system comprises a nucleic acid sequence provided in Table 20.

103. 1. A method of treating an HDV infection in a subject, comprising administering to the subject an epigenetic editing system; The epigenetic editing system a first DNA-binding domain, a first DNMT domain, and a transcriptional repressor domain, or one or more nucleic acid molecules encoding them Including, the first DNA-binding domain binds to a first target region of an HBV gene or genome; To bring into contact the number of HDV viral episomes, Replication of HDV genes or genomes, or Expression of the protein product encoded by said HDV gene or genome resulting in a reduction of wherein said reduction is at least about 20% as compared to administering a suitable control.

104. 1. A method of inhibiting viral replication in a cell infected with HDV, comprising administering an epigenetic editing system; The epigenetic editing system a first DNA-binding domain, a first DNMT domain, and a transcriptional repressor domain, or one or more nucleic acid molecules encoding them Including, the first DNA-binding domain binds to a first target region of an HBV gene or genome, and the epigenetic editing system targets the target region of the HBV gene or genome; the contacting results in a reduction in the number of HDV viral episomes or replication of HDV genes or genomes; wherein said reduction is at least about 20% as compared to administering a suitable control.

105. 105. The method of Claim 103 or 104, wherein the first DNA binding domain comprises a CRISPR-Cas protein.

106. 106. The method of Claim 105, wherein the epigenetic editing system further comprises a first guide RNA (gRNA) comprising a region complementary to a strand of the first target region.

107. 107. The method of Claim 106, wherein the gRNA comprises a sequence selected from the gRNAs provided herein, e.g., in Tables 14 and / or 15.

108. 105. The method of claim 103 or 104, wherein the first DNA binding domain comprises a zinc finger protein.

109. 109. The method of claim 108, wherein the zinc finger protein comprises a zinc finger motif having a sequence selected from any zinc finger or zinc finger motif provided herein, e.g., in Table 1.

110. 110. The method of claim 108 or 109, wherein the zinc finger protein comprises the sequence of any of the zinc finger epigenetic repressors provided herein.

111. 111. The method of any one of claims 103 to 110, wherein the transcriptional repressor domain comprises ZIM3.

112. 112. The method of any one of claims 103 to 111, wherein the first DNMT domain is a DNMT3A domain or a DNMT3L domain.

113. 113. The method of claim 112, wherein the first DNMT domain comprises the sequence of a DNMT domain provided herein.

114. 114. The method of any one of Claims 103 to 113, wherein the epigenetic editing system further comprises a second DNMT domain or a nucleic acid encoding same.

115. 115. The method of claim 114, wherein the second DNMT domain is a DNMT3A domain or a DNMT3L domain.

116. 116. The method of claim 115, wherein the second DNMT domain comprises the sequence of a DNMT domain provided herein.

117. 117. The method of any one of Claims 114 to 116, wherein the epigenetic editing system comprises a fusion protein or a nucleic acid encoding same, wherein the fusion protein comprises the first DNA binding domain, the first DNMT domain, the repressor domain, and the second DNMT domain.

118. 118. The method of claim 117, wherein the fusion protein further comprises a nuclear localization sequence (NLS).

119. 119. The method of claim 118, wherein the fusion protein comprises the sequence of a fusion protein provided herein.

120. 120. The method of any one of claims 103 to 119, wherein the first DNA-binding domain binds to a target region of an HBV gene or genome that encodes or controls the expression of an S antigen.

121. 1. A method comprising administering an epigenetic editing system to a subject in need thereof, The epigenetic editing system a first DNA-binding domain, a first DNMT domain, and a transcriptional repressor domain, or one or more nucleic acid molecules encoding them Including, the first DNA-binding domain binds to a first target region of an HBV gene or genome; To bring into contact the number of HBV viral episomes, Replication of the HBV gene or genome, or Expression of the protein product encoded by the HBV gene or genome resulting in a reduction in wherein said reduction is at least about 20% as compared to administering a suitable control.

122. 122. The method of any one of Claims 103-121, wherein the epigenetic editing system comprises a nucleic acid sequence provided in Table 20.

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